The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification

PubMed Central

2017-01-01

The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a “channel dye” into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation−π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation−π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation−π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors. PMID:28221782

The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.

PubMed

Liu, Yu; Miao, Kun; Dunham, Noah P; Liu, Hongbin; Fares, Matthew; Boal, Amie K; Li, Xiaosong; Zhang, Xin

2017-03-21

The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

PubMed

Hori, Yuichiro; Otomura, Norimichi; Nishida, Ayuko; Nishiura, Miyako; Umeno, Maho; Suetake, Isao; Kikuchi, Kazuya

2018-02-07

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

PubMed

Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

2017-08-01

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Synthetic biology for microbial heavy metal biosensors.

PubMed

Kim, Hyun Ju; Jeong, Haeyoung; Lee, Sang Jun

2018-02-01

Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

PubMed Central

Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

2012-01-01

Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

PubMed Central

Wang, Baojun; Barahona, Mauricio; Buck, Martin

2013-01-01

Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas. PMID:22981411

Wash-free and selective imaging of epithelial cell adhesion molecule (EpCAM) expressing cells with fluorogenic peptide ligands.

PubMed

K C, Tara Bahadur; Suga, Kanako; Isoshima, Takashi; Aigaki, Toshiro; Ito, Yoshihiro; Shiba, Kiyotaka; Uzawa, Takanori

2018-06-02

Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses. Copyright © 2018 Elsevier Inc. All rights reserved.

A photophysical study of two fluorogen-activating proteins bound to their cognate fluorogens

NASA Astrophysics Data System (ADS)

Gaiotto, Tiziano; Nguyen, Hau B.; Jung, Jaemyeong; Gnanakaran, Gnana S.; Schmidt, Jurgen G.; Waldo, Geoffrey S.; Bradbury, Andrew M.; Goodwin, Peter M.

2011-03-01

We are exploring the use of fluorogen-activating proteins (FAPs) as reporters for single-molecule imaging. FAPs are single-chain antibodies selected to specifically bind small chromophoric molecules termed fluorogens. Upon binding to its cognate FAP the fluorescence quantum yield of the fluorogen increases giving rise to a fluorescent complex. Based on the seminal work of Szent-Gyorgyi et al. (Nature Biotechnology, Volume 26, Number 2, pp 235-240, 2008) we have chosen to study two fluorogen-activating single-chain antibodies, HL1.0.1-TO1 and H6-MG, bound to their cognate fluorogens, thiazole orange and malachite green derivatives, respectively. Here we use fluorescence correlation spectroscopy to study the photophysics of these fluorescent complexes.

A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging.

PubMed

Komatsu, Naoki; Terai, Kenta; Imanishi, Ayako; Kamioka, Yuji; Sumiyama, Kenta; Jin, Takashi; Okada, Yasushi; Nagai, Takeharu; Matsuda, Michiyuki

2018-06-12

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.

[Receptor elements for biosensors in two ways of methylotrophic yeast immobilization].

PubMed

Zaĭtsev, M G; Arliapov, V A; Alferov, V A; Reshetilov, A N

2012-01-01

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 In yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05-1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2-1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzochinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Expanding the Use of a Fluorogenic Method to Determine Activity and Mode of Action of Bacillus thuringiensis Bacteriocins Against Gram-Positive and Gram-Negative Bacteria

PubMed Central

de la Fuente-Salcido, Norma M.; Barboza-Corona, J. Eleazar; Espino Monzón, A. N.; Pacheco Cano, R. D.; Balagurusamy, N.; Bideshi, Dennis K.; Salcedo-Hernández, Rubén

2012-01-01

Previously we described a rapid fluorogenic method to measure the activity of five bacteriocins produced by Mexican strains of Bacillus thuringiensis against B. cereus 183. Here we standardize this method to efficiently determine the activity of bacteriocins against both Gram-positive and Gram-negative bacteria. It was determined that the crucial parameter required to obtain reproducible results was the number of cells used in the assay, that is, ~4 × 108 cell/mL and ~7 × 108 cell/mL, respectively, for target Gram-positive and Gram-negative bacteria. Comparative analyses of the fluorogenic and traditional well-diffusion assays showed correlation coefficients of 0.88 to 0.99 and 0.83 to 0.99, respectively, for Gram-positive and Gram-negative bacteria. The fluorogenic method demonstrated that the five bacteriocins of B. thuringiensis have bacteriolytic and bacteriostatic activities against all microorganisms tested, including clinically significant bacteria such as Listeria monocytogenes, Proteus vulgaris, and Shigella flexneri reported previously to be resistant to the antimicrobials as determined using the well-diffusion protocol. These results demonstrate that the fluorogenic assay is a more sensitive, reliable, and rapid method when compared with the well-diffusion method and can easily be adapted in screening protocols for bacteriocin production by other microorganisms. PMID:22919330

Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

PubMed

Wynn, Daniel; Deo, Sapna; Daunert, Sylvia

2017-01-01

Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field. © 2017 Elsevier Inc. All rights reserved.

A hard-soft microfluidic-based biosensor flow cell for SPR imaging application.

PubMed

Liu, Changchun; Cui, Dafu; Li, Hui

2010-09-15

An ideal microfluidic-based biosensor flow cell should have not only a "soft" interface for high strength sealing with biosensing chips, but also "hard" macro-to-micro interface for tubing connection. Since these properties are exclusive of each other, no one material can provide the advantages of both. In this paper, we explore the application of a SiO(2) thin film, deposited by plasma-enhanced chemical vapor deposition (PECVD) technology, as an intermediate layer for irreversibly adhering polydimethylsiloxane (PDMS) to plastic substrate, and develop a hard-soft, compact, robust microfluidic-based biosensor flow cell for the multi-array immunoassay application of surface plasmon resonance (SPR) imaging. This hard-soft biosensor flow cell consists of one rigid, computer numerically controlled (CNC)-machined poly(methyl methacrylate) (PMMA) base coated with a 200 nm thick SiO(2) thin film, and one soft PDMS microfluidic layer. This novel microfluidic-based biosensor flow cell does not only keep the original advantage of conventional PDMS-based biosensor flow cell such as the intrinsically soft interface, easy-to-fabrication, and low cost, but also has a rigid, robust, easy-to-use interface to tubing connection and can be operated up to 185 kPa in aqueous environments without failure. Its application was successfully demonstrated with two types of experiments by coupling with SPR imaging biosensor: the real-time monitoring of the immunoglobulin G (IgG) interaction, as well as the detection of sulfamethoxazole (SMOZ) and sulfamethazine (SMZ) with the sensitivity of 3.5 and 0.6 ng/mL, respectively. This novel hard-soft microfluidic device is also useful for a variety of other biosensor flow cells. Copyright 2010 Elsevier B.V. All rights reserved.

Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor.

PubMed

Lau, Hui-Chong; Lee, In-Kyu; Ko, Pan-Woo; Lee, Ho-Won; Huh, Jeung-Soo; Cho, Won-Ju; Lim, Jeong-Ok

2015-01-01

Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer's disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor was built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), resulted in an increase in the sensitivity and reliability of detection. The biosensor was designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, we collected 20 saliva samples from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson's disease (PD), and a control group composed of healthy individuals. We then studied the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor. The cell-based biosensor significantly distinguished salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, we propose that salivary trehalose, might be a potential biomarker for AD and could be detected using our cell-based EG-ISFET biosensor. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and may be used in the future as a screening method for AD.

Twisted cyanines: a non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule.

PubMed

Shank, Nathaniel I; Pham, Ha H; Waggoner, Alan S; Armitage, Bruce A

2013-01-09

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.

Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule

PubMed Central

Shank, Nathaniel I.; Pham, Ha; Waggoner, Alan S.; Armitage, Bruce A.

2013-01-01

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates non-specific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules. PMID:23252842

Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

PubMed

Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

2017-01-01

Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

Immune biosensors based on the SPR and TIRE: efficiency of their application for bacteria determination

NASA Astrophysics Data System (ADS)

Starodub, N. F.; Ogorodniichuk, J.; Lebedeva, T.; Shpylovyy, P.

2013-11-01

In this work we have designed high-specific biosensors for Salmonella typhimurium detection based on the surface plasmon resonance (SPR) and total internal reflection ellipsometry (TIRE). It has been demonstrated high selectivity and sensitivity of analysis. As a registering part for our experiments the Spreeta (USA) and "Plasmonotest" (Ukraine) with flowing cell have been applied among of SPR device. Previous researches confirmed an efficiency of SPR biosensors using for detecting of specific antigen-antibody interactions therefore this type of reactions with some previous preparations of surface binding layer was used as reactive part. It has been defined that in case with Spreeta sensitivity was on the level 103 - 107 cells/ml. Another biosensor based on the SPR has shown the sensitivity within 101 - 106 cells/ml. Maximal sensitivity was on the level of several cells in 10 ml (up to the fact that less than 5 cells) which has been obtained using the biosensor based on TIRE.

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine).

PubMed

Akyilmaz, Erol; Yaşa, Ihsan; Dinçkaya, Erhan

2006-07-01

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.

A new pyrene based highly sensitive fluorescence probe for copper(II) and fluoride with living cell application.

PubMed

Goswami, Shyamaprosad; Chakraborty, Shampa; Paul, Sima; Halder, Sandipan; Panja, Sukanya; Mukhopadhyay, Subhra Kanti

2014-05-21

A new pyrene based fluorescence probe has been synthesized for fluorogenic detection of Cu(2+) in acetonitrile-aqueous media (7 : 3 CH3CN-HEPES buffer, v/v, at pH 7.5) with bioimaging in both prokaryotic (Candida albicans cells) and eukaryotic (Tecoma stans pollen cells) living cells. The anion recognition properties of the sensor have also been studied in acetonitrile by fluorescence methods which show remarkable sensitivity toward fluoride over other anions examined.

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

Bistetrazine-cyanines as double-clicking fluorogenic two-point binder or crosslinker probes.

PubMed

Kormos, Attila; Koehler, Christine; Fodor, Eszter; Rutkai, Zsófia; Martin, Maddison; Mező, Gábor; Lemke, Edward; Kele, Péter

2018-04-20

Fluorogenic probes are capable of minimizing background fluorescence of unreacted and non-specifically adsorbed reagents. The preceding years have brought substantial developments in the design and synthesis of bioorthogonally applicable fluorogenic systems mainly based on the quenching effects of azide and tetrazine moieties. The modulation power exerted by these bioorthogonal motifs typically becomes less efficient on more conjugated systems, i.e. on probes with red-shifted emission wavelength. In order to reach efficient quenching, i.e. fluorogenicity even in the red range of the spectrum, We present the synthesis, fluorogenic and conjugation characterization of bistetrazine-cyanine probes with emission maxima between 600-620 nm. The probes can bind to genetically altered proteins harboring an 11-amino acid peptide tag with two appending cyclooctyne motifs. Moreover, we also demonstrate the use of these bistetrazines as fluorogenic, covalent cross-linkers between monocyclooctynylated proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

PubMed Central

Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2013-01-01

Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

PubMed

Ku, Minhee; Hong, Yoochan; Heo, Dan; Lee, Eugene; Hwang, Seungyeon; Suh, Jin-Suck; Yang, Jaemoon

2016-03-15

Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model. Copyright © 2015 Elsevier B.V. All rights reserved.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

A vertically aligned carbon nanotube-based impedance sensing biosensor for rapid and high sensitive detection of cancer cells.

PubMed

Abdolahad, Mohammad; Taghinejad, Mohammad; Taghinejad, Hossein; Janmaleki, Mohsen; Mohajerzadeh, Shams

2012-03-21

A novel vertically aligned carbon nanotube based electrical cell impedance sensing biosensor (CNT-ECIS) was demonstrated for the first time as a more rapid, sensitive and specific device for the detection of cancer cells. This biosensor is based on the fast entrapment of cancer cells on vertically aligned carbon nanotube arrays and leads to mechanical and electrical interactions between CNT tips and entrapped cell membranes, changing the impedance of the biosensor. CNT-ECIS was fabricated through a photolithography process on Ni/SiO(2)/Si layers. Carbon nanotube arrays have been grown on 9 nm thick patterned Ni microelectrodes by DC-PECVD. SW48 colon cancer cells were passed over the surface of CNT covered electrodes to be specifically entrapped on elastic nanotube beams. CNT arrays act as both adhesive and conductive agents and impedance changes occurred as fast as 30 s (for whole entrapment and signaling processes). CNT-ECIS detected the cancer cells with the concentration as low as 4000 cells cm(-2) on its surface and a sensitivity of 1.7 × 10(-3)Ω cm(2). Time and cell efficiency factor (TEF and CEF) parameters were defined which describe the sensor's rapidness and resolution, respectively. TEF and CEF of CNT-ECIS were much higher than other cell based electrical biosensors which are compared in this paper.

Fluorogenic reaction-based prodrug conjugates as targeted cancer theranostics.

PubMed

Lee, Min Hee; Sharma, Amit; Chang, Min Jung; Lee, Jinju; Son, Subin; Sessler, Jonathan L; Kang, Chulhun; Kim, Jong Seung

2018-01-02

Theranostic systems are receiving ever-increasing attention due to their potential therapeutic utility, imaging enhancement capability, and promise for advancing the field of personalized medicine, particularly as it relates to the diagnosis, staging, and treatment of cancer. In this Tutorial Review, we provide an introduction to the concepts of theranostic drug delivery effected via use of conjugates that are able to target cancer cells selectively, provide cytotoxic chemotherapeutics, and produce readily monitored imaging signals in vitro and in vivo. The underlying design concepts, requiring the synthesis of conjugates composed of imaging reporters, masked chemotherapeutic drugs, cleavable linkers, and cancer targeting ligands, are discussed. Particular emphasis is placed on highlighting the potential benefits of fluorogenic reaction-based targeted systems that are activated for both imaging and therapy by cellular entities, e.g., thiols, reactive oxygen species and enzymes, which are present at relatively elevated levels in tumour environments, physiological characteristics of cancer, e.g., hypoxia and acidic pH. Also discussed are systems activated by an external stimulus, such as light. The work summarized in this Tutorial Review will help define the role fluorogenic reaction-based, cancer-targeting theranostics may have in advancing drug discovery efforts, as well as improving our understanding of cellular uptake and drug release mechanisms.

NASA Tech Briefs, January 2010

NASA Technical Reports Server (NTRS)

2010-01-01

Topics covered include: Cryogenic Flow Sensor; Multi-Sensor Mud Detection; Gas Flow Detection System; Mapping Capacitive Coupling Among Pixels in a Sensor Array; Fiber-Based Laser Transmitter for Oxygen A-Band Spectroscopy and Remote Sensing; Low-Profile, Dual-Wavelength, Dual-Polarized Antenna; Time-Separating Heating and Sensor Functions of Thermistors in Precision Thermal Control Applications; Cellular Reflectarray Antenna; A One-Dimensional Synthetic-Aperture Microwave Radiometer; Electrical Switching of Perovskite Thin-Film Resistors; Two-Dimensional Synthetic-Aperture Radiometer; Ethernet-Enabled Power and Communication Module for Embedded Processors; Electrically Variable Resistive Memory Devices; Improved Attachment in a Hybrid Inflatable Pressure Vessel; Electrostatic Separator for Beneficiation of Lunar Soil; Amorphous Rover; Space-Frame Antenna; Gear-Driven Turnbuckle Actuator; In-Situ Focusing Inside a Thermal Vacuum Chamber; Space-Frame Lunar Lander; Wider-Opening Dewar Flasks for Cryogenic Storage; Silicon Oxycarbide Aerogels for High-Temperature Thermal Insulation; Supercapacitor Electrolyte Solvents with Liquid Range Below -80 C; Designs and Materials for Better Coronagraph Occulting Masks; Fuel-Cell-Powered Vehicle with Hybrid Power Management; Fine-Water-Mist Multiple-Orientation-Discharge Fire Extinguisher; Fuel-Cell Water Separator; Turbulence and the Stabilization Principle; Improved Cloud Condensation Nucleus Spectrometer; Better Modeling of Electrostatic Discharge in an Insulator; Sub-Aperture Interferometers; Terahertz Mapping of Microstructure and Thickness Variations; Multiparallel Three-Dimensional Optical Microscopy; Stabilization of Phase of a Sinusoidal Signal Transmitted Over Optical Fiber; Vacuum-Compatible Wideband White Light and Laser Combiner Source System; Optical Tapers as White-Light WGM Resonators; EPR Imaging at a Few Megahertz Using SQUID Detectors; Reducing Field Distortion in Magnetic Resonance Imaging; Fluorogenic Cell-Based Biosensors for Monitoring Microbes; A Constant-Force Resistive Exercise Unit; GUI to Facilitate Research on Biological Damage from Radiation; On-Demand Urine Analyzer; More-Realistic Digital Modeling of a Human Body; and Advanced Liquid-Cooling Garment Using Highly Thermally Conductive Sheets.

FRET and BRET-based biosensors in live cell compound screens.

PubMed

Robinson, Katie Herbst; Yang, Jessica R; Zhang, Jin

2014-01-01

Live cell compound screening with genetically encoded fluorescence or bioluminescence-based biosensors offers a potentially powerful approach to identify novel regulators of a signaling event of interest. In particular, compound screening in living cells has the added benefit that the entire signaling network remains intact, and thus the screen is not just against a single molecule of interest but against any molecule within the signaling network that may modulate the distinct signaling event reported by the biosensor in use. Furthermore, only molecules that are cell permeable or act at cell surface receptors will be identified as "hits," thus reducing further optimization of the compound in terms of cell penetration. Here we discuss a detailed protocol for using genetically encoded biosensors in living cells in a 96-well format for the execution of high throughput compound screens and the identification of small molecules which modulate a signaling event of interest.

Biosensoric potential of microbial fuel cells.

PubMed

Schneider, György; Kovács, Tamás; Rákhely, Gábor; Czeller, Miklós

2016-08-01

Recent progress in microbial fuel cell (MFC) technology has highlighted the potential of these devices to be used as biosensors. The advantages of MFC-based biosensors are that they are phenotypic and can function in either assay- or flow-through formats. These features make them appropriate for contiguous on-line monitoring in laboratories and for in-field applications. The selectivity of an MFC biosensor depends on the applied microorganisms in the anodic compartment where electron transfer (ET) between the artificial surface (anode) and bacterium occurs. This process strongly determines the internal resistance of the sensoric system and thus influences signal outcome and response time. Despite their beneficial characteristics, the number of MFC-based biosensoric applications has been limited until now. The aim of this mini-review is to turn attention to the biosensoric potential of MFCs by summarizing ET mechanisms on which recently established and future sensoric devices are based.

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging.

PubMed

Chyan, Wen; Raines, Ronald T

2018-06-20

Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chemical biology. Those probes that respond to enzymatic activity illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, we review recent advances in enzyme-activated fluorogenic probes for biological imaging. We organize our survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside of therapeutic, diagnostic, and theranostic opportunities.

A photophysical study of two fluorogen-activating proteins bound to their cognate fluorogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaiotto, Tiziano; Nguyen, Hau B; Jung, Jaemyeong

We are exploring the feasibility of using recently developed flu orogen-activating proteins (FAPs) as reporters for single-molecule imaging. FAPs are single-chain antibodies choosen to specifically bind small chromophoric molecules termed f1uorogens. Upon binding to its cognate FAP the fluorescence quantum yield of the fluorogen can increase substantially giving rise to a fluorescent complex. Based on the seminal work of Szent-Gyorgyi et al. (Nature Biotechnology, Volume 26, Number 2, pp 235-240, 2008) we have chosen to study two fluorogen-activating single-chain antibodies, HL 1.0.1-TOI and H6-MG bound to their cognate fluorogens, thiazole orange and malachite green derivatives, respectively. Here we use fluorescencemore » correlation spectroscopy study the photophysics of these fluorescent complexes.« less

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

PubMed Central

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P.; Segal, Ester

2016-01-01

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps. PMID:27901131

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry.

PubMed

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P; Segal, Ester

2016-11-30

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >10 7  cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

NASA Astrophysics Data System (ADS)

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P.; Segal, Ester

2016-11-01

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

Electronic Biosensors Based on III-Nitride Semiconductors.

PubMed

Kirste, Ronny; Rohrbaugh, Nathaniel; Bryan, Isaac; Bryan, Zachary; Collazo, Ramon; Ivanisevic, Albena

2015-01-01

We review recent advances of AlGaN/GaN high-electron-mobility transistor (HEMT)-based electronic biosensors. We discuss properties and fabrication of III-nitride-based biosensors. Because of their superior biocompatibility and aqueous stability, GaN-based devices are ready to be implemented as next-generation biosensors. We review surface properties, cleaning, and passivation as well as different pathways toward functionalization, and critically analyze III-nitride-based biosensors demonstrated in the literature, including those detecting DNA, bacteria, cancer antibodies, and toxins. We also discuss the high potential of these biosensors for monitoring living cardiac, fibroblast, and nerve cells. Finally, we report on current developments of covalent chemical functionalization of III-nitride devices. Our review concludes with a short outlook on future challenges and projected implementation directions of GaN-based HEMT biosensors.

Recent progress on the development of biofuel cells for self-powered electrochemical biosensing and logic biosensing: A review

DOE PAGES

Zhou, Ming

2015-06-12

Biofuel cells (BFCs) based on enzymes and microorganisms have been recently received considerable attention because they are recognized as an attractive type of energy conversion technology. In addition to the research activities related to the application of BFCs as power source, we have witnessed recently a growing interest in using BFCs for self-powered electrochemical biosensing and electrochemical logic biosensing applications. Compared with traditional biosensors, one of the most significant advantages of the BFCs-based self-powered electrochemical biosensors and logic biosensors is their ability to detect targets integrated with chemical-to-electrochemical energy transformation, thus obviating the requirement of external power sources. Following mymore » previous review (Electroanalysis 2012, 24, 197-209), the present review summarizes, discusses and updates the most recent progress and latest advances on the design and construction of BFCs-based self-powered electrochemical biosensors and logic biosensors. In addition to the traditional approaches based on substrate effect, inhibition effect, blocking effect and gene regulation effect for BFCs-based self-powered electrochemical biosensors and logic biosensors design, some new principles including enzyme effect, co-stabilization effect, competition effect and hybrid effect are summarized and discussed by me in details. The outlook and recommendation of future directions of BFCs-based self-powered electrochemical biosensors and logic biosensors are discussed in the end.« less

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

PubMed

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Fluorescent nanoparticles based on AIE fluorogens for bioimaging.

PubMed

Yan, Lulin; Zhang, Yan; Xu, Bin; Tian, Wenjing

2016-02-07

Fluorescent nanoparticles (FNPs) have recently attracted increasing attention in the biomedical field because of their unique optical properties, easy fabrication and outstanding performance in imaging. Compared with conventional molecular probes including small organic dyes and fluorescent proteins, FNPs based on aggregation-induced emission (AIE) fluorogens have shown significant advantages in tunable emission and brightness, good biocompatibility, superb photo- and physical stability, potential biodegradability and facile surface functionalization. In this review, we summarize the latest advances in the development of fluorescent nanoparticles based on AIE fluorogens including polymer nanoparticles and silica nanoparticles over the past few years, and the various biomedical applications based on these fluorescent nanoparticles are also elaborated.

Microfluidic differential immunocapture biochip for specific leukocyte counting

PubMed Central

Hassan, Umer; Watkins, Nicholas N; Reddy, Bobby; Damhorst, Gregory; Bashir, Rashid

2016-01-01

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes—e.g., for counting of cD4 and cD8 t cells in HIV/aIDs diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 µl of blood. this paper provides a comprehensive stepwise protocol to replicate our biosensor for cD4 and cD8 cell counts. the biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. the biosensor production requires 24 h, a one-time cell capture optimization takes 6–9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete. PMID:26963632

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

PubMed

Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

2017-01-01

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

A Novel Cell-Based Hybrid Acoustic Wave Biosensor with Impedimetric Sensing Capabilities

PubMed Central

Liu, Fei; Li, Fang; Nordin, Anis Nurashikin; Voiculescu, Ioana

2013-01-01

A novel multiparametric biosensor system based on living cells will be presented. The biosensor system includes two biosensing techniques on a single device: resonant frequency measurements and electric cell-substrate impedance sensing (ECIS). The multiparametric sensor system is based on the innovative use of the upper electrode of a quartz crystal microbalance (QCM) resonator as working electrode for the ECIS technique. The QCM acoustic wave sensor consists of a thin AT-cut quartz substrate with two gold electrodes on opposite sides. For integration of the QCM with the ECIS technique a semicircular counter electrode was fabricated near the upper electrode on the same side of the quartz crystal. Bovine aortic endothelial live cells (BAECs) were successfully cultured on this hybrid biosensor. Finite element modeling of the bulk acoustic wave resonator using COMSOL simulations was performed. Simultaneous gravimetric and impedimetric measurements performed over a period of time on the same cell culture were conducted to validate the device's sensitivity. The time necessary for the BAEC cells to attach and form a compact monolayer on the biosensor was 35∼45 minutes for 1.5 × 104 cells/cm2 BAECs; 60 minutes for 2.0 × 104 cells/cm2 BAECs; 70 minutes for 3.0 × 104 cells/cm2 BAECs; and 100 minutes for 5.0 × 104 cells/cm2 BAECs. It was demonstrated that this time is the same for both gravimetric and impedimetric measurements. This hybrid biosensor will be employed in the future for water toxicity detection. PMID:23459387

A luminescent hybridoma-based biosensor for rapid detection of V. cholerae upon induction of calcium signaling pathway.

PubMed

Zamani, Parichehr; Sajedi, Reza H; Hosseinkhani, Saman; Zeinoddini, Mehdi; Bakhshi, Bita

2016-05-15

In this study, a hybridoma based biosensor was developed for rapid, sensitive and selective detection of Vibrio cholerae O1 which converts the antibody-antigen binding to bioluminescence light. After investigation on hybridoma performance, the biosensor was constructed by transfecting specific hybridoma cells with aequorin reporter gene and the bioluminescence activities of stable biosensor were measured. The sensitivity of biosensor was as few as 50 CFU/ml and it showed no responses to other entric bacteria. Moreover, the response time of biosensor was estimated in 7th second which means this method is considerably faster than many available detection assays. In addition, this biosensor was successfully applied to V. cholerae detection in environmental samples with no significant loss in sensitivity, demonstrating our proposed biosensor provides a sensitive and reliable method for detection of V. cholerae in natural samples. The application of whole hybridoma cell directly as a sensing element in biosensor construction which mentioned for the first time in present study suggests that hybridoma cells could provide a valuable tool for future studies in both basic and diagnostic sciences and could be considered as a fast and specific sensing element for detection of other pathogens in different applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Versatile graphene biosensors for enhancing human cell therapy.

PubMed

Vlăsceanu, George M; Amărandi, Roxana-Maria; Ioniță, Mariana; Tite, Teddy; Iovu, Horia; Pilan, Luisa; Burns, Jorge S

2018-05-01

Technological advances in engineering and cell biology stimulate novel approaches for medical treatment, in particular cell-based therapy. The first cell-based gene therapy against cancer was recently approved by the US Food and Drug Administration. Progress in cancer diagnosis includes a blood test detecting five cancer types. Numerous stem cell phase I/II clinical trials showing safety and efficacy will soon pursue qualifying criteria for advanced therapy medicinal products (ATMP), aspiring to join the first stem-cell therapy approved by the European Medicines Agency. Cell based therapy requires extensive preclinical characterisation of biomarkers indicating mechanisms of action crucial to the desired therapeutic effect. Quantitative analyses monitoring critical functions for the manufacture of optimal cell and tissue-based clinical products include successful potency assays for implementation. The challenge to achieve high quality measurement is increasingly met by progress in biosensor design. We adopt a cell therapy perspective to highlight recent examples of graphene-enhanced biointerfaces for measurement of biomarkers relevant to cancer treatment, diagnosis and tissue regeneration. Graphene based biosensor design problems can thwart their use for health care transformative point of care testing and real-time applications. We discuss concerns to be addressed and emerging solutions for establishing clinical grade biosensors to accelerate human cell therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

A general strategy to construct small molecule biosensors in eukaryotes.

PubMed

Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

2015-12-29

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments.

PubMed

Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren J

2006-02-28

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.

Microbial Fuels Cell-Based Biosensor for Toxicity Detection: A Review

PubMed Central

Zhou, Tuoyu; Han, Huawen; Liu, Pu; Xiong, Jian; Tian, Fake; Li, Xiangkai

2017-01-01

With the unprecedented deterioration of environmental quality, rapid recognition of toxic compounds is paramount for performing in situ real-time monitoring. Although several analytical techniques based on electrochemistry or biosensors have been developed for the detection of toxic compounds, most of them are time-consuming, inaccurate, or cumbersome for practical applications. More recently, microbial fuel cell (MFC)-based biosensors have drawn increasing interest due to their sustainability and cost-effectiveness, with applications ranging from the monitoring of anaerobic digestion process parameters (VFA) to water quality detection (e.g., COD, BOD). When a MFC runs under correct conditions, the voltage generated is correlated with the amount of a given substrate. Based on this linear relationship, several studies have demonstrated that MFC-based biosensors could detect heavy metals such as copper, chromium, or zinc, as well as organic compounds, including p-nitrophenol (PNP), formaldehyde and levofloxacin. Both bacterial consortia and single strains can be used to develop MFC-based biosensors. Biosensors with single strains show several advantages over systems integrating bacterial consortia, such as selectivity and stability. One of the limitations of such sensors is that the detection range usually exceeds the actual pollution level. Therefore, improving their sensitivity is the most important for widespread application. Nonetheless, MFC-based biosensors represent a promising approach towards single pollutant detection. PMID:28956857

Microbial Fuels Cell-Based Biosensor for Toxicity Detection: A Review.

PubMed

Zhou, Tuoyu; Han, Huawen; Liu, Pu; Xiong, Jian; Tian, Fake; Li, Xiangkai

2017-09-28

With the unprecedented deterioration of environmental quality, rapid recognition of toxic compounds is paramount for performing in situ real-time monitoring. Although several analytical techniques based on electrochemistry or biosensors have been developed for the detection of toxic compounds, most of them are time-consuming, inaccurate, or cumbersome for practical applications. More recently, microbial fuel cell (MFC)-based biosensors have drawn increasing interest due to their sustainability and cost-effectiveness, with applications ranging from the monitoring of anaerobic digestion process parameters (VFA) to water quality detection (e.g., COD, BOD). When a MFC runs under correct conditions, the voltage generated is correlated with the amount of a given substrate. Based on this linear relationship, several studies have demonstrated that MFC-based biosensors could detect heavy metals such as copper, chromium, or zinc, as well as organic compounds, including p -nitrophenol (PNP), formaldehyde and levofloxacin. Both bacterial consortia and single strains can be used to develop MFC-based biosensors. Biosensors with single strains show several advantages over systems integrating bacterial consortia, such as selectivity and stability. One of the limitations of such sensors is that the detection range usually exceeds the actual pollution level. Therefore, improving their sensitivity is the most important for widespread application. Nonetheless, MFC-based biosensors represent a promising approach towards single pollutant detection.

Recent Advances in Exosomal Protein Detection Via Liquid Biopsy Biosensors for Cancer Screening, Diagnosis, and Prognosis.

PubMed

Liu, Chang; Yang, Yunchen; Wu, Yun

2018-03-08

Current cancer diagnostic methods are challenged by low sensitivity, high false positive rate, limited tumor information, uncomfortable or invasive procedures, and high cost. Liquid biopsy that analyzes circulating biomarkers in body fluids represents a promising solution to these challenges. Exosomes are one of the promising cancer biomarkers for liquid biopsy because they are cell-secreted, nano-sized, extracellular vesicles that stably exist in all types of body fluids. Exosomes transfer DNAs, RNAs, proteins, and lipids from parent cells to recipient cells for intercellular communication and play important roles in cancer initiation, progression, and metastasis. Many liquid biopsy biosensors have been developed to offer non- or minimally-invasive, highly sensitive, simple, rapid, and cost-effective cancer diagnostics. This review summarized recent advances of liquid biopsy biosensors with a focus on the detection of exosomal proteins as biomarkers for cancer screening, diagnosis, and prognosis. We reviewed six major types of liquid biopsy biosensors including immunofluorescence biosensor, colorimetric biosensor, surface plasmon resonance (SPR) biosensor, surface-enhanced Raman scattering (SERS) biosensor, electrochemical biosensor, and nuclear magnetic resonance (NMR) biosensor. We shared our perspectives on future improvement of exosome-based liquid biopsy biosensors to accelerate their clinical translation.

Paper electrodes for bioelectrochemistry: Biosensors and biofuel cells.

PubMed

Desmet, Cloé; Marquette, Christophe A; Blum, Loïc J; Doumèche, Bastien

2016-02-15

Paper-based analytical devices (PAD) emerge in the scientific community since 2007 as low-cost, wearable and disposable devices for point-of-care diagnostic due to the widespread availability, long-time knowledge and easy manufacturing of cellulose. Rapidly, electrodes were introduced in PAD for electrochemical measurements. Together with biological components, a new generation of electrochemical biosensors was born. This review aims to take an inventory of existing electrochemical paper-based biosensors and biofuel cells and to identify, at the light of newly acquired data, suitable methodologies and crucial parameters in this field. Paper selection, electrode material, hydrophobization of cellulose, dedicated electrochemical devices and electrode configuration in biosensors and biofuel cells will be discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

New biosensor for detection of copper ions in water based on immobilized genetically modified yeast cells.

PubMed

Vopálenská, Irena; Váchová, Libuše; Palková, Zdena

2015-10-15

Contamination of water by heavy metals represents a potential risk for both aquatic and terrestrial organisms, including humans. Heavy metals in water resources can come from various industrial activities, and drinking water can be ex-post contaminated by heavy metals such as Cu(2+) from house fittings (e.g., water reservoirs) and pipes. Here, we present a new copper biosensor capable of detecting copper ions at concentrations of 1-100 μM. This biosensor is based on cells of a specifically modified Saccharomyces cerevisiae strain immobilized in alginate beads. Depending on the concentration of copper, the biosensor beads change color from white, when copper is present in concentrations below the detection limit, to pink or red based on the increase in copper concentration. The biosensor was successfully tested in the determination of copper concentrations in real samples of water contaminated with copper ions. In contrast to analytical methods or other biosensors based on fluorescent proteins, the newly designed biosensor does not require specific equipment and allows the quick detection of copper in many parallel samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Fiber Optic Surface Plasmon Resonance-Based Biosensor Technique: Fabrication, Advancement, and Application.

PubMed

Liang, Gaoling; Luo, Zewei; Liu, Kunping; Wang, Yimin; Dai, Jianxiong; Duan, Yixiang

2016-05-03

Fiber optic-based biosensors with surface plasmon resonance (SPR) technology are advanced label-free optical biosensing methods. They have brought tremendous progress in the sensing of various chemical and biological species. This review summarizes four sensing configurations (prism, grating, waveguide, and fiber optic) with two ways, attenuated total reflection (ATR) and diffraction, to excite the surface plasmons. Meanwhile, the designs of different probes (U-bent, tapered, and other probes) are also described. Finally, four major types of biosensors, immunosensor, DNA biosensor, enzyme biosensor, and living cell biosensor, are discussed in detail for their sensing principles and applications. Future prospects of fiber optic-based SPR sensor technology are discussed.

A biosensor generated via high throughput screening quantifies cell edge Src dynamics

PubMed Central

Gulyani, Akash; Vitriol, Eric; Allen, Richard; Wu, Jianrong; Gremyachinskiy, Dmitriy; Lewis, Steven; Dewar, Brian; Graves, Lee M.; Kay, Brian K.; Kuhlman, Brian; Elston, Tim; Hahn, Klaus M.

2011-01-01

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. PMID:21666688

Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

NASA Astrophysics Data System (ADS)

Gangadharan, Rajan

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

Selection of turning-on fluorogenic probe as protein-specific detector obtained via the 10BASEd-T

NASA Astrophysics Data System (ADS)

Uematsu, Shuta; Midorikawa, Taiki; Ito, Yuji; Taki, Masumi

2017-01-01

In order to obtain a molecular probe for specific protein detection, we have synthesized fluorogenic probe library of vast diversity on bacteriophage T7 via the gp10 based-thioetherification (10BASEd-T). A remarkable turning- on probe which is excitable by widely applicable visible light was selected from the library.

Targeted delivery of fluorogenic peptide aptamers into live microalgae by femtosecond laser photoporation at single-cell resolution.

PubMed

Maeno, Takanori; Uzawa, Takanori; Kono, Izumi; Okano, Kazunori; Iino, Takanori; Fukita, Keisuke; Oshikawa, Yuki; Ogawa, Taro; Iwata, Osamu; Ito, Takuro; Suzuki, Kengo; Goda, Keisuke; Hosokawa, Yoichiroh

2018-05-29

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells

PubMed Central

2016-01-01

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Human Neural Cell-Based Biosensor

DTIC Science & Technology

2010-06-11

stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E - 115 neuroblastoma cells. Exp Cell Res, 313 (9): p...developed methods for directed dopaminergic differentiation using defined medium conditions – all towards the goal of accelerating neuronal... differentiation for biosensor development. Moreover, we have begun an exploration of fluorescence-based assays as a new direction for ‘sensor element’ development

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

PubMed Central

Mitronova, Gyuzel Yu.; Sidenstein, Sven C.; Klocke, Jessica L.; Kamin, Dirk; Meineke, Dirk N. H.; D'Este, Elisa; Kraemer, Philip‐Tobias; Danzl, Johann G.

2016-01-01

Abstract A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm. PMID:26844929

A general strategy to construct small molecule biosensors in eukaryotes

DOE PAGES

Feng, Justin; Jester, Benjamin W.; Tinberg, Christine E.; ...

2015-12-29

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activatesmore » transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. As a result, this work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.« less

A general strategy to construct small molecule biosensors in eukaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Justin; Jester, Benjamin W.; Tinberg, Christine E.

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activatesmore » transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. As a result, this work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.« less

A general strategy to construct small molecule biosensors in eukaryotes

PubMed Central

Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

2015-01-01

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.10606.001 PMID:26714111

A cCPE-based xenon biosensor for magnetic resonance imaging of claudin-expressing cells.

PubMed

Piontek, Anna; Witte, Christopher; May Rose, Honor; Eichner, Miriam; Protze, Jonas; Krause, Gerd; Piontek, Jörg; Schröder, Leif

2017-06-01

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection. © 2017 New York Academy of Sciences.

Applications of Fluorogens with Rotor Structures in Solar Cells.

PubMed

Ong, Kok-Haw; Liu, Bin

2017-05-29

Solar cells are devices that convert light energy into electricity. To drive greater adoption of solar cell technologies, higher cell efficiencies and reductions in manufacturing cost are necessary. Fluorogens containing rotor structures may be helpful in addressing some of these challenges due to their unique twisted structures and photophysics. In this review, we discuss the applications of rotor-containing molecules as dyes for luminescent down-shifting layers and luminescent solar concentrators, where their aggregation-induced emission properties and large Stokes shifts are highly desirable. We also discuss the applications of molecules containing rotors in third-generation solar cell technologies, namely dye-sensitized solar cells and organic photovoltaics, where the twisted 3-dimensional rotor structures are used primarily for aggregation control. Finally, we discuss perspectives on the future role of molecules containing rotor structures in solar cell technologies.

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

PubMed

Fisher, Gregory W; Fuhrman, Margaret H; Adler, Sally A; Szent-Gyorgyi, Christopher; Waggoner, Alan S; Jarvik, Jonathan W

2014-09-01

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications. © 2014 Society for Laboratory Automation and Screening.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biosensor development.

PubMed

Ziegler, C; Göpel, W

1998-10-01

Current biosensor developments can be summarised by different trends. For traditional enzymatic biosensors such as glucose sensors, steady improvements of well known basic principles have been made in order to achieve better sensor stability. On the other hand, new affinity sensors such as nucleic acid sensors, transmembrane sensors, and sensors utilising whole cells or even cell networks have become of increasing interest. New ways to miniaturise biosensors and to control their interfaces down to the molecular level have been introduced (the bioelectronics approach). High-throughput screening based on various signal transduction principles has become of increasing importance.

SH2 Domain-Based FRET Biosensor for Measuring BCR-ABL Activity in Living CML Cells.

PubMed

Fujioka, Mari; Asano, Yumi; Nakada, Shigeyuki; Ohba, Yusuke

2017-01-01

Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

PubMed

Woronoff, Gabrielle; El Harrak, Abdeslam; Mayot, Estelle; Schicke, Olivier; Miller, Oliver J; Soumillion, Patrice; Griffiths, Andrew D; Ryckelynck, Michael

2011-04-15

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats. © 2011 American Chemical Society

Commercialisation of CMOS integrated circuit technology in multi-electrode arrays for neuroscience and cell-based biosensors.

PubMed

Graham, Anthony H D; Robbins, Jon; Bowen, Chris R; Taylor, John

2011-01-01

The adaptation of standard integrated circuit (IC) technology as a transducer in cell-based biosensors in drug discovery pharmacology, neural interface systems and electrophysiology requires electrodes that are electrochemically stable, biocompatible and affordable. Unfortunately, the ubiquitous Complementary Metal Oxide Semiconductor (CMOS) IC technology does not meet the first of these requirements. For devices intended only for research, modification of CMOS by post-processing using cleanroom facilities has been achieved. However, to enable adoption of CMOS as a basis for commercial biosensors, the economies of scale of CMOS fabrication must be maintained by using only low-cost post-processing techniques. This review highlights the methodologies employed in cell-based biosensor design where CMOS-based integrated circuits (ICs) form an integral part of the transducer system. Particular emphasis will be placed on the application of multi-electrode arrays for in vitro neuroscience applications. Identifying suitable IC packaging methods presents further significant challenges when considering specific applications. The various challenges and difficulties are reviewed and some potential solutions are presented.

Encapsulated Optically Responsive Cell Systems: Toward Smart Implants in Biomedicine.

PubMed

Boss, Christophe; Bouche, Nicolas; De Marchi, Umberto

2018-04-01

Managing increasingly prevalent chronic diseases will require close continuous monitoring of patients. Cell-based biosensors may be used for implantable diagnostic systems to monitor health status. Cells are indeed natural sensors in the body. Functional cellular systems can be maintained in the body for long-term implantation using cell encapsulation technology. By taking advantage of recent progress in miniaturized optoelectronic systems, the genetic engineering of optically responsive cells may be combined with cell encapsulation to generate smart implantable cell-based sensing systems. In biomedical research, cell-based biosensors may be used to study cell signaling, therapeutic effects, and dosing of bioactive molecules in preclinical models. Today, a wide variety of genetically encoded fluorescent sensors have been developed for real-time imaging of living cells. Here, recent developments in genetically encoded sensors, cell encapsulation, and ultrasmall optical systems are highlighted. The integration of these components in a new generation of biosensors is creating innovative smart in vivo cell-based systems, bringing novel perspectives for biomedical research and ultimately allowing unique health monitoring applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Small Microbial Three-Electrode Cell Based Biosensor for Online Detection of Acute Water Toxicity.

PubMed

Yu, Dengbin; Zhai, Junfeng; Liu, Changyu; Zhang, Xueping; Bai, Lu; Wang, Yizhe; Dong, Shaojun

2017-11-22

The monitoring of toxicity of water is very important to estimate the safety of drinking water and the level of water pollution. Herein, a small microbial three-electrode cell (M3C) biosensor filled with polystyrene particles was proposed for online monitoring of the acute water toxicity. The peak current of the biosensor related with the performance of the bioanode was regarded as the toxicity indicator, and thus the acute water toxicity could be determined in terms of inhibition ratio by comparing the peak current obtained with water sample to that obtained with nontoxic standard water. The incorporation of polystyrene particles in the electrochemical cell not only reduced the volume of the samples used, but also improved the sensitivity of the biosensor. Experimental conditions including washing time with PBS and the concentration of sodium acetate solution were optimized. The stability of the M3C biosensor under optimal conditions was also investigated. The M3C biosensor was further examined by formaldehyde at the concentration of 0.01%, 0.03%, and 0.05% (v/v), and the corresponding inhibition ratios were 14.6%, 21.6%, and 36.4%, respectively. This work provides a new insight into the development of an online toxicity detector based on M3C biosensor.

Functional nucleic acids as in vivo metabolite and ion biosensors.

PubMed

Alsaafin, Alaa; McKeague, Maureen

2017-08-15

Characterizing the role of metabolites, metals, and proteins is required to understand normal cell function, and ultimately, elucidate the mechanism of disease. Metabolite concentration and transformation results collected from cell lysates or fixed-cells conceal important dynamic information and differences between individual cells that often have profound functional consequences. Functional nucleic acid-based biosensors are emerging tools that are capable of monitoring ions and metabolites in cell populations or whole animals. Functional nucleic acids (FNAs) are a class of biomolecules that can exhibit either ligand binding or enzymatic activity. Unlike their protein analogues or the use of instrument-based analysis, FNA-based biosensors are capable of entering cells without disruption to the cellular environment and can report on the concentration, dynamics, and spatial localization of molecules in cells. Here, we review the types of FNAs that have been used as in vivo biosensors, and how FNAs can be coupled to transduction systems and delivered inside cells. We also provide examples from the literature that demonstrate their impact in practical applications. Finally, we comment on the critical limitations that need to be addressed to enable their use for single-cell dynamic tracking of metabolites and ions in vivo. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

PubMed

Kobras, Carolin Martina; Mascher, Thorsten; Gebhard, Susanne

2017-01-01

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

PubMed

Dutta, Subrata; Fülöp, Annabelle; Mokhir, Andriy

2013-09-18

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

Microbial fuel cell-based biosensor for toxic carbon monoxide monitoring.

PubMed

Zhou, Shaofeng; Huang, Shaobin; Li, Yi; Zhao, Nannan; Li, Han; Angelidaki, Irini; Zhang, Yifeng

2018-08-15

This study presents an innovative microbial fuel cell-based biosensor for carbon monoxide (CO) monitoring. The hypothesis for the function of the biosensor is that CO inhibits bacterial activity in the anode and thereby reduces electricity production. A mature electrochemically active biofilm on the anode was exposed to CO gas at varied concentrations. A proportional linear relationship (R 2 = 0.987) between CO concentration and voltage drop (0.8 to 24 mV) in the range of 10% and 70% of CO concentration was observed. Notably, no further decrease of voltage output was observed by with further increasing CO concentration over 70%. Besides, the response time of the biosensor was 1 h. The compact design and simple operation of the biosensor makes it easy to be integrated in existing CO-based industrial facilities either as a forewarning sensor for CO toxicity or even as an individual on-line monitoring device. Copyright © 2018 Elsevier B.V. All rights reserved.

The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

PubMed

Lu, Shaoying; Ouyang, Mingxing; Seong, Jihye; Zhang, Jin; Chien, Shu; Wang, Yingxiao

2008-07-25

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

Application of recombinant fluorescent mammalian cells as a toxicity biosensor.

PubMed

Kim, E J; Lee, Y; Lee, J E; Gu, M B

2002-01-01

With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

Zooming In on Plant Hormone Analysis: Tissue- and Cell-Specific Approaches.

PubMed

Novák, Ondřej; Napier, Richard; Ljung, Karin

2017-04-28

Plant hormones are a group of naturally occurring, low-abundance organic compounds that influence physiological processes in plants. Our knowledge of the distribution profiles of phytohormones in plant organs, tissues, and cells is still incomplete, but advances in mass spectrometry have enabled significant progress in tissue- and cell-type-specific analyses of phytohormones over the last decade. Mass spectrometry is able to simultaneously identify and quantify hormones and their related substances. Biosensors, on the other hand, offer continuous monitoring; can visualize local distributions and real-time quantification; and, in the case of genetically encoded biosensors, are noninvasive. Thus, biosensors offer additional, complementary technologies for determining temporal and spatial changes in phytohormone concentrations. In this review, we focus on recent advances in mass spectrometry-based quantification, describe monitoring systems based on biosensors, and discuss validations of the various methods before looking ahead at future developments for both approaches.

Label-free measurements on cell apoptosis using a terahertz metamaterial-based biosensor

NASA Astrophysics Data System (ADS)

Zhang, Caihong; Liang, Lanju; Ding, Liang; Jin, Biaobing; Hou, Yayi; Li, Chun; Jiang, Ling; Liu, Weiwei; Hu, Wei; Lu, Yanqing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

2016-06-01

Label-free, real-time, and in-situ measurement on cell apoptosis is highly desirable in cell biology. We propose here a design of terahertz (THz) metamaterial-based biosensor for meeting this requirement. This metamaterial consists of a planar array of five concentric subwavelength gold ring resonators on a 10 μm-thick polyimide substrate, which can sense the change of dielectric environment above the metamaterial. We employ this sensor to an oral cancer cell (SCC4) with and without cisplatin, a chemotherapy drug for cancer treatment, and find a linear relation between cell apoptosis measured by Flow Cytometry and the relative change of resonant frequencies of the metamaterial measured by THz time-domain spectroscopy. This implies that we can determine the cell apoptosis in a label-free manner. We believe that this metamaterial-based biosensor can be developed into a cheap, label-free, real-time, and in-situ detection tool, which is of significant impact on the study of cell biology.

Knowledge-based design of a biosensor to quantify localized ERK activation in living cells

PubMed Central

Kummer, Lutz; Hsu, Chia-Wen; Dagliyan, Onur; MacNevin, Christopher; Kaufholz, Melanie; Zimmermann, Bastian; Dokholyan, Nikolay V.; Hahn, Klaus M.; Plückthun, Andreas

2014-01-01

Summary Investigation of protein activation in living cells is fundamental to understand how proteins are influenced by the full complement of upstream regulators they experience. We describe here the generation of a biosensor based on the Designed Ankyrin Repeat Protein (DARPin) binding scaffold suited for intracellular applications. Combining selection and evolution from libraries, knowledge-based design and efficient and rapid testing of conjugate candidates, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK (pERK) with a solvatochromic merocyanine dye (mero87), whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to non-phosphorylated ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts incubated in 2% serum, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. Activity was greatly reduced by the MEK1/2 inhibitor U0126. The DARPin-based biosensor will serve as useful tool for studying biological functions of ERK in vitro and in vivo. PMID:23790495

Sensitive-cell-based fish chromatophore biosensor

NASA Astrophysics Data System (ADS)

Plant, Thomas K.; Chaplen, Frank W.; Jovanovic, Goran; Kolodziej, Wojtek; Trempy, Janine E.; Willard, Corwin; Liburdy, James A.; Pence, Deborah V.; Paul, Brian K.

2004-07-01

A sensitive biosensor (cytosensor) has been developed based on color changes in the toxin-sensitive colored living cells of fish. These chromatophores are highly sensitive to the presence of many known and unknown toxins produced by microbial pathogens and undergo visible color changes in a dose-dependent manner. The chromatophores are immobilized and maintained in a viable state while potential pathogens multiply and fish cell-microbe interactions are monitored. Low power LED lighting is used to illuminate the chromatophores which are magnified using standard optical lenses and imaged onto a CCD array. Reaction to toxins is detected by observing changes is the total area of color in the cells. These fish chromatophores are quite sensitive to cholera toxin, Staphococcus alpha toxin, and Bordatella pertussis toxin. Numerous other toxic chemical and biological agents besides bacterial toxins also cause readily detectable color effects in chromatophores. The ability of the chromatophore cell-based biosensor to distinguish between different bacterial pathogens was examined. Toxin producing strains of Salmonella enteritis, Vibrio parahaemolyticus, and Bacillus cereus induced movement of pigmented organelles in the chromatophore cells and this movement was measured by changes in the optical density over time. Each bacterial pathogen elicited this measurable response in a distinctive and signature fashion. These results suggest a chromatophore cell-based biosensor assay may be applicable for the detection and identification of virulence activities associated with certain air-, food-, and water-borne bacterial pathogens.

Optical biosensors.

PubMed

Damborský, Pavel; Švitel, Juraj; Katrlík, Jaroslav

2016-06-30

Optical biosensors represent the most common type of biosensor. Here we provide a brief classification, a description of underlying principles of operation and their bioanalytical applications. The main focus is placed on the most widely used optical biosensors which are surface plasmon resonance (SPR)-based biosensors including SPR imaging and localized SPR. In addition, other optical biosensor systems are described, such as evanescent wave fluorescence and bioluminescent optical fibre biosensors, as well as interferometric, ellipsometric and reflectometric interference spectroscopy and surface-enhanced Raman scattering biosensors. The optical biosensors discussed here allow the sensitive and selective detection of a wide range of analytes including viruses, toxins, drugs, antibodies, tumour biomarkers and tumour cells. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Targeted Molecular Imaging of Cancer Cells Using MS2-Based 129 Xe NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Keunhong; Netirojjanakul, Chawita; Munch, Henrik K.

Targeted, selective, and highly sensitive 129Xe NMR nanoscale biosensors have been synthesized using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized 129Xe NMR signal contrast and hyper-CEST 129Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized 129Xe NMR nanoscale biosensor for future applications in cancer detection in vivo.

Commercialisation of CMOS Integrated Circuit Technology in Multi-Electrode Arrays for Neuroscience and Cell-Based Biosensors

PubMed Central

Graham, Anthony H. D.; Robbins, Jon; Bowen, Chris R.; Taylor, John

2011-01-01

The adaptation of standard integrated circuit (IC) technology as a transducer in cell-based biosensors in drug discovery pharmacology, neural interface systems and electrophysiology requires electrodes that are electrochemically stable, biocompatible and affordable. Unfortunately, the ubiquitous Complementary Metal Oxide Semiconductor (CMOS) IC technology does not meet the first of these requirements. For devices intended only for research, modification of CMOS by post-processing using cleanroom facilities has been achieved. However, to enable adoption of CMOS as a basis for commercial biosensors, the economies of scale of CMOS fabrication must be maintained by using only low-cost post-processing techniques. This review highlights the methodologies employed in cell-based biosensor design where CMOS-based integrated circuits (ICs) form an integral part of the transducer system. Particular emphasis will be placed on the application of multi-electrode arrays for in vitro neuroscience applications. Identifying suitable IC packaging methods presents further significant challenges when considering specific applications. The various challenges and difficulties are reviewed and some potential solutions are presented. PMID:22163884

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

PubMed

Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Mammalian Cell-Based Sensor System

NASA Astrophysics Data System (ADS)

Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

Challenges in paper-based fluorogenic optical sensing with smartphones

NASA Astrophysics Data System (ADS)

Ulep, Tiffany-Heather; Yoon, Jeong-Yeol

2018-05-01

Application of optically superior, tunable fluorescent nanotechnologies have long been demonstrated throughout many chemical and biological sensing applications. Combined with microfluidics technologies, i.e. on lab-on-a-chip platforms, such fluorescent nanotechnologies have often enabled extreme sensitivity, sometimes down to single molecule level. Within recent years there has been a peak interest in translating fluorescent nanotechnology onto paper-based platforms for chemical and biological sensing, as a simple, low-cost, disposable alternative to conventional silicone-based microfluidic substrates. On the other hand, smartphone integration as an optical detection system as well as user interface and data processing component has been widely attempted, serving as a gateway to on-board quantitative processing, enhanced mobility, and interconnectivity with informational networks. Smartphone sensing can be integrated to these paper-based fluorogenic assays towards demonstrating extreme sensitivity as well as ease-of-use and low-cost. However, with these emerging technologies there are always technical limitations that must be addressed; for example, paper's autofluorescence that perturbs fluorogenic sensing; smartphone flash's limitations in fluorescent excitation; smartphone camera's limitations in detecting narrow-band fluorescent emission, etc. In this review, physical optical setups, digital enhancement algorithms, and various fluorescent measurement techniques are discussed and pinpointed as areas of opportunities to further improve paper-based fluorogenic optical sensing with smartphones.

Improved Photoinduced Fluorogenic Alkene-Tetrazole Reaction for Protein Labeling.

PubMed

Shang, Xin; Lai, Rui; Song, Xi; Li, Hui; Niu, Wei; Guo, Jiantao

2017-11-15

The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

PubMed

Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

2012-12-01

Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

Comparative analysis of microbial fuel cell based biosensors developed with a mixed culture and Shewanella loihica PV-4 and underlying biological mechanism.

PubMed

Yi, Yue; Xie, Beizhen; Zhao, Ting; Liu, Hong

2018-06-13

Microbial fuel cell based biosensors (MFC-biosensors) utilize anode biofilms as biological recognition elements to monitor biochemical oxygen demand (BOD) and biotoxicity. However, the relatively poor sensitivity constrains the application of MFC-biosensors. To address this limitation, this study provided a systematic comparison of sensitivity between the MFC-biosensors constructed with two inocula. Higher biomass density and viability were both observed in the anode biofilm of the mixed culture MFC, which resulted in better sensitivity for BOD assessment. Compared with using mixed culture as inoculum, the anode biofilm developed with Shewanella loihica PV-4 presented lower content of extracellular polymeric substances and poorer ability to secrete protein under toxic shocks. Moreover, the looser structure in the S. loihica PV-4 biofilm further facilitated its susceptibilities to toxic agents. Therefore, the MFC-biosensor with a pure culture of S. loihica PV-4 delivered higher sensitivity for biotoxicity monitoring. This study proposed a new perspective to enhance sensor performance. Copyright © 2018 Elsevier Ltd. All rights reserved.

A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

PubMed Central

Cox, Dianne; Hodgson, Louis

2014-01-01

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging.

PubMed

Hanna, Samer; Miskolci, Veronika; Cox, Dianne; Hodgson, Louis

2014-01-01

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.

Utility of Ochrobactrum anthropi YC152 in a Microbial Fuel Cell as an Early Warning Device for Hexavalent Chromium Determination.

PubMed

Wang, Guey-Horng; Cheng, Chiu-Yu; Liu, Man-Hai; Chen, Tzu-Yu; Hsieh, Min-Chi; Chung, Ying-Chien

2016-08-16

Fast hexavalent chromium (Cr(VI)) determination is important for environmental risk and health-related considerations. We used a microbial fuel cell-based biosensor inoculated with a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic Ochrobactrum anthropi YC152 to determine the Cr(VI) concentration in water. The results indicated that O. anthropi YC152 exhibited high adaptability to pH, temperature, salinity, and water quality under anaerobic conditions. The stable performance of the microbial fuel cell (MFC)-based biosensor indicated its potential as a reliable biosensor system. The MFC voltage decreased as the Cr(VI) concentration in the MFC increased. Two satisfactory linear relationships were observed between the Cr(VI) concentration and voltage output for various Cr(VI) concentration ranges (0.0125-0.3 mg/L and 0.3-5 mg/L). The MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in drinking water, groundwater, and electroplating wastewater in 45 min with low deviations (<10%). The use of the biosensor can help in preventing the violation of effluent regulations and the maximum allowable concentration of Cr(VI) in water. Thus, the developed MFC biosensor has potential as an early warning detection device for Cr(VI) determination even if O. anthropi YC152 is a possible opportunistic pathogen.

Autonomous electrochemical biosensors: A new vision to direct methanol fuel cells.

PubMed

Sales, M Goreti F; Brandão, Lúcia

2017-12-15

A new approach to biosensing devices is demonstrated aiming an easier and simpler application in routine health care systems. Our methodology considered a new concept for the biosensor transducing event that allows to obtain, simultaneously, an equipment-free, user-friendly, cheap electrical biosensor. The use of the anode triple-phase boundary (TPB) layer of a passive direct methanol fuel cell (DMFC) as biosensor transducer is herein proposed. For that, the ionomer present in the anode catalytic layer of the DMFC is partially replaced by an ionomer with molecular recognition capability working as the biorecognition element of the biosensor. In this approach, fuel cell anode catalysts are modified with a molecularly imprinted polymer (plastic antibody) capable of protein recognition (ferritin is used as model protein), inserted in a suitable membrane electrode assembly (MEA) and tested, as initial proof-of-concept, in a non-passive fuel cell operation environment. The anchoring of the ionomer-based plastic antibody on the catalyst surface follows a simple one-step grafting from approach through radical polymerization. Such modification increases fuel cell performance due to the proton conductivity and macroporosity characteristics of the polymer on the TPB. Finally, the response and selectivity of the bioreceptor inside the fuel cell showed a clear and selective signal from the biosensor. Moreover, such pioneering transducing approach allowed amplification of the electrochemical response and increased biosensor sensitivity by 2 orders of magnitude when compared to a 3-electrodes configuration system. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Biosensors for Cell Analysis.

PubMed

Zhou, Qing; Son, Kyungjin; Liu, Ying; Revzin, Alexander

2015-01-01

Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis.

REVIEW ARTICLE: Environmental applications of analytical biosensors

NASA Astrophysics Data System (ADS)

Marco, María-Pilar; Barceló, Damià

1996-11-01

A review of the fundamental aspects and environmental applications of biosensors is presented. The bases of different transducer principles such as electrochemical, optical and piezoelectric are discussed. Various examples are given of the applications of such principles to develop immunosensor devices to determine common environmental contaminants. Attention is also paid to catalytic biosensors, using enzymes as sensing elements. Biosensor devices based on the use of cholinesterase and various oxidase enzymes such as tyrosinase, laccase, peroxidase and aldehyde dehydrogenase are reported. Some examples are given of the applications of other biomolecules such as whole cells, DNA or proteins, to determine pollution. Validation studies are presented comparing biosensors with chromatographic techniques to determine organophosphorus pesticides and phenolic compounds in environmental samples.

Optical fiber-based biosensors.

PubMed

Monk, David J; Walt, David R

2004-08-01

This review outlines optical fiber-based biosensor research from January 2001 through September 2003 and was written to complement the previous review in this journal by Marazuela and Moreno-Bondi. Optical fiber-based biosensors combine the use of a biological recognition element with an optical fiber or optical fiber bundle. They are classified by the nature of the biological recognition element used for sensing: enzyme, antibody/antigen (immunoassay), nucleic acid, whole cell, and biomimetic, and may be used for a variety of analytes ranging from metals and chemicals to physiological materials.

Microbial Biosensor for the Detection of Protease-Virulent Factors from Pathogens

DTIC Science & Technology

2017-04-28

cleavage in the extracellular space. The cleavage of TCS receptor protein would abolish the kinase activity responsible for the phosphorylation of the...cytoplasmic response regulator, AgrA, which functions as a transcriptional activator . As the cell-based protease biosensor response requires over...to AIP; AgrC is a AIP receptor that phosphorylates AgrA, an activator for P2 and P3. Protein-based protease biosensor construction To facilitate

Photonic crystals: emerging biosensors and their promise for point-of-care applications.

PubMed

Inan, Hakan; Poyraz, Muhammet; Inci, Fatih; Lifson, Mark A; Baday, Murat; Cunningham, Brian T; Demirci, Utkan

2017-01-23

Biosensors are extensively employed for diagnosing a broad array of diseases and disorders in clinical settings worldwide. The implementation of biosensors at the point-of-care (POC), such as at primary clinics or the bedside, faces impediments because they may require highly trained personnel, have long assay times, large sizes, and high instrumental cost. Thus, there exists a need to develop inexpensive, reliable, user-friendly, and compact biosensing systems at the POC. Biosensors incorporated with photonic crystal (PC) structures hold promise to address many of the aforementioned challenges facing the development of new POC diagnostics. Currently, PC-based biosensors have been employed for detecting a variety of biotargets, such as cells, pathogens, proteins, antibodies, and nucleic acids, with high efficiency and selectivity. In this review, we provide a broad overview of PCs by explaining their structures, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-based biosensors incorporated with emerging technologies, including telemedicine, flexible and wearable sensing, smart materials and metamaterials. Finally, we discuss current challenges associated with existing biosensors, and provide an outlook for PC-based biosensors and their promise at the POC.

A novel and simple cell-based electrochemical biosensor for evaluating the antioxidant capacity of Lactobacillus plantarum strains isolated from Chinese dry-cured ham.

PubMed

Ge, Qingfeng; Ge, Panwei; Jiang, Donglei; Du, Nan; Chen, Jiahui; Yuan, Limin; Yu, Hai; Xu, Xin; Wu, Mangang; Zhang, Wangang; Zhou, Guanghong

2018-01-15

The analysis of antioxidants in foodstuffs has become an active area of research, leading to the recent development of numerous methods for assessing antioxidant capacity. Here we described the fabrication and validation of a novel and simple cell-based electrochemical biosensor for this purpose. The biosensor is used to assess the antioxidant capacity of cell-free extracts from Lactobacillus plantarum strains isolated from Chinese dry-cured ham. The biosensor relies on the determination of cellular reactive oxygen species (ROS) (the flux of H 2 O 2 released from RAW 264.7 macrophage cells) to indirectly assess changes in intracellular oxidative stress level as influenced by L. plantarum strains. A one-step acidified manganese dioxide (a-MnO 2 ) modified gold electrode (GE) was used to immobilize RAW 264.7 macrophage cells, which were then encapsulated in a 3D cell culture system consisting of alginate/ graphene oxide (NaAlg/GO). The biosensor exhibited a rapid and sensitive response for the detection of H 2 O 2 released from RAW264.7 cells. The detection limit was 0.02μM with a linear response from 0.05μM to 0.85μM and the biosensor was shown to have good stability and outstanding repeatability. This technique was then used for evaluating the antioxidant ability of extracts from L. plantarum NJAU-01. According to the electrochemical investigations and assays of SEM, TEM, and ROS, these cell-free extracts effectively reduced the oxidative stress levels in RAW264.7 cells under external stimulation. Extracts from L. plantarum strains at a dose of 10 10 CFU/mL showed the highest antioxidant activities with a relative antioxidant capacity (RAC) rate of 88.94%. Hence, this work provides a simple and efficient electrochemical biosensing platform based on RAW264.7 cells for fast, sensitive and quantitative assessment of antioxidant capacity of L. plantarum strains. The method demonstrates its potential for rapid screening for evaluating antioxidant properties of samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Biosensor-based engineering of biosynthetic pathways

DOE PAGES

Rogers, Jameson K.; Taylor, Noah D.; Church, George M.

2016-03-18

Biosynthetic pathways provide an enzymatic route from inexpensive renewable resources to valuable metabolic products such as pharmaceuticals and plastics. However, designing these pathways is challenging due to the complexities of biology. Advances in the design and construction of genetic variants has enabled billions of cells, each possessing a slightly different metabolic design, to be rapidly generated. However, our ability to measure the quality of these designs lags by several orders of magnitude. Recent research has enabled cells to report their own success in chemical production through the use of genetically encoded biosensors. A new engineering discipline is emerging around themore » creation and application of biosensors. Biosensors, implemented in selections and screens to identify productive cells, are paving the way for a new era of biotechnological progress.« less

A new respirometric endpoint-based biosensor to assess the relative toxicity of chemicals on immobilized human cells.

PubMed

Dragone, Roberto; Frazzoli, Chiara; Grappelli, Claudio; Campanella, Luigi

2009-01-01

Several functional and biochemical parameters have been proposed as biomarkers of effect of environmental pollutants. A rapid biosensor working with immobilized human U-937 cells was developed and applied to environmentally relevant chemicals with different structures and toxicological pathways, i.e. benzalkonium chloride, clofibric acid, diclofenac, mercury nitrate, ofloxacin, and sodium dodecyl sulphate. Respiration of cells was relied upon as a comprehensive biochemical effect for screening purposes. Analytical parameter (DeltappmO(2)) and toxicological index (respiratory inhibition, delta%) measured after 1h of exposure were utilized for dose-response relationship study. Results (toxicity rating scales based on delta(50)% and steepness) were compared with those obtained by the same approach previously optimized on Saccharomyces cerevisiae. The toxicity rating scale obtained by the biomarker based on human mitochondrial and cell metabolic activities compared well with previous scale obtained on yeast cells and with available in-vivo acute toxicity indexes; respiration was confirmed as toxicological endpoint reliably measurable by the biosensor.

Nanomaterials-based biosensors for detection of microorganisms and microbial toxins.

PubMed

Sutarlie, Laura; Ow, Sian Yang; Su, Xiaodi

2017-04-01

Detection of microorganisms and microbial toxins is important for health and safety. Due to their unique physical and chemical properties, nanomaterials have been extensively used to develop biosensors for rapid detection of microorganisms with microbial cells and toxins as target analytes. In this paper, the design principles of nanomaterials-based biosensors for four selected analyte categories (bacteria cells, toxins, mycotoxins, and protozoa cells), closely associated with the target analytes' properties is reviewed. Five signal transducing methods that are less equipment intensive (colorimetric, fluorimetric, surface enhanced Raman scattering, electrochemical, and magnetic relaxometry methods) is described and compared for their sensory performance (in term oflimit of detection, dynamic range, and response time) for all analyte categories. In the end, the suitability of these five sensing principles for on-site or field applications is discussed. With a comprehensive coverage of nanomaterials, design principles, sensing principles, and assessment on the sensory performance and suitability for on-site application, this review offers valuable insight and perspective for designing suitable nanomaterials-based microorganism biosensors for a given application. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

PubMed

Cevenini, Luca; Calabretta, Maria Maddalena; Lopreside, Antonia; Tarantino, Giuseppe; Tassoni, Annalisa; Ferri, Maura; Roda, Aldo; Michelini, Elisa

2016-12-01

The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

PubMed

Hernández-Ibáñez, Naiara; García-Cruz, Leticia; Montiel, Vicente; Foster, Christopher W; Banks, Craig E; Iniesta, Jesús

2016-03-15

l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9 µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6 µM and exhibits a sensitivity of 3417 ± 131 µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4 °C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols. Copyright © 2015 Elsevier B.V. All rights reserved.

Direct Measurement of Acetylesterase in Living Protist Cells1

PubMed Central

Medzon, Edward L.; Brady, Marilyn L.

1969-01-01

The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, “mesosome-like” bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10−5m. Eserine (10−5m) and Paraoxon (10−7m) inhibited B. megaterium enzyme. Sodium acetate at 10−2m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed. Images PMID:4974398

Fabrication and Evaluation of a Micro(Bio)Sensor Array Chip for Multiple Parallel Measurements of Important Cell Biomarkers

PubMed Central

Pemberton, Roy M.; Cox, Timothy; Tuffin, Rachel; Drago, Guido A.; Griffiths, John; Pittson, Robin; Johnson, Graham; Xu, Jinsheng; Sage, Ian C.; Davies, Rhodri; Jackson, Simon K.; Kenna, Gerry; Luxton, Richard; Hart, John P.

2014-01-01

This report describes the design and development of an integrated electrochemical cell culture monitoring system, based on enzyme-biosensors and chemical sensors, for monitoring indicators of mammalian cell metabolic status. MEMS technology was used to fabricate a microwell-format silicon platform including a thermometer, onto which chemical sensors (pH, O2) and screen-printed biosensors (glucose, lactate), were grafted/deposited. Microwells were formed over the fabricated sensors to give 5-well sensor strips which were interfaced with a multipotentiostat via a bespoke connector box interface. The operation of each sensor/biosensor type was examined individually, and examples of operating devices in five microwells in parallel, in either potentiometric (pH sensing) or amperometric (glucose biosensing) mode are shown. The performance characteristics of the sensors/biosensors indicate that the system could readily be applied to cell culture/toxicity studies. PMID:25360580

Developing Highly Sensitive Micro-Biosensors for in-situ Monitoring Mercury and Chromium(IV) Contaminants by Genetically-evolving and Computer-designing Metal-binding Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Qinghong; Fang, Xiangdong; Goddard, William

2013-10-17

Mercury has been well known as an environmental pollutant to the environment and to cause serious effects on human health for several decades. To effectively control mercury pollution and reduce mercury damages, the sensitive determination of mercury is essential. Currently, many different types of sensor-based assays have been developed, while the whole-cell biosensor has been gaining increasingly attentions due to its easy reproducibility and the possibility to greatly reduce the cost. However, significant improvements on the specificity, sensitivity, stability and simplicity of the whole-cell biosensor are still needed prior to its eventual commercialization. Sponsored by US Department of Energy undermore » the contract agreement DE-FG02-07ER64410, we applied the special synthetic biology and directed evolution strategies to improve the effectiveness and performance of whole-cell biosensors. We have constructed different whole-cell biosensors for the mercuric ion and methylmercury detection with metalloregulator MerR, fluorescent protein mCherry and organomercurial lyase MerB. By introducing the mercuric transporter MerT, we were able to increase the detection sensitivity of whole-cell biosensors by at least one fold. By introducing the bio-amplification genetic circuit based on the gene cascade expression system of PRM-cI from bacteriophage l and Pm-XylS2 from Pseudomonas putida, we have increased the detection sensitivity of whole-cell biosensors by 1~2 folds in our tested conditions. With the directed evolution of MerR and subsequent high-throughput screening via color assay and microplate screening, we have dramatically increased the detection sensitivity by up to 10 folds at low concentration of mercury (II) of 1-10nM. Structural modeling and computational analysis of the mutated MerR showed that many mutations could cause the change of a loop to helix, which could be responsible for the increased mercury sensitivity.« less

A silicon-based peptide biosensor for label-free detection of cancer cells

NASA Astrophysics Data System (ADS)

Martucci, Nicola M.; Rea, Ilaria; Ruggiero, Immacolata; Terracciano, Monica; De Stefano, Luca; Migliaccio, Nunzia; Dardano, Principia; Arcari, Paolo; Rendina, Ivo; Lamberti, Annalisa

2015-05-01

Sensitive and accurate detection of cancer cells plays a crucial role in diagnosis of cancer and minimal residual disease, so being one of the most hopeful approaches to reduce cancer death rates. In this paper, a strategy for highly selective and sensitive detection of lymphoma cells on planar silicon-based biosensor has been evaluated. In this setting an Idiotype peptide, able to specifically bind the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, has been covalently linked to the sensor active surface and used as molecular probe. The biochip here presented showed a coverage efficiency of 85% with a detection efficiency of 8.5×10-3 cells/μm2. The results obtained suggested an efficient way for specific label-free cell detection by using a silicon-based peptide biosensor. In addition, the present recognition strategy, besides being useful for the development of sensing devices capable of monitoring minimal residual disease, could be used to find and characterize new specific receptor-ligand interactions through the screening of a recombinant phage library.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Utility of Ochrobactrum anthropi YC152 in a Microbial Fuel Cell as an Early Warning Device for Hexavalent Chromium Determination

PubMed Central

Wang, Guey-Horng; Cheng, Chiu-Yu; Liu, Man-Hai; Chen, Tzu-Yu; Hsieh, Min-Chi; Chung, Ying-Chien

2016-01-01

Fast hexavalent chromium (Cr(VI)) determination is important for environmental risk and health-related considerations. We used a microbial fuel cell-based biosensor inoculated with a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic Ochrobactrum anthropi YC152 to determine the Cr(VI) concentration in water. The results indicated that O. anthropi YC152 exhibited high adaptability to pH, temperature, salinity, and water quality under anaerobic conditions. The stable performance of the microbial fuel cell (MFC)-based biosensor indicated its potential as a reliable biosensor system. The MFC voltage decreased as the Cr(VI) concentration in the MFC increased. Two satisfactory linear relationships were observed between the Cr(VI) concentration and voltage output for various Cr(VI) concentration ranges (0.0125–0.3 mg/L and 0.3–5 mg/L). The MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in drinking water, groundwater, and electroplating wastewater in 45 min with low deviations (<10%). The use of the biosensor can help in preventing the violation of effluent regulations and the maximum allowable concentration of Cr(VI) in water. Thus, the developed MFC biosensor has potential as an early warning detection device for Cr(VI) determination even if O. anthropi YC152 is a possible opportunistic pathogen. PMID:27537887

Functionalized nanopipettes: toward label-free, single cell biosensors.

PubMed

Actis, Paolo; Mak, Andy C; Pourmand, Nader

2010-08-01

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.

Functionalized nanopipettes: toward label-free, single cell biosensors

PubMed Central

Actis, Paolo; Mak, Andy C.

2010-01-01

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

Evaluation of a novel label-free photonic-crystal biosensor imaging system for the detection of prostate cancer cells

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Ding, XiaoFie; Sun, Lu-Zhe; Ye, Jing Yong

2017-02-01

Biomarker screening for prostate-specific antigen (PSA) is the current clinical standard for detection of prostate cancer. However this method has shown many limitations, mainly in its specificity, which can lead to a high false positive rate. Thus, there is a growing need in developing a more specific detection system for prostate cancer. Using a Photonic- Crystal-based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, we demonstrate the use of refractive index (RI) to accomplish label-free detection of prostate cancer cells against non-cancerous prostate epithelial cells. The PC-TIR biosensor possesses an open microcavity, which in contrast to traditional closed microcavities, allows for easier access of analyte molecules or cells to interact with its sensing surface. In this study, an imaging system was designed using the PC-TIR biosensor to quantify cell RI as the contrast parameter for prostate cancer detection. Non-cancerous BPH-1 prostate epithelial cells and prostate cancer PC-3 cells were placed on a single biosensor and measured concurrently. Recorded image data was then analyzed through a home-built MatLab program. Results demonstrate that RI is a suitable variable for differentiation between prostate cancer cells and non-cancerous prostate epithelial cells. Our study shows clinical potential in utilizing RI test for the detection of prostate cancer.

In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

PubMed Central

Edwards, Sarah K.; Ono, Toshikazu; Wang, Shenliang; Jiang, Wei; Franzini, Raphael M.; Jung, Jong Wha; Chan, Ke Min; Kool, Eric T.

2015-01-01

The repair of oxidative damage to DNA is essential to avoidance of mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a chief source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report on the design and properties of fluorogenic probes that directly report on OGG1 (and bacterial homologue Fpg) activity in real time as the oxidized base is excised. The probes are short modified DNA oligomers containing fluorescent DNA bases and are designed to utilize the damaged DNA base itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probe designs containing these fluorophores, and we found an optimized probe OGR1 that yields a 60-fold light-up signal in vitro with OGG1 and Fpg, and can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes may be useful in quantifying enzyme activity and performing competitive inhibition assays. PMID:26073452

Engineering the bioelectrochemical interface using functional nanomaterials and microchip technique toward sensitive and portable electrochemical biosensors.

PubMed

Jia, Xiaofang; Dong, Shaojun; Wang, Erkang

2016-02-15

Electrochemical biosensors have played active roles at the forefront of bioanalysis because they have the potential to achieve sensitive, specific and low-cost detection of biomolecules and many others. Engineering the electrochemical sensing interface with functional nanomaterials leads to novel electrochemical biosensors with improved performances in terms of sensitivity, selectivity, stability and simplicity. Functional nanomaterials possess good conductivity, catalytic activity, biocompatibility and high surface area. Coupled with bio-recognition elements, these features can amplify signal transduction and biorecognition events, resulting in highly sensitive biosensing. Additionally, microfluidic electrochemical biosensors have attracted considerable attention on account of their miniature, portable and low-cost systems as well as high fabrication throughput and ease of scaleup. For example, electrochemical enzymetic biosensors and aptamer biosensors (aptasensors) based on the integrated microchip can be used for portable point-of-care diagnostics and environmental monitoring. This review is a summary of our recent progress in the field of electrochemical biosensors, including aptasensors, cytosensors, enzymatic biosensors and self-powered biosensors based on biofuel cells. We presented the advantages that functional nanomaterials and microfluidic chip technology bring to the electrochemical biosensors, together with future prospects and possible challenges. Copyright © 2015 Elsevier B.V. All rights reserved.

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation.

PubMed

Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

2014-01-01

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

PubMed Central

Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S.

2017-01-01

ABSTRACT Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28112405

Recent Advances in Bioprinting and Applications for Biosensing

PubMed Central

Dias, Andrew D.; Kingsley, David M.; Corr, David T.

2014-01-01

Future biosensing applications will require high performance, including real-time monitoring of physiological events, incorporation of biosensors into feedback-based devices, detection of toxins, and advanced diagnostics. Such functionality will necessitate biosensors with increased sensitivity, specificity, and throughput, as well as the ability to simultaneously detect multiple analytes. While these demands have yet to be fully realized, recent advances in biofabrication may allow sensors to achieve the high spatial sensitivity required, and bring us closer to achieving devices with these capabilities. To this end, we review recent advances in biofabrication techniques that may enable cutting-edge biosensors. In particular, we focus on bioprinting techniques (e.g., microcontact printing, inkjet printing, and laser direct-write) that may prove pivotal to biosensor fabrication and scaling. Recent biosensors have employed these fabrication techniques with success, and further development may enable higher performance, including multiplexing multiple analytes or cell types within a single biosensor. We also review recent advances in 3D bioprinting, and explore their potential to create biosensors with live cells encapsulated in 3D microenvironments. Such advances in biofabrication will expand biosensor utility and availability, with impact realized in many interdisciplinary fields, as well as in the clinic. PMID:25587413

Mast cell-based electrochemical biosensor for quantification of the major shrimp allergen Pen a 1 (tropomyosin).

PubMed

Jiang, Donglei; Ji, Jian; An, Lu; Sun, Xiulan; Zhang, Yinzhi; Zhang, Genyi; Tang, Lili

2013-12-15

A novel cell-based electrochemical biosensor was developed to quantify major shrimp allergen Pen a 1 (tropomyosin) and to assess its immunoglobulin E (IgE)-mediated hypersensitivity. Rat basophilic leukemia (RBL-2H3) mast cells, encapsulated in type I collagen, were immobilized on a self-assembled l-cysteine/gold nanoparticle (AuNPsCys)-modified gold electrode to monitor IgE-mediated mast cell sensitization and activation. The exposure of dinitrophenol-bovine serum albumin (DNP-BSA), as a model antigen that stimulates mast cells, induced a robust and long-lasting electrochemical impedance signal in a dose-dependent manner which efficiently measured degranulation of anti-DNP IgE-stimulated mast cells. Then this mast cell-based biosensor was applied into quantification for the shrimp allergen with anti-shrimp tropomyosin IgE-sensitization. The electrochemical impedance spectroscopy (EIS) results showed that the impedance value (Ret) increased with the concentration of purified shrimp allergen Pen a 1 (tropomyosin) in range of 0.5-0.25 μg mL(-1) with the detection limit as 0.15 μg mL(-1), and the electrochemical result was confirmed by β-hexosaminidase assay and scanning electron microscopic morphological (SEM) analysis. Thus, a simple, label-free, and sensitive method for the determination of shrimp allergens was proposed and demonstrated here, implying a highly versatile biosensor for food allergen detection and prediction. Copyright © 2013 Elsevier B.V. All rights reserved.

Progress in chemical luminescence-based biosensors: A critical review.

PubMed

Roda, Aldo; Mirasoli, Mara; Michelini, Elisa; Di Fusco, Massimo; Zangheri, Martina; Cevenini, Luca; Roda, Barbara; Simoni, Patrizia

2016-02-15

Biosensors are a very active research field. They have the potential to lead to low-cost, rapid, sensitive, reproducible, and miniaturized bioanalytical devices, which exploit the high binding avidity and selectivity of biospecific binding molecules together with highly sensitive detection principles. Of the optical biosensors, those based on chemical luminescence detection (including chemiluminescence, bioluminescence, electrogenerated chemiluminescence, and thermochemiluminescence) are particularly attractive, due to their high-to-signal ratio and the simplicity of the required measurement equipment. Several biosensors based on chemical luminescence have been described for quantitative, and in some cases multiplex, analysis of organic molecules (such as hormones, drugs, pollutants), proteins, and nucleic acids. These exploit a variety of miniaturized analytical formats, such as microfluidics, microarrays, paper-based analytical devices, and whole-cell biosensors. Nevertheless, despite the high analytical performances described in the literature, the field of chemical luminescence biosensors has yet to demonstrate commercial success. This review presents the main recent advances in the field and discusses the approaches, challenges, and open issues, with the aim of stimulating a broader interest in developing chemical luminescence biosensors and improving their commercial exploitation. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent Developments in Enzyme, DNA and Immuno-Based Biosensors.

PubMed

Asal, Melis; Özen, Özlem; Şahinler, Mert; Polatoğlu, İlker

2018-06-13

Novel sensitive, rapid and economical biosensors are being developed in a wide range of medical environmental and food applications. In this paper, we review some of the main advances in the field over the past few years by discussing recent studies from literature. A biosensor, which is defined as an analytical device consisting of a biomolecule, a transducer and an output system, can be categorized according to the type of the incorporated biomolecule. The biomolecules can be enzymes, antibodies, ssDNA, organelles, cells etc. The main biosensor categories classified according to the biomolecules are enzymatic biosensors, immunosensors and DNA-based biosensors. These sensors can measure analytes produced or reduced during reactions at lower costs compared to the conventional detection techniques. Numerous types of biosensor studies conducted over the last decade have been explored here to reveal their key applications in medical, environmental and food industries which provide comprehensive perspective to the readers. Overviews of the working principles and applications of the reviewed sensors are also summarized.

A miniaturized electrochemical toxicity biosensor based on graphene oxide quantum dots/carboxylated carbon nanotubes for assessment of priority pollutants.

PubMed

Zhu, Xiaolin; Wu, Guanlan; Lu, Nan; Yuan, Xing; Li, Baikun

2017-02-15

The study presented a sensitive and miniaturized cell-based electrochemical biosensor to assess the toxicity of priority pollutants in the aquatic environment. Human hepatoma (HepG2) cells were used as the biological recognition agent to measure the changes of electrochemical signals and reflect the cell viability. The graphene oxide quantum dots/carboxylated carbon nanotubes hybrid was developed in a facile and green way. Based on the hybrid composite modified pencil graphite electrode, the cell culture and detection vessel was miniaturized to a 96-well plate instead of the traditional culture dish. In addition, three sensitive electrochemical signals attributed to guanine/xanthine, adenine, and hypoxanthine were detected simultaneously. The biosensor was used to evaluate the toxicity of six priority pollutants, including Cd, Hg, Pb, 2,4-dinitrophenol, 2,4,6-trichlorophenol, and pentachlorophenol. The 24h IC 50 values obtained by the electrochemical biosensor were lower than those of conventional MTT assay, suggesting the enhanced sensitivity of the electrochemical assay towards heavy metals and phenols. This platform enables the label-free and sensitive detection of cell physiological status with multi-parameters and constitutes a promising approach for toxicity detection of pollutants. It makes possible for automatical and high-throughput analysis on nucleotide catabolism, which may be critical for life science and toxicology. Copyright © 2016 Elsevier B.V. All rights reserved.

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

PubMed

Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

2018-06-01

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

1-D grating based SPR biosensor for the detection of lung cancer biomarkers using Vroman effect

NASA Astrophysics Data System (ADS)

Teotia, Pradeep Kumar; Kaler, R. S.

2018-01-01

Grating based surface plasmon resonance waveguide biosensor have been reported for the detection of lung cancer biomarkers using Vroman effect. The proposed grating based multilayered biosensor is designed with high detection accuracy for Epidermal growth factor receptor (EGFR) and also analysed to show high detection accuracy with acceptable sensitivity for both cancer biomarkers. The introduction of periodic grating with multilayer metals generates a good resonance that make it possible for early detection of cancerous cells. Using finite difference time domain method, it is observed wavelength of biosensor get red-shifted on variations of the refractive index due to the presence of both the cancerous bio-markers. The reported detection accuracy and sensitivity of proposed biosensor is quite acceptable for both lung cancer biomarkers i.e. Carcinoembryonic antigen (CEA) and Epidermal growth factor receptor (EGFR) which further offer us label free early detection of lung cancer using these biomarkers.

Biosensors for Whole-Cell Bacterial Detection

PubMed Central

Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

2014-01-01

SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

Fast and accurate detection of cancer cell using a versatile three-channel plasmonic sensor

NASA Astrophysics Data System (ADS)

Hoseinian, M.; Ahmadi, A. R.; Bolorizadeh, M. A.

2016-09-01

Surface Plasmon Resonance (SPR) optical fiber sensors can be used as cost-effective small sized biosensors that are relatively simple to operate. Additionally, these instruments are label-free, hence rendering them highly sensitive to biological measurements. In this study, a three-channel microstructure optical fiber plasmonic-based portable biosensor is designed and analyzed using Finite Element Method. The proposed system is capable of determining changes in sample's refractive index with precision of order one thousandth. The biosensor measures three absorption resonance wavelengths of the analytes simultaneously. This property is one of the main advantages of the proposed biosensor since it reduces the error in the measured wavelength and enhances the accuracy of the results up to 10-5 m/RIU by reducing noise. In this paper, Jurkat cell, an indicator cell for leukemia cancer, is considered as the analyte; and its absorption resonance wavelengths as well as sensitivity in each channel are determined.

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring

PubMed Central

Weisman, Itamar; Romano, Jacob; Ivics, Zoltán; Izsvák, Zsuzsanna; Barkai, Uriel

2017-01-01

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device. PMID:28949988

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring.

PubMed

Siska, Evangelia K; Weisman, Itamar; Romano, Jacob; Ivics, Zoltán; Izsvák, Zsuzsanna; Barkai, Uriel; Petrakis, Spyros; Koliakos, George

2017-01-01

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.

Bioelectrochemical interface engineering: toward the fabrication of electrochemical biosensors, biofuel cells, and self-powered logic biosensors.

PubMed

Zhou, Ming; Dong, Shaojun

2011-11-15

Over the past decade, researchers have devoted considerable attention to the integration of living organisms with electronic elements to yield bioelectronic devices. Not only is the integration of DNA, enzymes, or whole cells with electronics of scientific interest, but it has many versatile potential applications. Researchers are using these ideas to fabricate biosensors for analytical applications and to assemble biofuel cells (BFCs) and biomolecule-based devices. Other research efforts include the development of biocomputing systems for information processing. In this Account, we focus on our recent progress in engineering at the bioelectrochemical interface (BECI) for the rational design and construction of important bioelectronic devices, ranging from electrochemical (EC-) biosensors to BFCs, and self-powered logic biosensors. Hydrogels and sol-gels provide attractive materials for the immobilization of enzymes because they make EC-enzyme biosensors stable and even functional in extreme environments. We use a layer-by-layer (LBL) self-assembly technique to fabricate multicomponent thin films on the BECI at the nanometer scale. Additionally, we demonstrate how carbon nanomaterials have paved the way for new and improved EC-enzyme biosensors. In addition to the widely reported BECI-based electrochemical impedance spectroscopy (EIS)-type aptasensors, we integrate the LBL technique with our previously developed "solid-state probe" technique for redox probes immobilization on electrode surfaces to design and fabricate BECI-based differential pulse voltammetry (DPV)-type aptasensors. BFCs can directly harvest energy from ambient biofuels as green energy sources, which could lead to their application as simple, flexible, and portable power sources. Porous materials provide favorable microenvironments for enzyme immobilization, which can enhance BFC power output. Furthermore, by introducing aptamer-based logic systems to BFCs, such systems could be applied as self-powered and intelligent aptasensors for the logic detection. We have developed biocomputing keypad lock security systems which can be also used for intelligent medical diagnostics. BECI engineering provides a simple but effective approach toward the design and fabrication of EC-biosensors, BFCs, and self-powered logic biosensors, which will make essential contributions in the development of creative and practical bioelectronic devices. The exploration of novel interface engineering applications and the creation of new fabrication concepts or methods merit further attention.

Molecular signaling in live cells studied by FRET

NASA Astrophysics Data System (ADS)

Chien, Shu; Wang, Yingxiao

2011-11-01

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate molecular/cellular functions.

Molecular signaling in live cells studied by FRET

NASA Astrophysics Data System (ADS)

Chien, Shu; Wang, Yingxiao

2012-03-01

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate molecular/cellular functions.

Assessing activity of Hepatitis A virus 3C protease using a cyclized luciferase-based biosensor.

PubMed

Zhou, Junwei; Wang, Dang; Xi, Yongqiang; Zhu, Xinyu; Yang, Yuting; Lv, Mengting; Luo, Chuanzhen; Chen, Jiyao; Ye, Xu; Fang, Liurong; Xiao, Shaobo

2017-07-08

Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3C pro ), an important nonstructural protein, is responsible for most cleavage within the viral polyprotein and is critical for the processes of viral replication. Our group has previously demonstrated that HAV 3C pro cleaves human NF-κB essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQ↓ADIYKA) that is cleaved by HAV 3C pro and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3C pro in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3C pro recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferase-based biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3C pro . With this biosensor (233DH), we monitored HAV 3C pro activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3C pro and other virus-encoded proteases. The results showed that the activity of this luciferase biosensor is specifically dependent on HAV 3C pro . Collectively, our data demonstrate that the luciferase biosensor developed here might provide a rapid, sensitive, and efficient evaluation of HAV 3C pro activity, and should extend our better understanding of the biological relevance of HAV 3C pro . Copyright © 2017 Elsevier Inc. All rights reserved.

The development of graphene-based devices for cell biology research

NASA Astrophysics Data System (ADS)

Yan, Zhi-Qin; Zhang, Wei

2014-06-01

Graphene has emerged as a new carbon nanoform with great potential in many applications due to its exceptional physical and chemical properties. Especially, graphene and its derivatives are also gaining a lot of interest in the biomedical field as new components for biosensors, tissue engineering, and drug delivery. This review presents unique properties of graphene, the bio-effects of graphene and its derivatives, especially their interactions with cells and the development of graphene-based biosensors and nanomedicines for cancer diagnosis and treatment.

Fluorogenic DNA Sequencing in PDMS Microreactors

PubMed Central

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney

2012-01-01

We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization. PMID:21666670

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

PubMed

Orgovan, Norbert; Ungai-Salánki, Rita; Lukácsi, Szilvia; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Szabó, Bálint; Horvath, Robert

2016-09-01

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell-extracellular matrix interactions.

Investigating the effect of design parameters on the response time of a highly sensitive microbial hydrogen sulfide biosensor based on oxygen consumption.

PubMed

Vosoughi, Amin; Yazdian, Fatemeh; Amoabediny, Ghassem; Hakim, Maziar

2015-08-15

A novel hydrogen sulfide microbial biosensor was developed based on investigating the influence of four design parameters: cell concentration, immobilization bed type, hydrogen sulfide concentration, and geometrical shape of the biosensor. Thiobacillus thioparus was used as the recognition element and it was immobilized on sodium alginate as well as agarose bed. The results were optimized by the application of statistical optimization software based on response time of the system. Oxygen reduction was considered as the detection sign. Sodium alginate solution with a concentration of 2.3% (w/v) and optical density of 10 at 605 nm was found as the optimum conditions for immobilization with response time of 72s . Optimum response time of immobilized T. thioparus on agarose was also found equal to 120 s at agarose concentration of 1.2% (w/v) and optical density of 10.83. Performance of the biosensor in different temperatures, pH and agitation speeds was also analyzed. The designed biosensor could detect concentrations of hydrogen sulfide as low as 0.5 ppm. T. thioparus could retain 99% of the original activity in both systems, after ten days passing the fabrication. A fractal analysis was also done theoretically to investigate the diffusion of oxygen in immobilized cells which showed a satisfactory value of oxygen take up by the immobilized cells. Copyright © 2015 Elsevier B.V. All rights reserved.

A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.

PubMed

Slattery, Scott D; Hahn, Klaus M

2014-12-01

Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules. Copyright © 2014 John Wiley & Sons, Inc.

A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.

PubMed

Li, Chunjie; Imanishi, Ayako; Komatsu, Naoki; Terai, Kenta; Amano, Mutsuki; Kaibuchi, Kozo; Matsuda, Michiyuki

2017-01-11

Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

PubMed

Hirata, Eishu; Kiyokawa, Etsuko

2016-09-20

Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells.

PubMed

Bollella, Paolo; Gorton, Lo; Antiochia, Riccarda

2018-04-24

Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

Biosensor Architectures for High-Fidelity Reporting of Cellular Signaling

PubMed Central

Dushek, Omer; Lellouch, Annemarie C.; Vaux, David J.; Shahrezaei, Vahid

2014-01-01

Understanding mechanisms of information processing in cellular signaling networks requires quantitative measurements of protein activities in living cells. Biosensors are molecular probes that have been developed to directly track the activity of specific signaling proteins and their use is revolutionizing our understanding of signal transduction. The use of biosensors relies on the assumption that their activity is linearly proportional to the activity of the signaling protein they have been engineered to track. We use mechanistic mathematical models of common biosensor architectures (single-chain FRET-based biosensors), which include both intramolecular and intermolecular reactions, to study the validity of the linearity assumption. As a result of the classic mechanism of zero-order ultrasensitivity, we find that biosensor activity can be highly nonlinear so that small changes in signaling protein activity can give rise to large changes in biosensor activity and vice versa. This nonlinearity is abolished in architectures that favor the formation of biosensor oligomers, but oligomeric biosensors produce complicated FRET states. Based on this finding, we show that high-fidelity reporting is possible when a single-chain intermolecular biosensor is used that cannot undergo intramolecular reactions and is restricted to forming dimers. We provide phase diagrams that compare various trade-offs, including observer effects, which further highlight the utility of biosensor architectures that favor intermolecular over intramolecular binding. We discuss challenges in calibrating and constructing biosensors and highlight the utility of mathematical models in designing novel probes for cellular signaling. PMID:25099816

An optical microplate biosensor for the detection of methyl parathion pesticide using a biohybrid of Sphingomonas sp. cells-silica nanoparticles.

PubMed

Mishra, Archana; Kumar, Jitendra; Melo, Jose Savio

2017-01-15

The previously developed Sphingomonas sp. based optical microplate biosensor for methyl parathion (MP) was good as it detected multiple samples but had poor stability and low sensitivity. The present study aims to overcome these limitations. Silica nanoparticles (Si NP) were thus functionalized with polyethyleneimine (PEI) and the functionalized silica nanoparticles ( f Si NP) were then integrated with Sphingomonas sp. cells. The process was optimized for hydrolysis of MP into p-nitrophenol (PNP). Integration of f Si NP with cells was confirmed by FT-IR analysis. Biohybrid of Sphingomonas sp.- f Si NP was immobilized on the wells of microplate and associated directly with the optical transducer of microplate reader. Immobilized biohybrid of Sphingomonas sp.- f Si NP was characterized using SEM. A detection range of 0.1-1ppm MP was achieved from the linear range of calibration plot. After integration with f Si NP the storage stability of biohybrid was enhanced ten times from 18 to 180 days. This study proves that after interaction of cells with f Si NP, improved the sensitivity and stability of the biosensor. Spiked samples were also analyzed and correlated using this biohybrid based biosensor. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

PubMed

Castro-López, Vanessa; Harris, Leanne F; O'Donnell, James S; Killard, Anthony J

2011-01-01

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Dual functional extracellular recording using a light-addressable potentiometric sensor for bitter signal transduction.

PubMed

Du, Liping; Wang, Jian; Chen, Wei; Zhao, Luhang; Wu, Chunsheng; Wang, Ping

2018-08-31

This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

PubMed

Skjoedt, Mette L; Snoek, Tim; Kildegaard, Kanchana R; Arsovska, Dushica; Eichenberger, Michael; Goedecke, Tobias J; Rajkumar, Arun S; Zhang, Jie; Kristensen, Mette; Lehka, Beata J; Siedler, Solvej; Borodina, Irina; Jensen, Michael K; Keasling, Jay D

2016-11-01

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP

PubMed Central

Wang, Xin C.; Wilson, Stephen C.; Hammond, Ming C.

2016-01-01

Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host–microbial and microbial–microbial interactions through small molecule signals. PMID:27382070

Application of the SSB biosensor to study in vitro transcription.

PubMed

Cook, Alexander; Hari-Gupta, Yukti; Toseland, Christopher P

2018-02-12

Gene expression, catalysed by RNA polymerases (RNAP), is one of the most fundamental processes in living cells. The majority of methods to quantify mRNA are based upon purification of the nucleic acid which leads to experimental inaccuracies and loss of product, or use of high cost dyes and sensitive spectrophotometers. Here, we describe the use of a fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, the SSB biosensor showed similar binding properties to mRNA, to that of its native substrate, single-stranded DNA (ssDNA). We found the biosensor to be reproducible with no associated loss of product through purification, or the requirement for expensive dyes. Therefore, we propose that the SSB biosensor is a useful tool for comparative measurement of mRNA yield following in vitro transcription. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A bio-inspired design of live cell biosensors

NASA Astrophysics Data System (ADS)

Marcek Chorvatova, A.; Teplicky, T.; Pavlinska, Z.; Kronekova, Z.; Trelova, D.; Razga, F.; Nemethova, V.; Uhelska, L.; Lacik, I.; Chorvat, D.

2018-02-01

The last decade has witnessed a rapid growth of nanoscale-oriented biosensors that becomes one of the most promising and rapidly growing areas of modern research. Despite significant advancements in conception of such biosensors, most are based at evaluation of molecular, or protein interactions. It is however becoming increasingly evident that functionality of a living system does not reside in genome or in individual proteins, as no real biological functionality is expressed at these levels. Instead, to comprehend the true functioning of a biological system, it is essential to understand the integrative physiological behavior of the complex molecular interactions in their natural environment and precise spatio-temporal topology. In this contribution we therefore present a new concept for creation of biosensors, bio-inspired from true functioning of living cells, while monitoring their endogenous fluorescence, or autofluorescence.

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.

PubMed

Lang, Kathrin; Davis, Lloyd; Wallace, Stephen; Mahesh, Mohan; Cox, Daniel J; Blackman, Melissa L; Fox, Joseph M; Chin, Jason W

2012-06-27

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds†

PubMed Central

Stiner, Lawrence; Halverson, Larry J.

2002-01-01

A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with Kapp values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants. PMID:11916719

Optical biosensor optimized for continuous in-line glucose monitoring in animal cell culture.

PubMed

Tric, Mircea; Lederle, Mario; Neuner, Lisa; Dolgowjasow, Igor; Wiedemann, Philipp; Wölfl, Stefan; Werner, Tobias

2017-09-01

Biosensors for continuous glucose monitoring in bioreactors could provide a valuable tool for optimizing culture conditions in biotechnological applications. We have developed an optical biosensor for long-term continuous glucose monitoring and demonstrated a tight glucose level control during cell culture in disposable bioreactors. The in-line sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase (GOD). The dynamic range of the sensor was tuned by a hydrophilic perforated diffusion membrane with an optimized permeability for glucose and oxygen. The biosensor was thoroughly characterized by experimental data and numerical simulations, which enabled insights into the internal concentration profile of the deactivating by-product hydrogen peroxide. The simulations were carried out with a one-dimensional biosensor model and revealed that, in addition to the internal hydrogen peroxide concentration, the turnover rate of the enzyme GOD plays a crucial role for biosensor stability. In the light of this finding, the glucose sensor was optimized to reach a long functional stability (>52 days) under continuous glucose monitoring conditions with a dynamic range of 0-20 mM and a response time of t 90  ≤ 10 min. In addition, we demonstrated that the sensor was sterilizable with beta and UV irradiation and only subjected to minor cross sensitivity to oxygen, when an oxygen reference sensor was applied. Graphical abstract Measuring setup of a glucose biosensor in a shake flask for continuous glucose monitoring in mammalian cell culture.

Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer

PubMed Central

Komatsubara, Akira T.; Matsuda, Michiyuki; Aoki, Kazuhiro

2015-01-01

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002–0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

In situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell imaging probes.

PubMed

Wu, Haoxing; Yang, Jun; Šečkutė, Jolita; Devaraj, Neal K

2014-06-02

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted 6-alkenyl-1,2,4,5-tetrazine derivatives through an elimination-Heck cascade reaction. By using this strategy, we provide 24 examples of π-conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta-1,3-diene-substituted tetrazines, and a diverse array of fluorescent probes suitable for live-cell imaging. These highly conjugated probes show very strong fluorescence turn-on (up to 400-fold) when reacted with dienophiles such as cyclopropenes and trans-cyclooctenes, and we demonstrate their application for live-cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly Sensitive Electrochemical Biosensor for Evaluation of Oxidative Stress Based on the Nanointerface of Graphene Nanocomposites Blended with Gold, Fe3O4, and Platinum Nanoparticles.

PubMed

Wang, Le; Zhang, Yuanyuan; Cheng, Chuansheng; Liu, Xiaoli; Jiang, Hui; Wang, Xuemei

2015-08-26

High levels of H2O2 pertain to high oxidative stress and are associated with cancer, autoimmune, and neurodegenerative disease, and other related diseases. In this study, a sensitive H2O2 biosensor for evaluation of oxidative stress was fabricated on the basis of the reduced graphene oxide (RGO) nanocomposites decorated with Au, Fe3O4, and Pt nanoparticles (RGO/AuFe3O4/Pt) modified glassy carbon electrode (GCE) and used to detect the released H2O2 from cancer cells and assess the oxidative stress elicited from H2O2 in living cells. Electrochemical behavior of RGO/AuFe3O4/Pt nanocomposites exhibits excellent catalytic activity toward the relevant reduction with high selection and sensitivity, low overpotential of 0 V, low detection limit of ∼0.1 μM, large linear range from 0.5 μM to 11.5 mM, and outstanding reproducibility. The as-prepared biosensor was applied in the measurement of efflux of H2O2 from living cells including healthy normal cells and tumor cells under the external stimulation. The results display that this new nanocomposites-based biosensor is a promising candidate of nonenzymatic H2O2 sensor which has the possibility of application in clinical diagnostics to assess oxidative stress of different kinds of living cells.

Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

PubMed

Wang, Hongwei; Lang, Qiaolin; Li, Liang; Liang, Bo; Tang, Xiangjiang; Kong, Lingrang; Mascini, Marco; Liu, Aihua

2013-06-18

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

How to design cell-based biosensors using the sol-gel process.

PubMed

Depagne, Christophe; Roux, Cécile; Coradin, Thibaud

2011-05-01

Inorganic gels formed using the sol-gel process are promising hosts for the encapsulation of living organisms and the design of cell-based biosensors. However, the possibility to use the biological activity of entrapped cells as a biological signal requires a good understanding and careful control of the chemical and physical conditions in which the organisms are placed before, during, and after gel formation, and their impact on cell viability. Moreover, it is important to examine the possible transduction methods that are compatible with sol-gel encapsulated cells. Through an updated presentation of the current knowledge in this field and based on selected examples, this review shows how it has been possible to convert a chemical technology initially developed for the glass industry into a biotechnological tool, with current limitations and promising specificities.

Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues.

PubMed

Collot, Mayeul; Fam, Tkhe Kyong; Ashokkumar, Pichandi; Faklaris, Orestis; Galli, Thierry; Danglot, Lydia; Klymchenko, Andrey S

2018-04-25

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M -1 cm -1 ) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly.

PubMed

Sun, Qing; Chen, Qi; Blackstock, Daniel; Chen, Wilfred

2015-08-25

Context driven biosensor assembly with modular targeting and detection moieties is gaining significant attentions. Although protein-based nanoparticles have emerged as an excellent platform for biosensor assembly, current strategies of decorating bionanoparticles with targeting and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. Herein, we report a highly modular post-translational modification approach for biosensor assembly based on sortase A-mediated ligation. This approach enables the simultaneous modifications of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA aptamer, and dye decoration. The resulting easy-purification platform offers a high degree of targeting and detection modularity with signal amplification. This flexibility is demonstrated for the detection of both immobilized antigens and cancer cells.

A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction.

PubMed

Zhang, Jing; Wang, Liang-Liang; Hou, Mei-Feng; Xia, Yao-Kun; He, Wen-Hui; Yan, An; Weng, Yun-Ping; Zeng, Lu-Peng; Chen, Jing-Hua

2018-04-15

Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

PubMed Central

van Unen, Jakobus; Woolard, Jeanette; Rinken, Ago; Hoffmann, Carsten; Hill, Stephen J.; Goedhart, Joachim; Bruchas, Michael R.; Bouvier, Michel

2015-01-01

The last frontier for a complete understanding of G-protein–coupled receptor (GPCR) biology is to be able to assess GPCR activity, interactions, and signaling in vivo, in real time within biologically intact systems. This includes the ability to detect GPCR activity, trafficking, dimerization, protein-protein interactions, second messenger production, and downstream signaling events with high spatial resolution and fast kinetic readouts. Resonance energy transfer (RET)–based biosensors allow for all of these possibilities in vitro and in cell-based assays, but moving RET into intact animals has proven difficult. Here, we provide perspectives on the optimization of biosensor design, of signal detection in living organisms, and the multidisciplinary development of in vitro and cell-based assays that more appropriately reflect the physiologic situation. In short, further development of RET-based probes, optical microscopy techniques, and mouse genome editing hold great potential over the next decade to bring real-time in vivo GPCR imaging to the forefront of pharmacology. PMID:25972446

Sensitivity of cell-based biosensors to environmental variables.

PubMed

Gilchrist, Kristin H; Giovangrandi, Laurent; Whittington, R Hollis; Kovacs, Gregory T A

2005-01-15

Electrically active living cells cultured on extracellular electrode arrays are utilized to detect biologically active agents. Because cells are highly sensitive to environmental conditions, environmental fluctuations can elicit cellular responses that contribute to the noise in a cell-based biosensor system. Therefore, the characterization and control of environmental factors such as temperature, pH, and osmolarity is critical in such a system. The cell-based biosensor platform described here utilizes the measurement of action potentials from cardiac cells cultured on electrode arrays. A recirculating fluid flow system is presented for use in dose-response experiments that regulates temperature within +/-0.2 degrees C, pH to within +/-0.05 units, and allows no significant change in osmolarity. Using this system, the relationship between the sensor output parameters and environmental variation was quantified. Under typical experimental conditions, beat rate varied approximately 10% per degree change in temperature or per 0.1 unit change in pH. Similar relationships were measured for action potential amplitude, duration, and conduction velocity. For the specific flow system used in this work, the measured environmental sensitivity resulted in an overall beat rate variation of +/-4.7% and an overall amplitude variation of +/-3.3%. The magnitude of the noise due to environmental sensitivity has a large impact on the detection capability of the cell-based system. The significant responses to temperature, pH, and osmolarity have important implications for the use of living cells in detection systems and should be considered in the design and evaluation of such systems.

Measurement of Biologically Available Naphthalene in Gas and Aqueous Phases by Use of a Pseudomonas putida Biosensor

PubMed Central

Werlen, Christoph; Jaspers, Marco C. M.; van der Meer, Jan Roelof

2004-01-01

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 μM). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices. PMID:14711624

Immobilization free electrochemical biosensor for folate receptor in cancer cells based on terminal protection.

PubMed

Ni, Jiancong; Wang, Qingxiang; Yang, Weiqiang; Zhao, Mengmeng; Zhang, Ying; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Yang, Huang-Hao

2016-12-15

The determination of folate receptor (FR) that over expressed in vast quantity of cancerous cells frequently is significant for the clinical diagnosis and treatment of cancers. Many DNA-based electrochemical biosensors have been developed for FR detection with high selectivity and sensitivity, but most of them need complicated immobilization of DNA on the electrode surface firstly, which is tedious and therefore results in the poor reproducibility. In this study, a simple, sensitive, and selective electrochemical FR biosensor in cancer cells has been proposed, which combines the advantages of the convenient immobilization-free homogeneous indium tin oxide (ITO)-based electrochemical detection strategy and the high selectivity of the terminal protection of small molecule linked DNA. The small molecule of folic acid (FA) and an electroactive molecule of ferrocence (Fc) were tethered to 3'- and 5'-end of an arbitrary single-stranded DNA (ssDNA), respectively, forming the FA-ssDNA-Fc complex. In the absence of the target FR, the FA-ssDNA-Fc was degraded by exonuclease I (Exo I) from 3'-end and produced a free Fc, diffusing freely to the ITO electrode surface and resulting in strong electrochemical signal. When the target FR was present, the FA-ssDNA-Fc was bound to FR through specific interaction with FA anchored at the 3'-end, effectively protecting the ssDNA strand from hydrolysis by Exo I. The FR-FA-ssDNA-Fc could not diffuse easily to the negatively charged ITO electrode surface due to the electrostatic repulsion between the DNA strand and the negatively charged ITO electrode, so electrochemical signal reduced. The decreased electrochemical signal has a linear relationship with the logarithm of FR concentration in range of 10fM to 10nM with a detection limit of 3.8fM (S/N=3). The proposed biosensor has been applied to detect FR in HeLa cancer cells, and the decreased electrochemical signal has a linear relationship with the logarithm of cell concentration ranging from 100-10000cell/mL. Compared with the traditional heterogeneous electrochemical FR biosensors, the proposed biosensor owns the merits of the simplicity and high specificity, presenting the great potential application in the area of early diagnosis of cancers. Copyright © 2016 Elsevier B.V. All rights reserved.

Thin Hydrogel Films for Optical Biosensor Applications

PubMed Central

Mateescu, Anca; Wang, Yi; Dostalek, Jakub; Jonas, Ulrich

2012-01-01

Hydrogel materials consisting of water-swollen polymer networks exhibit a large number of specific properties highly attractive for a variety of optical biosensor applications. This properties profile embraces the aqueous swelling medium as the basis of biocompatibility, non-fouling behavior, and being not cell toxic, while providing high optical quality and transparency. The present review focuses on some of the most interesting aspects of surface-attached hydrogel films as active binding matrices in optical biosensors based on surface plasmon resonance and optical waveguide mode spectroscopy. In particular, the chemical nature, specific properties, and applications of such hydrogel surface architectures for highly sensitive affinity biosensors based on evanescent wave optics are discussed. The specific class of responsive hydrogel systems, which can change their physical state in response to externally applied stimuli, have found large interest as sophisticated materials that provide a complex behavior to hydrogel-based sensing devices. PMID:24957962

A heparin-functionalized carbon nanotube-based affinity biosensor for dengue virus.

PubMed

Wasik, Daniel; Mulchandani, Ashok; Yates, Marylynn V

2017-05-15

Dengue virus is an arthropod-borne virus transmitted primarily by Aedes mosquitos and is major cause of disease in tropical and subtropical regions. Colloquially known as Dengue Fever, infection can cause hemorrhagic disorders and death in humans and non-human primates. We report a novel electronic biosensor based on a single-walled carbon nanotube network chemiresistive transducer that is functionalized with heparin for low-cost, label-free, ultra-sensitive, and rapid detection of whole dengue virus (DENV). Heparin, an analog of the heparan sulfate proteoglycans that are receptors for dengue virus during infection of Vero cells and hepatocytes, was used for the first time in a biosensor as a biorecognition element instead of traditional antibody. Detection of DENV in viral culture supernatant has similar sensitivity as the corresponding viral titer in phosphate buffer despite the presence of growth media and Vero cell lysate. The biosensor demonstrated sensitivity within the clinically relevant range for humans and infected Aedes aegypti. It has potential application in clinical diagnosis and can improve point-of-care diagnostics of dengue infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays.

PubMed

Kintzios, Spiridon; Papageorgiou, Katerina; Yiakoumettis, Iakovos; Baricevic, Dea; Kusar, Anita

2010-11-02

We investigated the antioxidant activity of methanolic and water extracts of Slovene accessions of four medicinal plant species (Salvia officinalis, Achillea millefolium, Origanum vulgare subsp. vulgare and Gentiana lutea). Their free radical-scavenging activity against the DPPH. free radical was studied with a spectrophotometric assay, while their biological activity with the help of a laboratory-made biosensor based on immobilized fibroblast cells (assay duration: 3 min). The observed antioxidant activity of the extracts from the four investigated medicinal plant species was dependent on both the solvent used for extraction and the assay method (conventional or biosensor-based). Independently from the assay method and the solvent used for extraction, the lowest scavenging activity was observed in root extracts of G. lutea. Treatment of the immobilized cells with the plant extracts resulted in an increase of the cell membrane potential (membrane hyperpolarization), possibly due to the reduction of membrane damage due to oxidation. The novel cell biosensor could be utilized as a rapid, high throughput tool for screening the antioxidant properties of plant-derived compounds. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

PubMed

Rana, Subinoy; Elci, S Gokhan; Mout, Rubul; Singla, Arvind K; Yazdani, Mahdieh; Bender, Markus; Bajaj, Avinash; Saha, Krishnendu; Bunz, Uwe H F; Jirik, Frank R; Rotello, Vincent M

2016-04-06

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

Engineering and characterization of fluorogenic glycine riboswitches

PubMed Central

Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

2016-01-01

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3s−1 and 10−2s−1, respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. PMID:27220466

Nomad Biosensors: A New Multiplexed Technology for the Screening of GPCR Ligands.

PubMed

Mella, Rosa M; Kortazar, Danel; Roura-Ferrer, Meritxell; Salado, Clarisa; Valcárcel, María; Castilla, Amaia; Villacé, Patricia

2018-06-01

Nomad Technology (Innoprot [Innovative Technologies in Biological Systems], Derio, Spain), a novel tool for multiplexing high-throughput cell-based G protein-coupled receptor (GPCR) assays, is described in this work. This new technology comprises a family of fluorescent biosensors called Nomad Biosensors that allow for the measurement of responses mediated by G proteins through their interactions with second-messenger transduction proteins. GPCRs are one of the largest protein families of receptors in eukaryotes, and their signaling mediates important physiological processes within cells. Thus, GPCRs are associated with a wide variety of diseases, and considered major targets in therapeutic research. Nomad constitutes a novel tool for unraveling the mechanism of GPCR signal transduction by simultaneously tracing different pathways. GPCR activation changes the structural folding of the biosensor and promotes its vesicularization, as well as an increase in the fluorescence intensity. Based on this technology, the MPX Nomad cellular model was developed to discriminate between the Ca 2+ -mediated pathway and the cyclic adenosine monophosphate (cAMP)-mediated pathway. To validate this model, endothelin receptor B (ET B R) was coexpressed into the MPX Nomad cell line and assessed with a specific agonist, an antagonist, and a chemical library of compounds. Nomad Technology optimizes the identification of novel GPCR ligands and enables the testing of large numbers of compounds.

A novel thermal biosensor based on enzyme reaction for pesticides measurement.

PubMed

Zheng, Yi-Hu; Hua, Tse-Chao; Xu, Fei

2005-01-01

A novel thermal biosensor based on enzyme reaction for pesticides detection has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and identical reference column, which is set for eliminating the unspecific heat. The enzyme reaction takes place in the enzyme reaction column at a constant temperature (40 degrees C) realized by a thermoelectric thermostat. Thermosensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two effluents from enzyme reaction column and reference column. The ability of this biosensor to detect pesticides is demonstrated by the decreased degree of the hydrolytic heat in two types of thermosensor mode. The hydrolytic reaction is inhibited by 36% at 1 mg/L DDVP and 50% at 10 mg/L DDVP when cell-typed thermosensor is used. The percent inhibition is 30% at 1 mg/L DDVP and 42% at 10 mg/L DDVP in tube-typed thermosensor mode. The detection for real sample shows that this biosensor can be used for detection of organophosphate pesticides residue.

Establishment of Genetically Encoded Biosensors for Cytosolic Boric Acid in Plant Cells.

PubMed

Fukuda, Makiha; Wakuta, Shinji; Kamiyo, Jio; Fujiwara, Toru; Takano, Junpei

2018-06-08

Boron (B) is an essential micronutrient for plants. To maintain B concentration in tissues at appropriate levels, plants use boric acid channels belonging to the NIP subfamily of aquaporins and BOR borate exporters. To regulate B transport, these transporters exhibit different cell-type specific expression, polar localization, and B-dependent post-transcriptional regulation. Here, we describe the development of genetically encoded biosensors for cytosolic boric acid to visualize the spatial distribution and temporal dynamics of B in plant tissues. The biosensors were designed based on the function of the NIP5;1 5'-untranslated region (UTR), which promotes mRNA degradation in response to an elevated cytosolic boric acid concentration. The signal intensities of the biosensor coupled with Venus fluorescent protein and a nuclear localization signal (uNIP5;1-Venus) showed a negative correlation with intracellular B concentrations in cultured tobacco BY-2 cells. When expressed in Arabidopsis thaliana, uNIP5;1-Venus enabled quantification of the B distribution in roots at single-cell resolution. In mature roots, cytosolic B levels in stele were maintained under low-B supply, while those in epidermal, cortical, and endodermal cells were influenced by external B concentrations. Another biosensor coupled with a luciferase protein fused to a destabilization PEST sequence (uNIP5;1-Luc) was used to visualize changes in cytosolic boric acid concentrations. Thus, uNIP5;1-Venus/Luc enables visualization of B transport in various plant cells/tissues. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

PubMed Central

Edmondson, Rasheena; Broglie, Jessica Jenkins; Adcock, Audrey F.

2014-01-01

Abstract Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this review, we discuss the characteristics of 3D cell culture systems in comparison to the two-dimensional (2D) monolayer culture, focusing on cell growth conditions, cell proliferation, population, and gene and protein expression profiles. The innovations and development in 3D culture systems for drug discovery over the past 5 years are also reviewed in the article, emphasizing the cellular response to different classes of anticancer drugs, focusing particularly on similarities and differences between 3D and 2D models across the field. The progression and advancement in the application of 3D cell cultures in cell-based biosensors is another focal point of this review. PMID:24831787

A portable bioluminescence engineered cell-based biosensor for on-site applications.

PubMed

Roda, Aldo; Cevenini, Luca; Michelini, Elisa; Branchini, Bruce R

2011-04-15

We have developed a portable biosensing device based on genetically engineered bioluminescent (BL) cells. Cells were immobilized on a 4 × 3 multiwell cartridge using a new biocompatible matrix that preserved their vitality. Using a fiber optic taper, the cartridge was placed in direct contact with a cooled CCD sensor to image and quantify the BL signals. Yeast and bacterial cells were engineered to express recognition elements, whose interaction with the analyte led to luciferase expression, via reporter gene technology. Three different biosensors were developed. The first detects androgenic compounds using yeast cells carrying a green-emitting P. pyralis luciferase regulated by the human androgen receptor and a red mutant of the same species as internal vitality control. The second biosensor detects two classes of compounds (androgens and estrogens) using yeast strains engineered to express green-or red-emitting mutant firefly luciferases in response to androgens or estrogens, respectively. The third biosensor detects lactose analogue isopropyl β-d-1-thiogalactopyranoside using two E. coli strains. One strain exploits the lac operon as recognition element for the expression of P. pyralis luciferase. The other strain serves as a vitality control expressing Gaussia princeps luciferase, which requires a different luciferin substrate. The immobilized cells were stable for up to 1 month. The analytes could be detected at nanomolar levels with good precision and accuracy when the specific signal was corrected using the internal vitality control. This portable device can be used for on-site multiplexed bioassays for different compound classes. Copyright © 2011 Elsevier B.V. All rights reserved.

A novel electrochemical biosensor based on polyadenine modified aptamer for label-free and ultrasensitive detection of human breast cancer cells.

PubMed

Wang, Kun; He, Meng-Qi; Zhai, Fu-Heng; He, Rong-Huan; Yu, Yong-Liang

2017-05-01

Simple, rapid, sensitive, and specific detection of cancer cells plays a pivotal role in the diagnosis and prognosis of cancer. A sandwich electrochemical biosensor was developed based on polyadenine (polydA)-aptamer modified gold electrode (GE) and polydA-aptamer functionalized gold nanoparticles/graphene oxide (AuNPs/GO) hybrid for the label-free and selective detection of breast cancer cells (MCF-7) via a differential pulse voltammetry (DPV) technique. Due to the intrinsic affinity between multiple consecutive adenines of polydA sequences and gold, polydA modified aptamer instead of thiol terminated aptamer was immobilized on the surface of GE and AuNPs/GO. The label-free MCF-7 cells could be recognized by polydA-aptamer and self-assembled onto the surface of GE. The polydA-aptamer functionalized AuNPs/GO hybrid could further bind to MCF-7 cells to form a sandwich sensing system. Characterization of the surface modified GE was carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using Fe(CN) 6 3-/4- as a redox probe. Under the optimized experimental conditions, a detection limit of 8 cellsmL -1 (3σ/slope) was obtained for MCF-7 cells by the present electrochemical biosensor, along with a linear range of 10-10 5 cellsmL -1 . By virtue of excellent sensitivity, specificity and repeatability, the present electrochemical biosensor provides a potential application in point-of-care cancer diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

A Novel Early Warning System Based on a Sediment Microbial Fuel Cell for In Situ and Real Time Hexavalent Chromium Detection in Industrial Wastewater.

PubMed

Zhao, Shuai; Liu, Pu; Niu, Yongyan; Chen, Zhengjun; Khan, Aman; Zhang, Pengyun; Li, Xiangkai

2018-02-22

Hexavalent chromium (Cr(VI)) is a well-known toxic heavy metal in industrial wastewater, but in situ and real time monitoring cannot be achieved by current methods used during industrial wastewater treatment processes. In this study, a Sediment Microbial Fuel Cell (SMFC) was used as a biosensor for in situ real-time monitoring of Cr(VI), which was the organic substrate is oxidized in the anode and Cr(VI) is reduced at the cathode simultaneously. The pH 6.4 and temperature 25 °C were optimal conditions for the operation. Under the optimal conditions, linearity (R² = 0.9935) of the generated voltage was observed in the Cr(VI) concentration range from 0.2 to 0.7 mg/L. The system showed high specificity for Cr(VI), as other co-existing ions such as Cu 2+ , Zn 2+ , and Pb 2+ did not interfere with Cr(VI) detection. In addition, when the sediment MFC-based biosensor was applied for measuring Cr(VI) in actual wastewater samples, a low deviation (<8%) was obtained, which indicated its potential as a reliable biosensor device. MiSeq sequencing results showed that electrochemically active bacteria ( Geobacter and Pseudomonas ) were enriched at least two-fold on the biofilm of the anode in the biosensor as compared to the SMFC without Cr(VI). Cyclic voltammetry curves indicated that a pair of oxidation/reduction peaks appeared at -111 mV and 581 mV, respectively. These results demonstrated that the proposed sediment microbial fuel cell-based biosensor can be applied as an early warning device for real time in situ detection of Cr(VI) in industrial wastewaters.

A Novel Early Warning System Based on a Sediment Microbial Fuel Cell for In Situ and Real Time Hexavalent Chromium Detection in Industrial Wastewater

PubMed Central

Zhao, Shuai; Liu, Pu; Niu, Yongyan; Chen, Zhengjun; Khan, Aman; Zhang, Pengyun; Li, Xiangkai

2018-01-01

Hexavalent chromium (Cr(VI)) is a well-known toxic heavy metal in industrial wastewater, but in situ and real time monitoring cannot be achieved by current methods used during industrial wastewater treatment processes. In this study, a Sediment Microbial Fuel Cell (SMFC) was used as a biosensor for in situ real-time monitoring of Cr(VI), which was the organic substrate is oxidized in the anode and Cr(VI) is reduced at the cathode simultaneously. The pH 6.4 and temperature 25 °C were optimal conditions for the operation. Under the optimal conditions, linearity (R2 = 0.9935) of the generated voltage was observed in the Cr(VI) concentration range from 0.2 to 0.7 mg/L. The system showed high specificity for Cr(VI), as other co-existing ions such as Cu2+, Zn2+, and Pb2+ did not interfere with Cr(VI) detection. In addition, when the sediment MFC-based biosensor was applied for measuring Cr(VI) in actual wastewater samples, a low deviation (<8%) was obtained, which indicated its potential as a reliable biosensor device. MiSeq sequencing results showed that electrochemically active bacteria (Geobacter and Pseudomonas) were enriched at least two-fold on the biofilm of the anode in the biosensor as compared to the SMFC without Cr(VI). Cyclic voltammetry curves indicated that a pair of oxidation/reduction peaks appeared at −111 mV and 581 mV, respectively. These results demonstrated that the proposed sediment microbial fuel cell-based biosensor can be applied as an early warning device for real time in situ detection of Cr(VI) in industrial wastewaters. PMID:29470394

A low cost color-based bacterial biosensor for measuring arsenic in groundwater.

PubMed

Huang, Chi-Wei; Wei, Chia-Cheng; Liao, Vivian Hsiu-Chuan

2015-12-01

Using arsenic (As) contaminated groundwater for drinking or irrigation has caused major health problems for humans around the world, raising a need to monitor As level efficiently and economically. This study developed a color-based bacterial biosensor which is easy-to-use and inexpensive for measuring As and could be complementary to current As detecting techniques. The arsR-lacZ recombinant gene cassette in nonpathogenic strain Escherichia coli DH5α was used in the color-based biosensor which could be observed by eyes or measured by spectrometer. The developed bacterial biosensor demonstrates a quantitative range from 10 to 500μgL(-1) of As in 3-h reaction time. Furthermore, the biosensor was able to successfully detect and estimate As concentration in groundwater sample by measuring optical density at 595nm (OD595). Among different storage methods used in this study, biosensor in liquid at 4°C showed the longest shelf life about 9d, and liquid storage at RT and cell pellet could also be stored for about 3-5d. In conclusion, this study showed that the As biosensor with reliable color signal and economical preservation methods is useful for rapid screening of As pollutant, providing the potential for large scale screening and better management strategies for environmental quality control. Copyright © 2015 Elsevier Ltd. All rights reserved.

Live-cell MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface glycans.

PubMed

Witte, Christopher; Martos, Vera; Rose, Honor May; Reinke, Stefan; Klippel, Stefan; Schröder, Leif; Hackenberger, Christian P R

2015-02-23

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Building a minimal and generalizable model of transcription factor-based biosensors: Showcasing flavonoids.

PubMed

Trabelsi, Heykel; Koch, Mathilde; Faulon, Jean-Loup

2018-05-07

Progress in synthetic biology tools has transformed the way we engineer living cells. Applications of circuit design have reached a new level, offering solutions for metabolic engineering challenges that include developing screening approaches for libraries of pathway variants. The use of transcription-factor-based biosensors for screening has shown promising results, but the quantitative relationship between the sensors and the sensed molecules still needs more rational understanding. Herein, we have successfully developed a novel biosensor to detect pinocembrin based on a transcriptional regulator. The FdeR transcription factor (TF), known to respond to naringenin, was combined with a fluorescent reporter protein. By varying the copy number of its plasmid and the concentration of the biosensor TF through a combinatorial library, different responses have been recorded and modeled. The fitted model provides a tool to understand the impact of these parameters on the biosensor behavior in terms of dose-response and time curves and offers guidelines to build constructs oriented to increased sensitivity and or ability of linear detection at higher titers. Our model, the first to explicitly take into account the impact of plasmid copy number on biosensor sensitivity using Hill-based formalism, is able to explain uncharacterized systems without extensive knowledge of the properties of the TF. Moreover, it can be used to model the response of the biosensor to different compounds (here naringenin and pinocembrin) with minimal parameter refitting. © 2018 Wiley Periodicals, Inc.

Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering.

PubMed

Liu, Yang; Liu, Ye; Wang, Meng

2017-01-01

The development of synthetic biology and metabolic engineering has painted a great future for the bio-based economy, including fuels, chemicals, and drugs produced from renewable feedstocks. With the rapid advance of genome-scale modeling, pathway assembling and genome engineering/editing, our ability to design and generate microbial cell factories with various phenotype becomes almost limitless. However, our lack of ability to measure and exert precise control over metabolite concentration related phenotypes becomes a bottleneck in metabolic engineering. Genetically encoded small molecule biosensors, which provide the means to couple metabolite concentration to measurable or actionable outputs, are highly promising solutions to the bottleneck. Here we review recent advances in the design, optimization and application of small molecule biosensor in metabolic engineering, with particular focus on optimization strategies for transcription factor (TF) based biosensors.

Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering

PubMed Central

Liu, Yang; Liu, Ye; Wang, Meng

2017-01-01

The development of synthetic biology and metabolic engineering has painted a great future for the bio-based economy, including fuels, chemicals, and drugs produced from renewable feedstocks. With the rapid advance of genome-scale modeling, pathway assembling and genome engineering/editing, our ability to design and generate microbial cell factories with various phenotype becomes almost limitless. However, our lack of ability to measure and exert precise control over metabolite concentration related phenotypes becomes a bottleneck in metabolic engineering. Genetically encoded small molecule biosensors, which provide the means to couple metabolite concentration to measurable or actionable outputs, are highly promising solutions to the bottleneck. Here we review recent advances in the design, optimization and application of small molecule biosensor in metabolic engineering, with particular focus on optimization strategies for transcription factor (TF) based biosensors. PMID:29089935

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

NASA Astrophysics Data System (ADS)

Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

2013-07-01

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes. Electronic supplementary information (ESI) available: Experimental details; characterization of probes; the influence of electrolyte pH; probe concentration and glucose concentration on the electrode ECL effect. See DOI: 10.1039/c3nr01598j

Toxicity assessment using different bioassays and microbial biosensors.

PubMed

Hassan, Sedky H A; Van Ginkel, Steven W; Hussein, Mohamed A M; Abskharon, Romany; Oh, Sang-Eun

2016-01-01

Toxicity assessment of water streams, wastewater, and contaminated sediments, is a very important part of environmental pollution monitoring. Evaluation of biological effects using a rapid, sensitive and cost effective method can indicate specific information on ecotoxicity assessment. Recently, different biological assays for toxicity assessment based on higher and lower organisms such as fish, invertebrates, plants and algal cells, and microbial bioassays have been used. This review focuses on microbial biosensors as an analytical device for environmental, food, and biomedical applications. Different techniques which are commonly used in microbial biosensing include amperometry, potentiometry, conductometry, voltammetry, microbial fuel cells, fluorescence, bioluminescence, and colorimetry. Examples of the use of different microbial biosensors in assessing a variety of environments are summarized. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glycoprofiling of cancer biomarkers: Label-free electrochemical lectin-based biosensors

PubMed Central

Pihíková, Dominika; Kasák, Peter

2016-01-01

Glycosylation of biomolecules is one of the most prevalent post- and co-translational modification in a human body, with more than half of all human proteins being glycosylated. Malignant transformation of cells influences glycosylation machinery resulting in subtle changes of the glycosylation pattern within the cell populations as a result of cancer. Thus, an altered terminal glycan motif on glycoproteins could provide a warning signal about disease development and progression and could be applied as a reliable biomarker in cancer diagnostics. Among all highly effective glycoprofiling tools, label-free electrochemical impedance spectroscopy (EIS)-based biosensors have emerged as especially suitable tool for point-of-care early-stage cancer detection. Herein, we highlight the current challenges in glycoprofiling of various cancer biomarkers by ultrasensitive impedimetric-based biosensors with low sample consumption, low cost fabrication and simple miniaturization. Additionally, this review provides a short introduction to the field of glycomics and lectinomics and gives a brief overview of glycan alterations in different types of cancer. PMID:27275016

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.

PubMed

Pasquato, Antonella; Pullikotil, Philomena; Asselin, Marie-Claude; Vacatello, Manuela; Paolillo, Livio; Ghezzo, Francesca; Basso, Federica; Di Bello, Carlo; Dettin, Monica; Seidah, Nabil G

2006-08-18

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor

PubMed Central

Khalilzadeh, Balal; Shadjou, Nasrin; Afsharan, Hadi; Eskandani, Morteza; Nozad Charoudeh, Hojjatollah; Rashidi, Mohammad-Reza

2016-01-01

Introduction:Growing demands for ultrasensitive biosensing have led to the development of numerous signal amplification strategies. In this report, a novel electrochemiluminescence (ECL) method was developed for the detection and determination of caspase-3 activity based on reduced graphene oxide sheets decorated by gold nanoparticles as signal amplification element and horseradish peroxidase enzyme (HRP) as ECL intensity enhancing agent. Methods: The ECL intensity of the luminol was improved by using the streptavidin coated magnetic beads and HRP in the presence of hydrogen peroxide. The cleavage behavior of caspase-3 was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using biotinylated peptide (DEVD containing peptide) which was coated on reduced graphene oxide decorated with gold nanoparticle. The surface modification of graphene oxide was successfully confirmed by FTIR, UV-vis and x-ray spectroscopy. Results: ECL based biosensor showed that the linear dynamic range (LDR) and the lower limit of quantification (LLOQ) were 0.5-100 and 0.5 femtomolar (fM), respectively. Finally, the performance of the engineered peptide based biosensor was validated in the A549 cell line as real samples. Conclusion: The prepared peptide based biosensor could be considered as an excellent candidate for early detection of apoptosis, cell turnover, and cancer related diseases. PMID:27853677

A non-toxic fluorogenic dye for mitochondria labeling.

PubMed

Han, Junyan; Han, Myung Shin; Tung, Ching-Hsuan

2013-11-01

Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging. © 2013.

A Non-Toxic Fluorogenic Dye for Mitochondria Labeling

PubMed Central

Han, Junyan; Han, Myung Shin; Tung, Ching-Hsuan

2013-01-01

Background Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. Methods A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. Results AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. Conclusions A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. General Significance The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging. PMID:23850639

Graphene-interfaced electrical biosensor for label-free and sensitive detection of foodborne pathogenic E. coli O157:H7.

PubMed

Pandey, Ashish; Gurbuz, Yasar; Ozguz, Volkan; Niazi, Javed H; Qureshi, Anjum

2017-05-15

E. coli O157:H7 is an enterohemorrhagic bacteria responsible for serious foodborne outbreaks that causes diarrhoea, fever and vomiting in humans. Recent foodborne E. coli outbreaks has left a serious concern to public health. Therefore, there is an increasing demand for a simple, rapid and sensitive method for pathogen detection in contaminated foods. In this study, we developed a label-free electrical biosensor interfaced with graphene for sensitive detection of pathogenic bacteria. This biosensor was fabricated by interfacing graphene with interdigitated microelectrodes of capacitors that were biofunctionalized with E. coli O157:H7 specific antibodies for sensitive pathogenic bacteria detection. Here, graphene nanostructures on the sensor surface provided superior chemical properties such as high carrier mobility and biocompatibility with antibodies and bacteria. The sensors transduced the signal based on changes in dielectric properties (capacitance) through (i) polarization of captured cell-surface charges, (ii) cells' internal bioactivity, (iii) cell-wall's electronegativity or dipole moment and their relaxation and (iv) charge carrier mobility of graphene that modulated the electrical properties once the pathogenic E. coli O157:H7 captured on the sensor surface. Sensitive capacitance changes thus observed with graphene based capacitors were specific to E. coli O157:H7 strain with a sensitivity as low as 10-100 cells/ml. The proposed graphene based electrical biosensor provided advantages of speed, sensitivity, specificity and in-situ bacterial detection with no chemical mediators, represents a versatile approach for detection of a wide variety of other pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

PubMed

Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

PubMed

Yerramilli, V Siddartha; Kim, Kyung Hyuk

2018-03-16

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

PubMed Central

Xie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.

2018-01-01

Abstract Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains. PMID:29575702

Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates

NASA Astrophysics Data System (ADS)

Gates, R. D.; Muscatine, L.

1992-09-01

Three maceration methods are described for the isolation of single endoderm cells from marine cnidarians. Two are enzymatic treatments suitable for fleshy anthozoans such as sea anemones and zoanthids. The third employs calcium free sea water and is suitable for stony corals. The viability and morphology of the endoderm cells is described using fluorogenic dyes and scanning and transmission electron microscopy.

Rapid, portable detection of endocrine disrupting chemicals through ligand-nuclear hormone receptor interactions.

PubMed

Hunt, J Porter; Schinn, Song-Min; Jones, Matthew D; Bundy, Bradley C

2017-12-04

Endocrine disrupting chemicals (EDC) are structurally diverse compounds that can interact with nuclear hormone receptors, posing significant risk to human and ecological health. Unfortunately, many conventional biosensors have been too structure-specific, labor-intensive or laboratory-oriented to detect broad ranges of EDC effectively. Recently, several technological advances are providing more rapid, portable, and affordable detection of endocrine-disrupting activity through ligand-nuclear hormone receptor interactions. Here, we overview these recent advances applied to EDC biosensors - including cell lyophilization, cell immobilization, cell-free systems, smartphone-based signal detection, and improved competitive binding assays.

Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

PubMed

Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

2015-01-01

The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

PubMed Central

Geng, Deyu; Zhang, Zhixia; Guo, Huarong

2012-01-01

p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

MEMS-based power generation techniques for implantable biosensing applications.

PubMed

Lueke, Jonathan; Moussa, Walied A

2011-01-01

Implantable biosensing is attractive for both medical monitoring and diagnostic applications. It is possible to monitor phenomena such as physical loads on joints or implants, vital signs, or osseointegration in vivo and in real time. Microelectromechanical (MEMS)-based generation techniques can allow for the autonomous operation of implantable biosensors by generating electrical power to replace or supplement existing battery-based power systems. By supplementing existing battery-based power systems for implantable biosensors, the operational lifetime of the sensor is increased. In addition, the potential for a greater amount of available power allows additional components to be added to the biosensing module, such as computational and wireless and components, improving functionality and performance of the biosensor. Photovoltaic, thermovoltaic, micro fuel cell, electrostatic, electromagnetic, and piezoelectric based generation schemes are evaluated in this paper for applicability for implantable biosensing. MEMS-based generation techniques that harvest ambient energy, such as vibration, are much better suited for implantable biosensing applications than fuel-based approaches, producing up to milliwatts of electrical power. High power density MEMS-based approaches, such as piezoelectric and electromagnetic schemes, allow for supplemental and replacement power schemes for biosensing applications to improve device capabilities and performance. In addition, this may allow for the biosensor to be further miniaturized, reducing the need for relatively large batteries with respect to device size. This would cause the implanted biosensor to be less invasive, increasing the quality of care received by the patient.

Co-immobilization of glucoamylase and glucose oxidase for electrochemical sequential enzyme electrode for starch biosensor and biofuel cell.

PubMed

Lang, Qiaolin; Yin, Long; Shi, Jianguo; Li, Liang; Xia, Lin; Liu, Aihua

2014-01-15

A novel electrochemical sequential biosensor was constructed by co-immobilizing glucoamylase (GA) and glucose oxidase (GOD) on the multi-walled carbon nanotubes (MWNTs)-modified glassy carbon electrode (GCE) by chemical crosslinking method, where glutaraldehyde and bovine serum albumin was used as crosslinking and blocking agent, respectively. The proposed biosensor (GA/GOD/MWNTs/GCE) is capable of determining starch without using extra sensors such as Clark-type oxygen sensor or H2O2 sensor. The current linearly decreased with the increasing concentration of starch ranging from 0.005% to 0.7% (w/w) with the limit of detection of 0.003% (w/w) starch. The as-fabricated sequential biosensor can be applicable to the detection of the content of starch in real samples, which are in good accordance with traditional Fehling's titration. Finally, a stable starch/O2 biofuel cell was assembled using the GA/GOD/MWNTs/GCE as bioanode and laccase/MWNTs/GCE as biocathode, which exhibited open circuit voltage of ca. 0.53 V and the maximum power density of 8.15 μW cm(-2) at 0.31 V, comparable with the other glucose/O2 based biofuel cells reported recently. Therefore, the proposed biosensor exhibited attractive features such as good stability in weak acidic buffer, good operational stability, wide linear range and capable of determination of starch in real samples as well as optimal bioanode for the biofuel cell. Copyright © 2013 Elsevier B.V. All rights reserved.

A Perspective on Studying G-Protein-Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms.

PubMed

van Unen, Jakobus; Woolard, Jeanette; Rinken, Ago; Hoffmann, Carsten; Hill, Stephen J; Goedhart, Joachim; Bruchas, Michael R; Bouvier, Michel; Adjobo-Hermans, Merel J W

2015-09-01

The last frontier for a complete understanding of G-protein-coupled receptor (GPCR) biology is to be able to assess GPCR activity, interactions, and signaling in vivo, in real time within biologically intact systems. This includes the ability to detect GPCR activity, trafficking, dimerization, protein-protein interactions, second messenger production, and downstream signaling events with high spatial resolution and fast kinetic readouts. Resonance energy transfer (RET)-based biosensors allow for all of these possibilities in vitro and in cell-based assays, but moving RET into intact animals has proven difficult. Here, we provide perspectives on the optimization of biosensor design, of signal detection in living organisms, and the multidisciplinary development of in vitro and cell-based assays that more appropriately reflect the physiologic situation. In short, further development of RET-based probes, optical microscopy techniques, and mouse genome editing hold great potential over the next decade to bring real-time in vivo GPCR imaging to the forefront of pharmacology. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect.

PubMed

Compan, Vincent; Pierredon, Sandra; Vanderperre, Benoît; Krznar, Petra; Marchiq, Ibtissam; Zamboni, Nicola; Pouyssegur, Jacques; Martinou, Jean-Claude

2015-08-06

The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

The Application of Whole Cell-Based Biosensors for Use in Environmental Analysis and in Medical Diagnostics

PubMed Central

Gui, Qingyuan; Lawson, Tom; Shan, Suyan; Yan, Lu; Liu, Yong

2017-01-01

Various whole cell-based biosensors have been reported in the literature for the last 20 years and these reports have shown great potential for their use in the areas of pollution detection in environmental and in biomedical diagnostics. Unlike other reviews of this growing field, this mini-review argues that: (1) the selection of reporter genes and their regulatory proteins are directly linked to the performance of celllular biosensors; (2) broad enhancements in microelectronics and information technologies have also led to improvements in the performance of these sensors; (3) their future potential is most apparent in their use in the areas of medical diagnostics and in environmental monitoring; and (4) currently the most promising work is focused on the better integration of cellular sensors with nano and micro scaled integrated chips. With better integration it may become practical to see these cells used as (5) real-time portable devices for diagnostics at the bedside and for remote environmental toxin detection and this in situ application will make the technology commonplace and thus as unremarkable as other ubiquitous technologies. PMID:28703749

[Acute toxicity analysis performance of CellSense biosensor with E. coli].

PubMed

Wang, Xue-Jiang; Wang, Hong; Zhao, Jian-Fu; Xia, Si-Qing; Zhao, Hong-Ning

2009-04-15

E. coli microbial electrodes for CellSense biosensor were prepared by polycarbonate membrane immobilization process, and their performance for heavy metals and toxic organic compounds acute toxicity determination were studied. The results showed that when E. coli was in logarithmic and stationary phase, the CellSense biosensor with E. coli showed good performance in heavy metal ions and organic pollutants acute toxicity analysis, when E. coli was in its decline phase, the stability and sensitivity of the CellSense biosensor was poor. The EC50 values of Hg2+, Cu2+, Zn2+, o-chlorophenol (2-CP) and p-nitrophenol (4-NP) detected by CellSense biosensor with E. coli were 0.6, 3.1, 5.8, 180 and 94 microg/mL, respectively. The immobilized E. coli electrodes could still suit for acute toxicity assessment after 2 months storage at 4 degrees C.

Engineering and characterization of fluorogenic glycine riboswitches.

PubMed

Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

2016-07-08

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-1), respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization.

PubMed

Bommana, Rupesh; Mozziconacci, Olivier; John Wang, Y; Schöneich, Christian

2017-07-01

The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

Caco-2 cell-based electrochemical biosensor for evaluating the antioxidant capacity of Asp-Leu-Glu-Glu isolated from dry-cured Xuanwei ham.

PubMed

Xing, Lujuan; Ge, Qingfeng; Jiang, Donglei; Gao, Xiaoge; Liu, Rui; Cao, Songmin; Zhuang, Xinbo; Zhou, Guanghong; Zhang, Wangang

2018-05-15

A cell-based electrochemical biosensor was developed to determine the antioxidant activity of Asp-Leu-Glu-Glu (DLEE) isolated from dry-cured Chinese Xuanwei ham. A platinized gold electrode (Pt NPs/GE) covered with silver nanowires (Ag NWs) was fabricated to detect H 2 O 2 using redox signaling via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Under optimal condition, the detection limit of the modified electrode was 0.12μM with a linear relationship from 0.2 to 2μM, which showed relatively outstanding catalytic effects towards the reduction of H 2 O 2 . Furthermore, the generation of reactive oxygen species (ROS) in the cell can be used to indirectly assess changes in intercellular oxidative stress by detecting variations in electrochemical signals. A 3D cell culture of alginate/graphene oxide (NaAlg/GO) was used to encapsulate and immobilize Caco-2 cells. Based on ROS generation and electrochemical results, we found that DLEE could effectively reduce oxidative stress level in Caco-2 cells under external stimulation. DLEE showed high antioxidant activity with a relative antioxidant capacity (RAC) rate of 88.17% at 1.5mg/mL. Finally, an efficient electrochemical biosensor was established using the active 3D Caco-2 cell platform. This system is sensitive and simple to operate with the property to evaluate the antioxidant activity of peptides by the detection of H 2 O 2 in cell membrane. In summary, this work describes a new method for assessing antioxidant capacity of peptide DLEE using cell-based electrochemical signaling with a rapid screening pattern. Copyright © 2018 Elsevier B.V. All rights reserved.

A universal fluorogenic switch for Fe(ii) ion based on N-oxide chemistry permits the visualization of intracellular redox equilibrium shift towards labile iron in hypoxic tumor cells.

PubMed

Hirayama, Tasuku; Tsuboi, Hitomi; Niwa, Masato; Miki, Ayaji; Kadota, Satoki; Ikeshita, Yukie; Okuda, Kensuke; Nagasawa, Hideko

2017-07-01

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe 2+ . In this study, a universal fluorogenic switching system is established, which is responsive to Fe 2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe 2+ to generate various fluorescent probes of Fe 2+ -CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe 2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe 2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe 2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.

Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors.

PubMed

Descamps, Elodie C T; Meunier, Damien; Brutesco, Catherine; Prévéral, Sandra; Franche, Nathalie; Bazin, Ingrid; Miclot, Bertrand; Larosa, Philippe; Escoffier, Camille; Fantino, Jean-Raphael; Garcia, Daniel; Ansaldi, Mireille; Rodrigue, Agnès; Pignol, David; Cholat, Pierre; Ginet, Nicolas

2017-01-01

The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

Human Neural Cell-Based Biosensor

DTIC Science & Technology

2011-03-11

following areas: (1) neural progenitor isolation from induced pluripotent stem cells , (2) directed differentiation of progenitors into dopaminergic...from induced pluripotent stem cells , (2) directed differentiation of progenitors into dopaminergic neurons, motoneurons and astrocytes using defined...progenitors from mixed populations, such as induced pluripotent stem cells (iPSCs). We also developed lentiviral based methods to generate iPSCs in

Progress of new label-free techniques for biosensors: a review.

PubMed

Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

2016-01-01

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

A photoelectrochemical biosensor for fibroblast-like synoviocyte cell using visible light-activated NCQDs sensitized-ZnO/CH3NH3PbI3 heterojunction.

PubMed

Pang, Xuehui; Zhang, Yong; Pan, Jihong; Zhao, Yanxia; Chen, Yao; Ren, Xiang; Ma, Hongmin; Wei, Qin; Du, Bin

2016-03-15

Based on ZnO nanorods (NRs)/CH3NH3PbI3/nitrogen-doped carbon quantum dots (NCQDs) nanocomposites, the highly sensitive detection of fibroblast-like synoviocyte (FLS) cell was realized by a photoelectrochemical (PEC) biosensor. ZnO/CH3NH3PbI3/NCQDs nanocomposites were exploited as the photo-to-electron generator to produce the signal. CH3NH3PbI3 was spin-coated on ZnO surface after ZnO NRs grew on ITO electrode then by dropping on the modified electrode, NCQDs were diffused and adhered to the surface of ZnO and CH3NH3PbI3. In the presence of EDC/NHS, the combination of CH3NH3PbI3 and NCQDs was achieved by the carboxyl groups (-COOH) and amino groups (-NH2) in the preparation process. Furthermore, the capture probe of FLS cell, CD95 antibody, can be anchored by -COOH and -NH2 groups through EDC/NHS. The specific recognition between the antibody capture probes and cell targets gained high-sensitive detection for FLS cell for the first time. The developed biosensor showed a wide linear range from 1.0 × 10(4)cell/mL to 10 cell/mL and a low detection limit of 2 cell/mL. This kind of biosensor would provide a novel detection strategy for FLS cell. Copyright © 2015 Elsevier B.V. All rights reserved.

A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors

NASA Astrophysics Data System (ADS)

Guo, Jingxing; Zhang, Tao; Hu, Chengguo; Fu, Lei

2015-01-01

In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells. Electronic supplementary information (ESI) available: Procedures for CVD growth of 3D-NG, XRD and TEM measurements, a comparison with other graphene-based biosensors, a detailed study on the universality of 3D-NG as an enzyme carrier and more CV data on selectivity and stability. See DOI: 10.1039/c4nr05325g

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions †

PubMed Central

Herrero-Foncubierta, Pilar; Cuerva, Juan M.; Miguel, Delia

2018-01-01

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants. PMID:29315248

Biosensors of bacterial cells.

PubMed

Burlage, Robert S; Tillmann, Joshua

2017-07-01

Biosensors are devices which utilize both an electrical component (transducer) and a biological component to study an environment. They are typically used to examine biological structures, organisms and processes. The field of biosensors has now become so large and varied that the technology can often seem impenetrable. Yet the principles which underlie the technology are uncomplicated, even if the details of the mechanisms are elusive. In this review we confine our analysis to relatively current advancements in biosensors for the detection of whole bacterial cells. This includes biosensors which rely on an added labeled component and biosensors which do not have a labeled component and instead detect the binding event or bound structure on the transducer. Methods to concentrate the bacteria prior to biosensor analysis are also described. The variety of biosensor types and their actual and potential uses are described. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of genetically engineered microbial whole-cell biosensors for combined chemosensing.

PubMed

He, Wei; Yuan, Sheng; Zhong, Wen-Hui; Siddikee, Md Ashaduzzaman; Dai, Chuan-Chao

2016-02-01

The progress of genetically engineered microbial whole-cell biosensors for chemosensing and monitoring has been developed in the last 20 years. Those biosensors respond to target chemicals and produce output signals, which offer a simple and alternative way of assessment approaches. As actual pollution caused by human activities usually contains a combination of different chemical substances, how to employ those biosensors to accurately detect real contaminant samples and evaluate biological effects of the combined chemicals has become a realistic object of environmental researches. In this review, we outlined different types of the recent method of genetically engineered microbial whole-cell biosensors for combined chemical evaluation, epitomized their detection performance, threshold, specificity, and application progress that have been achieved up to now. We also discussed the applicability and limitations of this biosensor technology and analyzed the optimum conditions for their environmental assessment in a combined way.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer.

PubMed

Li, Yan; Wen, Yanli; Wang, Lele; Liang, Wen; Xu, Li; Ren, Shuzhen; Zou, Ziying; Zuo, Xiaolei; Fan, Chunhai; Huang, Qing; Liu, Gang; Jia, Nengqin

2015-05-15

The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Recent Trends in Rapid Environmental Monitoring of Pathogens and Toxicants: Potential of Nanoparticle-Based Biosensor and Applications

PubMed Central

Koedrith, Preeyaporn; Thasiphu, Thalisa; Weon, Jong-Il; Boonprasert, Rattana; Tuitemwong, Kooranee; Tuitemwong, Pravate

2015-01-01

Of global concern, environmental pollution adversely affects human health and socioeconomic development. The presence of environmental contaminants, especially bacterial, viral, and parasitic pathogens and their toxins as well as chemical substances, poses serious public health concerns. Nanoparticle-based biosensors are considered as potential tools for rapid, specific, and highly sensitive detection of the analyte of interest (both biotic and abiotic contaminants). In particular, there are several limitations of conventional detection methods for water-borne pathogens due to low concentrations and interference with various enzymatic inhibitors in the environmental samples. The increase of cells to detection levels requires long incubation time. This review describes current state of biosensor nanotechnology, the advantage over conventional detection methods, and the challenges due to testing of environmental samples. The major approach is to use nanoparticles as signal reporter to increase output rather than spending time to increase cell concentrations. Trends in future development of novel detection devices and their advantages over other environmental monitoring methodologies are also discussed. PMID:25884032

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor.

PubMed

Potocký, Martin; Pleskot, Roman; Pejchar, Přemysl; Vitale, Nicolas; Kost, Benedikt; Zárský, Viktor

2014-07-01

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor.

PubMed

Tang, Ho Man; Fung, Ming Chiu; Tang, Ho Lam

2018-02-01

Anastasis (Greek for "rising to life") is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Labatiuk, C.W.; Finch, G.R.; Belosevic, M.

1991-11-01

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation were detected for inactivations upmore » to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.« less

Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging

PubMed Central

Bhargava, Yogesh; Hampden-Smith, Kathryn; Chachlaki, Konstantina; Wood, Katherine C.; Vernon, Jeffrey; Allerston, Charles K.; Batchelor, Andrew M.; Garthwaite, John

2013-01-01

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations. PMID:24068983

MEMS-Based Power Generation Techniques for Implantable Biosensing Applications

PubMed Central

Lueke, Jonathan; Moussa, Walied A.

2011-01-01

Implantable biosensing is attractive for both medical monitoring and diagnostic applications. It is possible to monitor phenomena such as physical loads on joints or implants, vital signs, or osseointegration in vivo and in real time. Microelectromechanical (MEMS)-based generation techniques can allow for the autonomous operation of implantable biosensors by generating electrical power to replace or supplement existing battery-based power systems. By supplementing existing battery-based power systems for implantable biosensors, the operational lifetime of the sensor is increased. In addition, the potential for a greater amount of available power allows additional components to be added to the biosensing module, such as computational and wireless and components, improving functionality and performance of the biosensor. Photovoltaic, thermovoltaic, micro fuel cell, electrostatic, electromagnetic, and piezoelectric based generation schemes are evaluated in this paper for applicability for implantable biosensing. MEMS-based generation techniques that harvest ambient energy, such as vibration, are much better suited for implantable biosensing applications than fuel-based approaches, producing up to milliwatts of electrical power. High power density MEMS-based approaches, such as piezoelectric and electromagnetic schemes, allow for supplemental and replacement power schemes for biosensing applications to improve device capabilities and performance. In addition, this may allow for the biosensor to be further miniaturized, reducing the need for relatively large batteries with respect to device size. This would cause the implanted biosensor to be less invasive, increasing the quality of care received by the patient. PMID:22319362

Theoretical analysis and simulation study of low-power CMOS electrochemical impedance spectroscopy biosensor in 55 nm deeply depleted channel technology for cell-state monitoring

NASA Astrophysics Data System (ADS)

Itakura, Keisuke; Kayano, Keisuke; Nakazato, Kazuo; Niitsu, Kiichi

2018-01-01

We present an impedance-detection complementary metal oxide semiconductor (CMOS) biosensor circuit for cell-state observation. The proposed biosensor can measure the expected impedance values encountered by a cell-state observation measurement system within a 0.1-200 MHz frequency range. The proposed device is capable of monitoring the intracellular conditions necessary for real-time cell-state observation, and can be fabricated using a 55 nm deeply depleted channel CMOS process. Operation of the biosensor circuit with 0.9 and 1.7 V supply voltages is verified via a simulated program with integrated circuit emphasis (SPICE) simulation. The power consumption is 300 µW. Further, the standby power consumption is 290 µW, indicating that this biosensor is a low-power instrument suitable for use in Internet of Things (IoT) devices.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Increasing cell-device adherence using cultured insect cells for receptor-based biosensors

NASA Astrophysics Data System (ADS)

Terutsuki, Daigo; Mitsuno, Hidefumi; Sakurai, Takeshi; Okamoto, Yuki; Tixier-Mita, Agnès; Toshiyoshi, Hiroshi; Mita, Yoshio; Kanzaki, Ryohei

2018-03-01

Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell-device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell-device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell-device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs.

Rapid and sensitive Nitrosomonas europaea biosensor assay for quantification of bioavailable ammonium sensu strictu in soil.

PubMed

Nguyen, Minh Dong; Risgaard-Petersen, Nils; Sørensen, Jan; Brandt, Kristian K

2011-02-01

Knowledge on bioavailable ammonium sensu strictu (i.e., immediately available for cellular uptake) in soil is required to understand nutrient uptake processes in microorganisms and thus of vital importance for plant production. We here present a novel ammonium biosensor approach based on the lithoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea transformed with a luxAB sensor plasmid. Bioluminescence-based ammonium detection was achieved within 10 min with a quantification limit in liquid samples of ∼20 μM and a linear response range up to 400 μM. Biosensor and conventional chemical quantification of ammonium in soil solutions agreed well across a range of sample and assay conditions. The biosensor was subsequently applied for a solid phase-contact assay allowing for direct interaction of biosensor cells with soil particle-associated (i.e., exchangeable plus fixed) ammonium. The assay successfully quantified bioavailable ammonium even in unfertilized soil and demonstrated markedly higher ratios of bioavailable ammonium to water- or 2 M KCl-exchangeable ammonium in anoxic soil than in corresponding oxic soil. Particle-associated ammonium contributed by at least 74% and 93% of the total bioavailable pool in oxic and anoxic soil, respectively. The N. europaea biosensor should have broad relevance for environmental monitoring of bioavailable ammonium and processes depending on ammonium bioavailability.

On-Line Monitoring the Growth of E. coli or HeLa Cells Using an Annular Microelectrode Piezoelectric Biosensor.

PubMed

Tong, Feifei; Lian, Yan; Han, Junliang

2016-12-18

Biological information is obtained from the interaction between the series detection electrode and the organism or the physical field of biological cultures in the non-mass responsive piezoelectric biosensor. Therefore, electric parameter of the electrode will affect the biosensor signal. The electric field distribution of the microelectrode used in this study was simulated using the COMSOL Multiphysics analytical tool. This process showed that the electric field spatial distribution is affected by the width of the electrode finger or the space between the electrodes. In addition, the characteristic response of the piezoelectric sensor constructed serially with an annular microelectrode was tested and applied for the continuous detection of Escherichia coli culture or HeLa cell culture. Results indicated that the piezoelectric biosensor with an annular microelectrode meets the requirements for the real-time detection of E. coli or HeLa cells in culture. Moreover, this kind of piezoelectric biosensor is more sensitive than the sensor with an interdigital microelectrode. Thus, the piezoelectric biosensor acts as an effective analysis tool for acquiring online cell or microbial culture information.

A new fluorogenic small molecule labeling tool for surface diffusion analysis and advanced fluorescence imaging of β-site amyloid precursor protein (APP)-cleaving enzyme 1 based on silicone rhodamine: SiR-BACE1.

PubMed

Karch, Sandra; Broichhagen, Johannes; Schneider, Julia; Böning, Daniel; Hartmann, Stephanie; Schmid, Benjamin; Tripal, Philipp; Palmisano, Ralf; Alzheimer, Christian; Johnsson, Kai; Huth, Tobias

2018-06-25

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo-physical and pharmacologic properties, and evaluate its behavior in solution, in over-expression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

Tracing the pH dependent activation of autophagy in cancer cells by silicon nanowire-based impedance biosensor.

PubMed

Alikhani, Alireza; Gharooni, Milad; Abiri, Hamed; Farokhmanesh, Fatemeh; Abdolahad, Mohammad

2018-05-30

Monitoring the pH dependent behavior of normal and cancer cells by impedimetric biosensor based on Silicon Nanowires (SiNWs) was introduced to diagnose the invasive cancer cells. Autophagy as a biologically activated process in invasive cancer cells during acidosis, protect them from apoptosis in lower pH which presented in our work. As the autophagy is the only activated pathways which can maintain cellular proliferation in acidic media, responses of SiNW-ECIS in acidified cells could be correlated to the probability of autophagy activation in normal or cancer cells. In contrast, cell survival pathway wasn't activated in low-grade cancer cells which resulted in their acidosis. The measured electrical resistance of MCF10, MCF7, and MDA-MB468 cell lines, by SiNW sensor, in normal and acidic media were matched by the biological analyses of their vital functions. Invasive cancer cells exhibited increased electrical resistance in pH 6.5 meanwhile the two other types of the breast cells exhibited sharp (MCF10) and moderate (MCF7) decrease in their resistance. This procedure would be a new trend in microenvironment based cancer investigation. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

PubMed

Kucherenko, I S; Soldatkin, O O; Lagarde, F; Jaffrezic-Renault, N; Dzyadevych, S V; Soldatkin, A P

2015-11-01

Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes. Copyright © 2015 Elsevier B.V. All rights reserved.

Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.

PubMed

Chen, Lizhen; Fu, Ying; Wang, Naixiang; Yang, Anneng; Li, Yuanzhe; Wu, Jie; Ju, Huangxian; Yan, Feng

2018-05-23

Cell surface glycans play critical roles in diverse biological processes, such as cell-cell communication, immunity, infection, development, and differentiation. Their expressions are closely related to cancer growth and metastasis. This work demonstrates an organic electrochemical transistor (OECT)-based biosensor for the detection of glycan expression on living cancer cells. Herein, mannose on human breast cancer cells (MCF-7) as the target glycan model, poly dimethyl diallyl ammonium chloride-multiwall carbon nanotubes (PDDA-MWCNTs) as the loading interface, concanavalin A (Con A) with active mannose binding sites, aptamer and horseradish peroxidase co-immobilized gold nanoparticles (HRP-aptamer-Au NPs) as specific nanoprobes are used to fabricate the OECT biosensor. In this strategy, PDDA-MWCNT interfaces can enhance the loading of Con A, and the target cells can be captured through Con A via active mannose binding sites. Thus, the expression of cell surface can be reflected by the amount of cells captured on the gate. Specific nanoprobes are introduced to the captured cells to produce an OECT signal because of the reduction of hydrogen peroxide catalyzed by HRP conjugated on Au nanoparticles, while the aptamer on nanoprobes can selectively recognize the MCF-7 cells. It is reasonable that more target cells are captured on the gate electrode, more HRP-nanoprobes are loaded thus a larger signal response. The device shows an obvious response to MCF-7 cells down to 10 cells/μL and can be used to selectively monitor the change of mannose expression on cell surfaces upon a treatment with the N-glycan inhibitor. The OECT-based biosensor is promising for the analysis of glycan expressions on the surfaces of different types of cells.

Recent advances in material science for developing enzyme electrodes.

PubMed

Sarma, Anil Kumar; Vatsyayan, Preety; Goswami, Pranab; Minteer, Shelley D

2009-04-15

The enzyme-modified electrode is the fundamental component of amperometric biosensors and biofuel cells. The selection of appropriate combinations of materials, such as: enzyme, electron transport mediator, binding and encapsulation materials, conductive support matrix and solid support, for construction of enzyme-modified electrodes governs the efficiency of the electrodes in terms of electron transfer kinetics, mass transport, stability, and reproducibility. This review investigates the varieties of materials that can be used for these purposes. Recent innovation in conductive electro-active polymers, functionalized polymers, biocompatible composite materials, composites of transition metal-based complexes and organometallic compounds, sol-gel and hydro-gel materials, nanomaterials, other nano-metal composites, and nano-metal oxides are reviewed and discussed here. In addition, the critical issues related to the construction of enzyme electrodes and their application for biosensor and biofuel cell applications are also highlighted in this article. Effort has been made to cover the recent literature on the advancement of materials sciences to develop enzyme electrodes and their potential applications for the construction of biosensors and biofuel cells.

Electrochemical lectin based biosensors as a label-free tool in glycomics

PubMed Central

Bertók, Tomáš; Katrlík, Jaroslav; Gemeiner, Peter; Tkac, Jan

2016-01-01

Glycans and other saccharide moieties attached to proteins and lipids, or present on the surface of a cell, are actively involved in numerous physiological or pathological processes. Their structural flexibility (that is based on the formation of various kinds of linkages between saccharides) is making glycans superb “identity cards”. In fact, glycans can form more “words” or “codes” (i.e., unique sequences) from the same number of “letters” (building blocks) than DNA or proteins. Glycans are physicochemically similar and it is not a trivial task to identify their sequence, or - even more challenging - to link a given glycan to a particular physiological or pathological process. Lectins can recognise differences in glycan compositions even in their bound state and therefore are most useful tools in the task to decipher the “glycocode”. Thus, lectin-based biosensors working in a label-free mode can effectively complement the current weaponry of analytical tools in glycomics. This review gives an introduction into the area of glycomics and then focuses on the design, analytical performance, and practical utility of lectin-based electrochemical label-free biosensors for the detection of isolated glycoproteins or intact cells. PMID:27239071

Low-cost fluorimetric determination of radicals based on fluorogenic dimerization of the natural phenol sesamol.

PubMed

Makino, Yumi; Uchiyama, Seiichi; Ohno, Ken-ichi; Arakawa, Hidetoshi

2010-02-15

A novel fluorimetric method for determining radicals using the natural phenol sesamol as a fluorogenic reagent is reported. In this assay, sesamol was reacted with aqueous radicals to yield one isomer of a sesamol dimer exclusively. The dimer emitted purple fluorescence near 400 nm around neutral pH, where it assumed the monoanionic form. This method was applied to the straightforward detection of radical nitric oxide (NO). The ready availability of sesamol should enable rapid implementation of applications utilizing this new assay, particularly in high-throughput analysis or screening.

A screen-printed microband glucose biosensor system for real-time monitoring of toxicity in cell culture.

PubMed

Pemberton, R M; Xu, J; Pittson, R; Drago, G A; Griffiths, J; Jackson, S K; Hart, J P

2011-01-15

Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (E(app) of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24 h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10 mM (dynamic range 30 mM). Within the linear region, a correlation coefficient (R(2)) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24 h, an estimated 0.15% (89 nmol) of the starting glucose concentration (24 mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4. Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 10(6) cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1 mM paracetamol, glucose uptake was inhibited by 95% after 6 h in culture; at 500 μM, around 15% inhibition was observed after 16 h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system. Copyright © 2010 Elsevier B.V. All rights reserved.

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

PubMed Central

Pratt, Christopher P.; Kuljis, Dika A.; Homanics, Gregg E.; He, Jianjun; Kolodieznyi, Dmytro; Dudem, Srikanth; Hollywood, Mark A.; Barth, Alison L.; Bruchez, Marcel P.

2017-01-01

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. PMID:29163049

A novel self-powered and sensitive label-free DNA biosensor in microbial fuel cell.

PubMed

Asghary, Maryam; Raoof, Jahan Bakhsh; Rahimnejad, Mostafa; Ojani, Reza

2016-08-15

In this work, a novel self-powered, sensitive, low-cost, and label-free DNA biosensor is reported by applying a two-chambered microbial fuel cell (MFC) as a power supply. A graphite electrode and an Au nanoparticles modified graphite electrode (AuNP/graphite electrode) were used as anode and cathode in the MFC system, respectively. The active biocatalyst in the anodic chamber was a mixed culture of microorganisms. The sensing element of the biosensor was fabricated by the well-known Au-thiol binding the ssDNA probe on the surface of an AuNP/graphite cathode. Electrons produced by microorganisms were transported from the anode to the cathode through an external circuit, which could be detected by the terminal multi-meter detector. The difference between power densities of the ssDNA probe modified cathode in the absence and presence of complementary sequence served as the detection signal of the DNA hybridization with detection limit of 3.1nM. Thereafter, this biosensor was employed for diagnosis and determination of complementary sequence in a human serum sample. The hybridization specificity studies further revealed that the developed DNA biosensor could distinguish fully complementary sequences from one-base mismatched and non-complementary sequences. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

PubMed

Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

2001-01-01

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

Imaging mycobacterial growth and division with a fluorogenic probe.

PubMed

Hodges, Heather L; Brown, Robert A; Crooks, John A; Weibel, Douglas B; Kiessling, Laura L

2018-05-15

Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

A Dual Anticancer Efficacy Molecule: A Selective Dark Cytotoxicity Photosensitizer.

PubMed

Chen, Jyun-Wei; Chang, Cheng-Chung

2016-11-09

Unlike traditional binary nanostructures that construct chemotherapy drugs and photodynamic therapy photosensitizers, we introduce a molecule with a chemo-photodynamic dual therapy function. A water-soluble aggregation-induced emission enhancement (AIEE) fluorogen, NV-12P, was designed and synthesized based on asymmetric 1,6-disubstituted naphthalene and can generate particular reactive oxygen species to undergo type I photodynamic therapy under irradiation. Furthermore, this compound can specifically localize in mitochondria and, after biological evaluation, can cause mitochondrial dysfunction and potent cytotoxicity to cancer cells but not normal cells. We conclude that this compound is a potential dual-toxic efficacy molecule because it exhibits selective dark cytotoxicity and efficient photodamage in cancer cells. Additionally, we also supported the optimal combinational treatment course for the best chemo-phototherapy efficacy.

Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

PubMed Central

Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

2014-01-01

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

Label-free optical biosensor for real-time monitoring the cytotoxicity of xenobiotics: A proof of principle study on glyphosate.

PubMed

Farkas, Eniko; Szekacs, Andras; Kovacs, Boglarka; Olah, Marianna; Horvath, Robert; Szekacs, Inna

2018-06-05

Rapid and inexpensive biosensor technologies allowing real-time analysis of biomolecular and cellular events have become the basis of next-generation cell-based screening techniques. Our work opens up novel opportunities in the application of the high-throughput label-free Epic BenchTop optical biosensor in cell toxicity studies. The Epic technology records integrated cellular responses about changes in cell morphology and dynamic mass redistribution of cellular contents at the 100-150 nm layer above the sensor surface. The aim of the present study was to apply this novel technology to identify the effect of the herbicide Roundup Classic, its co-formulant polyethoxylated tallow amine (POEA), and its active ingredient glyphosate, on MC3T3-E1 cells adhered on the biosensor surface. The half maximal inhibitory concentrations of Roundup Classic, POEA and glyphosate upon 1 h of exposure were found to be 0.024%, 0.021% and 0.163% in serum-containing medium and 0.028%, 0.019% and 0.538% in serum-free conditions, respectively (at concentrations equivalent to the diluted Roundup solution). These results showed a good correlation with parallel end-point assays, demonstrating the outstanding utility of the Epic technique in cytotoxicity screening, allowing not only high-throughput, real-time detection, but also reduced assay run time and cytotoxicity assessment at end-points far before cell death would occur. Copyright © 2018 Elsevier B.V. All rights reserved.

Construction of a ColD cda promoter-based SOS-green fluorescent protein whole-cell biosensor with higher sensitivity toward genotoxic compounds than constructs based on recA, umuDC, or sulA promoters.

PubMed

Norman, Anders; Hestbjerg Hansen, Lars; Sørensen, Søren J

2005-05-01

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 microM, 1.1 microM, and 141 microM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.

Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry

PubMed Central

Hansen, Lars Hestbjerg; Ferrari, Belinda; Sørensen, Anders Hay; Veal, Duncan; Sørensen, Søren Johannes

2001-01-01

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated Ptet promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil. PMID:11133451

Potentiometric Biosensor for Studying Hydroquinone Cytotoxicity in vitro

PubMed Central

Wang, Yanyan; Chen, Qiang; Zeng, Xiangqun

2009-01-01

Many processes in living cells have electrochemical characteristics that are suitable for measurement by potentiometric biosensors. Potentiometric biosensors allow non invasive, real-time monitoring of the extracellular environment changes by measuring the potential at cell/sensor interface. This can be used as an indicator for overall cell cytotoxicity. The present work employs a potentiometric sensor array to investigate the cytotoxicity of hydroquinone to cultured mammalian V79 cells. Various electrode substrates (Au, PPy-HQ and PPy-PS) used for cell growth were designed and characterized. The controllable release of hydroquinone from PPy substrates was studied. Our results showed that hydroquinone exposure affected cell proliferation and delayed cell growth and attachment in a dose-dependent manner. Additionally, we have shown that exposure of V79 cells to hydroquinone at low doses (i.e 5μM) for more than 15 hours allows V79 cells to gain enhanced adaptability to survive exposure to high toxic HQ doses afterwards. Compared with traditional methods, the potentiometric biosensor not only provides non-invasive and real time monitoring of the cellular reactions but also is more sensitive for in vitro cytotoxicity study. By real time and non-invasive monitoring of the extracellular potential in vitro, the potentiometric sensor system represents a promising biosensor system for drug discovery. PMID:19926470

The blocking reagent optimization for the magnetoelastic biosensor

NASA Astrophysics Data System (ADS)

Hu, Jiajia; Chai, Yating; Horikawa, Shin; Wikle, Howard C.; Wang, Feng'en; Du, Songtao; Chin, Bryan A.; Hu, Jing

2015-06-01

The wireless phage-based magnetoelastic (ME) biosensor has proven to be promising for real-time detection of pathogenic bacteria on fresh produces. The ME biosensor consists of a freestanding ME resonator as the signal transducer and filamentous phage as the biomolecular-recognition element, which can specifically bind to a pathogen of interest. Due to the Joule magnetostriction effect, the biosensors can be placed into mechanical resonance when subjected to a time-varying magnetic field alternating at the sensor's resonant frequency. Upon the attachment of the target pathogen, the mass of the biosensor increases, thereby decreasing its resonant frequency. This paper presents an investigation of blocking reagents immobilization for detecting Salmonella Typhimurium on fresh food surfaces. Three different blocking reagents (BSA, SuperBlock blocking buffer, and blocker BLOTTO) were used and compared. The optical microscope was used for bacterial cells binding observation. Student t-test was used to statistically analysis the experiment results. The results shows that SuperBlock blocking buffer and blocker BLOTTO have much better blocking performance than usually used BSA.

Progress in the biosensing techniques for trace-level heavy metals.

PubMed

Mehta, Jyotsana; Bhardwaj, Sanjeev K; Bhardwaj, Neha; Paul, A K; Kumar, Pawan; Kim, Ki-Hyun; Deep, Akash

2016-01-01

Diverse classes of sensors have been developed over the past few decades for on-site detections of heavy metals. Most of these sensor systems have exploited optical, electrochemical, piezoelectric, ion-selective (electrode), and electrochemical measurement techniques. As such, numerous efforts have been made to explore the role of biosensors in the detection of heavy metals based on well-known interactions between heavy metals and biomolecules (e.g. proteins, peptides, enzymes, antibodies, whole cells, and nucleic acids). In this review, we cover the recent progress made on different types of biosensors for the detection of heavy metals. Our major focus was examining the use of biomolecules for constructing these biosensors. The discussion is extended further to cover the biosensors' performance along with challenges and opportunities for practical utilization. Copyright © 2015 Elsevier Inc. All rights reserved.

Advances in arsenic biosensor development--a comprehensive review.

PubMed

Kaur, Hardeep; Kumar, Rabindra; Babu, J Nagendra; Mittal, Sunil

2015-01-15

Biosensors are analytical devices having high sensitivity, portability, small sample requirement and ease of use for qualitative and quantitative monitoring of various analytes of human importance. Arsenic (As), owing to its widespread presence in nature and high toxicity to living creatures, requires frequent determination in water, soil, agricultural and food samples. The present review is an effort to highlight the various advancements made so far in the development of arsenic biosensors based either on recombinant whole cells or on certain arsenic-binding oligonucleotides or proteins. The role of futuristic approaches like surface plasmon resonance (SPR) and aptamer technology has also been discussed. The biomethods employed and their general mechanisms, advantages and limitations in relevance to arsenic biosensors developed so far are intended to be discussed in this review. Copyright © 2014 Elsevier B.V. All rights reserved.

A thiourea-appended rhodamine chemodosimeter for mercury(II) and its bioimaging application

NASA Astrophysics Data System (ADS)

Tantipanjaporn, Ajcharapan; Prabpai, Samran; Suksen, Kanoknetr; Kongsaeree, Palangpon

2018-03-01

A rhodamine-thiourea conjugate RTP with an o-phenylenediamine linker was developed as a fluorogenic chemodosimeter for Hg2+ detection. In the presence of Hg2+, a colorless solution of RTP turned pink with a maximum absorption band at 555 nm and with a 62-fold fluorescence enhancement at 578 nm (Φ = 0.34). RTP is highly selective to Hg2+ among other metal ions with a detection limit of 1.6 nM (0.3 ppb). A similar rhodamine analog with a flexible ethylenediamine spacer was less selective and less sensitive than RTP. Hg2+ induced cyclic guanylation to yield a benzimidazole moiety and a subsequent ring-opening of the spirolactam unit resulted in chromogenic and fluorogenic changes. The membrane-permeable RTP probe was successfully demonstrated in monitoring of Hg2+ in cultured HeLa cells.

Norepinephrine is coreleased with serotonin in mouse taste buds.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Roper, Stephen D

2008-12-03

ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT.

Basics of DNA biosensors and cancer diagnosis.

PubMed

Sohrabi, Nasrin; Valizadeh, Alireza; Farkhani, Samad Mussa; Akbarzadeh, Abolfazl

2016-01-01

The human genome is exposed to mutations during the life cycle because of many types of changes in the DNA. Viruses, radiation, transposons, mutagenic chemicals, or any errors that happen during DNA replication or the meiotic process in the cell, may cause the mutation. Many mutations have no effect on phenotype or health, while some mutations cause crucial diseases such as cancer or cardiac diseases; therefore, a better understanding of the effects of mutation on phenotype is a very important part of genetic studies. Biosensors based on DNA, RNA, and peptide nucleic acids are the most sensitive tools, due to a strong pairing of lined up nucleotide strands between bases in their complementary parts. These methods can provide information to assist clinicians in making successful treatment decisions and increase the patient survival rate. In this review, we discuss DNA biosensors based on peptide nucleic acids that have an important role in cancer diagnosis.

Development of a novel microbial sensor with baker's yeast cells for monitoring temperature control during cold food chain.

PubMed

Kogure, H; Kawasaki, S; Nakajima, K; Sakai, N; Futase, K; Inatsu, Y; Bari, M L; Isshiki, K; Kawamoto, S

2005-01-01

A novel microbial sensor containing a commercial baker's yeast with a high freeze tolerance was developed for visibly detecting inappropriate temperature control of food. When the yeast cells fermented glucose, the resulting gas production triggered the microbial sensor. The biosensor was a simple, small bag containing a solution of yeast cells, yeast extract, glucose, and glycerol sealed up with multilayer transparent film with barriers against oxygen and humidity. Fine adjustment of gas productivity in the biosensor at low temperatures was achieved by changing either or both concentrations of glucose and yeast cells. Moreover, the amount of time that food was exposed to inappropriate temperatures could be deduced by the amount of gas produced in the biosensor. The biosensor was stable without any functional loss for up to 1 week in frozen storage. The biosensor could offer a useful tool for securing food safety by maintaining low-temperature control in every stage from farm to fork, including during transportation, in the store, and at home.

Increasing cell–device adherence using cultured insect cells for receptor-based biosensors

PubMed Central

Mitsuno, Hidefumi; Sakurai, Takeshi; Okamoto, Yuki; Tixier-Mita, Agnès; Toshiyoshi, Hiroshi; Mita, Yoshio; Kanzaki, Ryohei

2018-01-01

Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell–device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell–device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell–device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs. PMID:29657822

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

PubMed

Devillers, Marion; Ahmad, Lama; Korri-Youssoufi, Hafsa; Salmon, Laurent

2017-10-15

Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation. In this study, a sensitive and specific carbohydrate-based electrochemical biosensor was designed for the detection and quantification of a protein model of AMF, namely phosphoglucose isomerase from rabbit muscle (RmPGI). Indeed, RmPGI displays high homology with AMF and has been shown to have AMF activity. The biosensor was constructed by covalent binding of the enzyme substrate d-fructose 6-phosphate (F6P). Immobilization was achieved on a gold surface electrode following a bottom-up approach through an aminated surface obtained by electrochemical patterning of ethylene diamine and terminal amine polyethylene glycol chain to prevent non-specific interactions. Carbohydrate-protein interactions were quantified in a range of 10 fM to 100nM. Complex formation was analyzed through monitoring of the redox couple Fe 2+ /Fe 3+ by electrochemical impedance spectroscopy and square wave voltammetry. The F6P-biosensor demonstrates a detection limit of 6.6 fM and high selectivity when compared to other non-specific glycolytic proteins such as d-glucose-6-phosphate dehydrogenase. Detection of protein in spiked plasma was demonstrated and accuracy of 95% is obtained compared to result obtained in PBS (phosphate buffered saline). F6P-biosensor is a very promising proof of concept required for the design of a carbohydrate-based electrochemical biosensor using the enzyme substrate as bioreceptor. Such biosensor could be generalized to detect other protein biomarkers of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH.

PubMed

Rupprecht, Christian; Wingen, Marcus; Potzkei, Janko; Gensch, Thomas; Jaeger, Karl-Erich; Drepper, Thomas

2017-09-20

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution. We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pK a ∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pK a values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress. Copyright © 2017 Elsevier B.V. All rights reserved.

[Development of a Fluorescence Probe for Live Cell Imaging].

PubMed

Shibata, Aya

2017-01-01

 Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Human Neural Cell-Based Biosensor

DTIC Science & Technology

2013-05-28

ionotropic receptors in human embryonic stem cell derived neural progenitors. Neuroscience. 2011 Sep 29;192:793-805. Krishnamoorthy M, Gerwe BA, Scharer...coupled receptors . Pharmacol Ther. 2011 Mar;129(3):290-306. Workshop/conference abstracts, presentations, posters, and papers Powe, A, et al

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates.

PubMed

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

2015-09-07

E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

Tyramine Hydrochloride Based Label-Free System for Operating Various DNA Logic Gates and a DNA Caliper for Base Number Measurements.

PubMed

Fan, Daoqing; Zhu, Xiaoqing; Dong, Shaojun; Wang, Erkang

2017-07-05

DNA is believed to be a promising candidate for molecular logic computation, and the fluorogenic/colorimetric substrates of G-quadruplex DNAzyme (G4zyme) are broadly used as label-free output reporters of DNA logic circuits. Herein, for the first time, tyramine-HCl (a fluorogenic substrate of G4zyme) is applied to DNA logic computation and a series of label-free DNA-input logic gates, including elementary AND, OR, and INHIBIT logic gates, as well as a two to one encoder, are constructed. Furthermore, a DNA caliper that can measure the base number of target DNA as low as three bases is also fabricated. This DNA caliper can also perform concatenated AND-AND logic computation to fulfil the requirements of sophisticated logic computing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid and label-free bioanalytical method of alpha fetoprotein detection using LSPR chip

NASA Astrophysics Data System (ADS)

Kim, Dongjoo; Kim, Jinwoon; Kwak, Cheol Hwan; Heo, Nam Su; Oh, Seo Yeong; Lee, Hoomin; Lee, Go-Woon; Vilian, A. T. Ezhil; Han, Young-Kyu; Kim, Woo-Sik; Kim, Gi-bum; Kwon, Soonjo; Huh, Yun Suk

2017-07-01

Alpha fetoprotein (AFP) is a cancer marker, particularly for hepatocellular carcinoma. Normal levels of AFP are less than 20 ng/mL; however, its levels can reach more than 400 ng/mL in patients with HCC. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been employed for clinical diagnosis of AFP; however, these methods are time consuming and labor intensive. In this study, we developed a localized surface plasmon resonance (LSPR) based biosensor for simple and rapid detection of AFP. This biosensor consists of a UV-Vis spectrometer, a cuvette cell, and a biosensor chip nanopatterned with gold nanoparticles (AuNPs). In our LSPR biosensor, binding of AFP to the surface of the sensor chip led to an increasing magnitude of the LSPR signals, which was measured by an ultraviolet-visible (UV-Vis) spectrometer. Our LSPR biosensor showed sufficient detectability of AFP at concentrations of 1 ng/mL to 1 μg/mL. Moreover, the overall procedure for detection of AFP was completed within 20 min. This biosensor could also be utilized for a point of care test (POCT) by employing a portable UV-Vis spectrometer. Owing to the simplicity and rapidity of the detection process, our LSPR biosensor is expected to replace traditional diagnostic methods for the early detection of diseases.

Bioanalytical and chemical sensors using living taste, olfactory, and neural cells and tissues: a short review.

PubMed

Wu, Chunsheng; Lillehoj, Peter B; Wang, Ping

2015-11-07

Biosensors utilizing living tissues and cells have recently gained significant attention as functional devices for chemical sensing and biochemical analysis. These devices integrate biological components (i.e. single cells, cell networks, tissues) with micro-electro-mechanical systems (MEMS)-based sensors and transducers. Various types of cells and tissues derived from natural and bioengineered sources have been used as recognition and sensing elements, which are generally characterized by high sensitivity and specificity. This review summarizes the state of the art in tissue- and cell-based biosensing platforms with an emphasis on those using taste, olfactory, and neural cells and tissues. Many of these devices employ unique integration strategies and sensing schemes based on sensitive transducers including microelectrode arrays (MEAs), field effect transistors (FETs), and light-addressable potentiometric sensors (LAPSs). Several groups have coupled these hybrid biosensors with microfluidics which offers added benefits of small sample volumes and enhanced automation. While this technology is currently limited to lab settings due to the limited stability of living biological components, further research to enhance their robustness will enable these devices to be employed in field and clinical settings.

Silicon nanowire based biosensing platform for electrochemical sensing of Mebendazole drug activity on breast cancer cells.

PubMed

Shashaani, Hani; Faramarzpour, Mahsa; Hassanpour, Morteza; Namdar, Nasser; Alikhani, Alireza; Abdolahad, Mohammad

2016-11-15

Electrochemical approaches have played crucial roles in bio sensing because of their Potential in achieving sensitive, specific and low-cost detection of biomolecules and other bio evidences. Engineering the electrochemical sensing interface with nanomaterials tends to new generations of label-free biosensors with improved performances in terms of sensitive area and response signals. Here we applied Silicon Nanowire (SiNW) array electrodes (in an integrated architecture of working, counter and reference electrodes) grown by low pressure chemical vapor deposition (LPCVD) system with VLS procedure to electrochemically diagnose the presence of breast cancer cells as well as their response to anticancer drugs. Mebendazole (MBZ), has been used as antitubulin drug. It perturbs the anodic/cathodic response of the cell covered biosensor by releasing Cytochrome C in cytoplasm. Reduction of cytochrome C would change the ionic state of the cells monitored by SiNW biosensor. By applying well direct bioelectrical contacts with cancer cells, SiNWs can detect minor signal transduction and bio recognition events, resulting in precise biosensing. Our device detected the trace of MBZ drugs (with the concentration of 2nM) on electrochemical activity MCF-7 cells. Also, experimented biological analysis such as confocal and Flowcytometry assays confirmed the electrochemical results. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free electrochemiluminescence biosensor for ultrasensitive detection of telomerase activity in HeLa cells based on extension reaction and intercalation of Ru(phen)3 (2.).

PubMed

Lin, Yue; Yang, Linlin; Yue, Guiyin; Chen, Lifen; Qiu, Bin; Guo, Longhua; Lin, Zhenyu; Chen, Guonan

2016-10-01

Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were <5 % at 100 HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.

Biosensor and chemical sensor probes for calcium and other metal ions

DOEpatents

Vo-Dinh, Tuan; Viallet, Pierre

1996-01-01

The present invention relates to chemical sensor and biosensor probes for measuring low concentration of metals and metal ions in complex samples such as biological fluids, living cells, and environmental samples. More particularly the present invention relates to a gel-based Indo-1 and Fura-2 chemical sensor probes for the measurement of low concentrations of calcium, cadmium, magnesium and the like. Also disclosed is a detector device using the sensors of the present invention.

Graphene-based field-effect transistor biosensors

DOEpatents

Chen; , Junhong; Mao, Shun; Lu, Ganhua

2017-06-14

The disclosure provides a field-effect transistor (FET)-based biosensor and uses thereof. In particular, to FET-based biosensors using thermally reduced graphene-based sheets as a conducting channel decorated with nanoparticle-biomolecule conjugates. The present disclosure also relates to FET-based biosensors using metal nitride/graphene hybrid sheets. The disclosure provides a method for detecting a target biomolecule in a sample using the FET-based biosensor described herein.

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

PubMed Central

Shadpour, Hamed; Zawistowski, Jon S.; Herman, Annadele; Hahn, Klaus; Allbritton, Nancy L.

2011-01-01

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties. PMID:21621038

Towards a subcutaneous optical biosensor based on thermally hydrocarbonised porous silicon.

PubMed

Tong, Wing Yin; Sweetman, Martin J; Marzouk, Ezzat R; Fraser, Cara; Kuchel, Tim; Voelcker, Nicolas H

2016-01-01

Advanced biosensors in future medicine hinge on the evolvement of biomaterials. Porous silicon (pSi), a generally biodegradable and biocompatible material that can be fabricated to include environment-responsive optical characteristics, is an excellent candidate for in vivo biosensors. However, the feasibility of using this material as a subcutaneously implanted optical biosensor has never been demonstrated. Here, we investigated the stability and biocompatibility of a thermally-hydrocarbonised (THC) pSi optical rugate filter, and demonstrated its optical functionality in vitro and in vivo. We first compared pSi films with different surface chemistries and observed that the material was cytotoxic despite the outstanding stability of the THC pSi films. We then showed that the cytotoxicity correlates with reactive oxygen species levels, which could be mitigated by pre-incubation of THC pSi (PITHC pSi). PITHC pSi facilitates normal cellular phenotypes and is biocompatible in vivo. Importantly, the material also possesses optical properties capable of responding to microenvironmental changes that are readable non-invasively in cell culture and subcutaneous settings. Collectively, we demonstrate, for the first time, that PITHC pSi rugate filters are both biocompatible and optically functional for lab-on-a-chip and subcutaneous biosensing scenarios. We believe that this study will deepen our understanding of cell-pSi interactions and foster the development of implantable biosensors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Monitoring Phosphatidic Acid Signaling in Breast Cancer Cells Using Genetically Encoded Biosensors.

PubMed

Lu, Maryia; Tay, Li Wei Rachel; He, Jingquan; Du, Guangwei

2016-01-01

Phospholipids are important signaling molecules that regulate cell proliferation, death, migration, and metabolism. Many phospholipid signaling cascades are altered in breast cancer. To understand the functions of phospholipid signaling molecules, genetically encoded phospholipid biosensors have been developed to monitor their spatiotemporal dynamics. Compared to other phospholipids, much less is known about the subcellular production and cellular functions of phosphatidic acid (PA), partially due to the lack of a specific and sensitive PA biosensor in the past. This chapter describes the use of a newly developed PA biosensor, PASS, in two applications: regular fluorescent microscopy and fluorescence lifetime imaging microscopy-Förster/fluorescence resonance energy transfer (FLIM-FRET). These protocols can be also used with other phospholipid biosensors.

Biosensor for the detection of Listeria monocytogenes: emerging trends.

PubMed

Soni, Dharmendra Kumar; Ahmad, Rafiq; Dubey, Suresh Kumar

2018-05-23

The early detection of Listeria monocytogenes (L. monocytogenes) and understanding the disease burden is of paramount interest. The failure to detect pathogenic bacteria in the food industry may have terrible consequences, and poses deleterious effects on human health. Therefore, integration of methods to detect and trace the route of pathogens along the entire food supply network might facilitate elucidation of the main contamination sources. Recent research interest has been oriented towards the development of rapid and affordable pathogen detection tools/techniques. An innovative and new approach like biosensors has been quite promising in revealing the foodborne pathogens. In spite of the existing knowledge, advanced research is still needed to substantiate the expeditious nature and sensitivity of biosensors for rapid and in situ analysis of foodborne pathogens. This review summarizes recent developments in optical, piezoelectric, cell-based, and electrochemical biosensors for Listeria sp. detection in clinical diagnostics, food analysis, and environmental monitoring, and also lists their drawbacks and advantages.

The Need and Potential of Biosensors to Detect Dioxins and Dioxin-Like Polychlorinated Biphenyls along the Milk, Eggs and Meat Food Chain

PubMed Central

Chobtang, Jeerasak; de Boer, Imke J. M.; Hoogenboom, Ron L. A. P.; Haasnoot, Willem; Kijlstra, Aize; Meerburg, Bastiaan G.

2011-01-01

Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain. PMID:22247688

The need and potential of biosensors to detect dioxins and dioxin-like polychlorinated biphenyls along the milk, eggs and meat food chain.

PubMed

Chobtang, Jeerasak; de Boer, Imke J M; Hoogenboom, Ron L A P; Haasnoot, Willem; Kijlstra, Aize; Meerburg, Bastiaan G

2011-01-01

Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain.

A sensitive biosensor using double-layer capillary based immunomagnetic separation and invertase-nanocluster based signal amplification for rapid detection of foodborne pathogen.

PubMed

Huang, Fengchun; Zhang, Huilin; Wang, Lei; Lai, Weihua; Lin, Jianhan

2018-02-15

Combining double-layer capillary based high gradient immunomagnetic separation, invertase-nanocluster based signal amplification and glucose meter based signal detection, a novel biosensor was developed for sensitive and rapid detection of E. coli O157:H7 in this study. The streptavidin modified magnetic nanobeads (MNBs) were conjugated with the biotinylated polyclonal antibodies against E. coli O157:H7 to form the immune MNBs, which were captured by the high gradient magnetic field in the double-layer capillary to specifically separate and efficiently concentrate the target bacteria. Calcium chloride was used with the monoclonal antibodies against E. coli O157:H7 and the invertase to form the immune invertase-nanoclusters (INCs), which were used to react with the target bacteria to form the MNB-bacteria-INC complexes in the capillary. The sucrose was then injected into the capillary and catalyzed by the invertase on the complexes into the glucose, which was detected using the glucose meter to obtain the concentration of the glucose for final determination of the E. coli O157:H7 cells in the sample. A linear relationship between the readout of the glucose meter and the concentration of the E. coli O157:H7 cells (from 10 2 to 10 7 CFU/mL) was found and the lower detection limit of this biosensor was 79 CFU/mL. This biosensor might be extended for the detection of other foodborne pathogens by changing the antibodies and has shown the potential for the detection of foodborne pathogens in a large volume of sample to further increase the sensitivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Future of biosensors: a personal view.

PubMed

Scheller, Frieder W; Yarman, Aysu; Bachmann, Till; Hirsch, Thomas; Kubick, Stefan; Renneberg, Reinhard; Schumacher, Soeren; Wollenberger, Ulla; Teller, Carsten; Bier, Frank F

2014-01-01

Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.

Defining an additivity framework for mixture research in inducible whole-cell biosensors

NASA Astrophysics Data System (ADS)

Martin-Betancor, K.; Ritz, C.; Fernández-Piñas, F.; Leganés, F.; Rodea-Palomares, I.

2015-11-01

A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks

Determination of glucose in human stomach cancer cell extracts and single cells by capillary electrophoresis with a micro-biosensor.

PubMed

Wang, Xiaolei; Ma, Yanfang; Zhao, Man; Zhou, Minfeng; Xiao, Yan; Sun, Zifei; Tong, Lili

2016-10-21

Bioactive species in cells can provide information about signal transduction, cell function, and the effects of disease treatment. In this article, a novel micro-biosensor was fabricated to detect glucose in individual human stomach cancer cells (MGC80-3 cells) with capillary electrophoresis (CE). We fabricated the micro-biosensors by immobilizing a single-walled carbon nanotube-glucose oxidase (GOx)-glutaraldehyde (GA) bio-composite at the palladium nanoparticle (PdNPs) modified Pt electrode. The linear concentration of glucose ranged from 2.0μM to 1.0mM, with a detection limit of 0.5μM. Using this method, the mean amount of glucose in MGC80-3 cell extracts and in single cells was 20.0 fmol and 20±6 fmol (n=10), respectively. The micro-biosensor exhibited high sensitivity, stability, and a long operating life, which are likely due to the biocompatible environment provided by BSA and GA, and the adsorption and faster electron transfer of SWNTs and PdNPs to GOx. Copyright © 2016. Published by Elsevier B.V.

Development of Amperometric Glucose Biosensor Based on Prussian Blue Functionlized TiO2 Nanotube Arrays

PubMed Central

Gao, Zhi-Da; Qu, Yongfang; Li, Tongtong; Shrestha, Nabeen K.; Song, Yan-Yan

2014-01-01

Amperometric biosensors consisting of oxidase and peroxidase have attracted great attention because of their wide application. The current work demonstrates a novel approach to construct an enzymatic biosensor based on TiO2 nanotube arrays (TiNTs) as a supporting electrode on which Prussian Blue (PB)-an “artificial enzyme peroxidase” and enzyme glucose oxidase (GOx) have been immobilized. For this, PB nanocrystals are deposited onto the nanotube wall photocatalytically using the intrinsic photocatalytical property of TiO2, and the GOx/AuNPs nanobiocomposites are subsequently immobilized into the nanotubes via the electrodeposition of polymer. The resulting electrode exhibits a fast response, wide linear range, and good stability for glucose sensing. The sensitivity of the sensor is as high as 248 mA M−1 cm−2, and the detection limit is about 3.2 μM. These findings demonstrate a promising strategy to integrate enzymes and TiNTs, which could provide an analytical access to a large group of enzymes for bioelectrochemical applications including biosensors and biofuel cells. PMID:25367086

A colorimetric and fluorogenic probe for bisulfite using benzopyrylium as the recognition unit.

PubMed

Zhang, Yun; Zhang, Xiangwen; Yang, Xiao-Feng; Zhang, Juan

2017-11-01

A coumarin-benzopyrylium (CB) platform has been developed for the colorimetric and fluorogenic detection of bisulfite. The proposed probe utilizes coumarin as the fluorophore and positively charged benzopyrylium as the reaction site. The method employs the nucleophilic addition of bisulfite to the benzopyrylium moiety of CB to inactivate the electron-deficient oxonium ion. The driving force for photo-induced electron transfer is considerably diminished, thereby promoting the emission intensity of the coumarin fluorophore. The fluorescence intensity at 510 nm is linear with bisulfite concentration over a range of 0.2-7.5 μM with a detection limit of 42 nM (3δ). CB shows a rapid response (within 30 s) and high selectivity and sensitivity for bisulfite. Preliminary studies show that CB has great potential for bisulfite detection in real samples and in living cells. Copyright © 2017 John Wiley & Sons, Ltd.

Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase.

PubMed

Liang, Bo; Zhang, Shu; Lang, Qiaolin; Song, Jianxia; Han, Lihui; Liu, Aihua

2015-07-16

A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP(+) involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current-time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM-1 mM and 2-10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N=3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection. Copyright © 2015 Elsevier B.V. All rights reserved.

Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

PubMed

Kapp, Nikki; Barnes, William J; Richard, Tom L; Anderson, Charles T

2015-07-01

Lignin is a complex polyphenolic heteropolymer that is abundant in the secondary cell walls of plants and functions in growth and defence. It is also a major barrier to the deconstruction of plant biomass for bioenergy production, but the spatiotemporal details of how lignin is deposited in actively lignifying tissues and the precise relationships between wall lignification in different cell types and developmental events, such as flowering, are incompletely understood. Here, the lignin-detecting fluorogenic dye, Basic Fuchsin, was adapted to enable comparative fluorescence-based imaging of lignin in the basal internodes of three Brachypodium distachyon ecotypes that display divergent flowering times. It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls. Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon. Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses. This study provides a new comparative tool for imaging lignin in plants and helps inform our views of how lignification proceeds in grasses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon

PubMed Central

Kapp, Nikki; Barnes, William J.; Richard, Tom L.; Anderson, Charles T.

2015-01-01

Lignin is a complex polyphenolic heteropolymer that is abundant in the secondary cell walls of plants and functions in growth and defence. It is also a major barrier to the deconstruction of plant biomass for bioenergy production, but the spatiotemporal details of how lignin is deposited in actively lignifying tissues and the precise relationships between wall lignification in different cell types and developmental events, such as flowering, are incompletely understood. Here, the lignin-detecting fluorogenic dye, Basic Fuchsin, was adapted to enable comparative fluorescence-based imaging of lignin in the basal internodes of three Brachypodium distachyon ecotypes that display divergent flowering times. It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls. Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon. Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses. This study provides a new comparative tool for imaging lignin in plants and helps inform our views of how lignification proceeds in grasses. PMID:25922482

Guiding pancreatic beta cells to target electrodes in a whole-cell biosensor for diabetes.

PubMed

Pedraza, Eileen; Karajić, Aleksandar; Raoux, Matthieu; Perrier, Romain; Pirog, Antoine; Lebreton, Fanny; Arbault, Stéphane; Gaitan, Julien; Renaud, Sylvie; Kuhn, Alexander; Lang, Jochen

2015-10-07

We are developing a cell-based bioelectronic glucose sensor that exploits the multi-parametric sensing ability of pancreatic islet cells for the treatment of diabetes. These cells sense changes in the concentration of glucose and physiological hormones and immediately react by generating electrical signals. In our sensor, signals from multiple cells are recorded as field potentials by a micro-electrode array (MEA). Thus, cell response to various factors can be assessed rapidly and with high throughput. However, signal quality and consequently overall sensor performance rely critically on close cell-electrode proximity. Therefore, we present here a non-invasive method of further exploiting the electrical properties of these cells to guide them towards multiple micro-electrodes via electrophoresis. Parameters were optimized by measuring the cell's zeta potential and modeling the electric field distribution. Clonal and primary mouse or human β-cells migrated directly to target electrodes during the application of a 1 V potential between MEA electrodes for 3 minutes. The morphology, insulin secretion, and electrophysiological characteristics were not altered compared to controls. Thus, cell manipulation on standard MEAs was achieved without introducing any external components and while maintaining the performance of the biosensor. Since the analysis of the cells' electrical activity was performed in real time via on-chip recording and processing, this work demonstrates that our biosensor is operational from the first step of electrically guiding cells to the final step of automatic recognition. Our favorable results with pancreatic islets, which are highly sensitive and fragile cells, are encouraging for the extension of this technique to other cell types and microarray devices.

A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells

PubMed Central

Paulmurugan, Ramasamy; Afjei, Rayhaneh; Sekar, Thillai V.; Babikir, Husam A.; Massoud, Tarik F.

2018-01-01

Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53. PMID:29765555

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis.

PubMed

Tarantola, Marco; Marel, Anna-Kristina; Sunnick, Eva; Adam, Holger; Wegener, Joachim; Janshoff, Andreas

2010-03-01

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

Wearable Sensor System Powered by a Biofuel Cell for Detection of Lactate Levels in Sweat (Postprint)

DTIC Science & Technology

2016-05-04

attractive for development of sensing technology for the monitoring of human performance. Amperometric biosensors are known to be inexpensive, repro...biofuel cells for self-powered biosensors was first discussed in 2001 and has gained momentum in recent years.32–34 Information technology has...lactate biosensor ,35,36 a glucose oxidase BFC power source, an energy har- vester and a micropotentiostat. The following sections describe the development

Surface stress-based biosensors.

PubMed

Sang, Shengbo; Zhao, Yuan; Zhang, Wendong; Li, Pengwei; Hu, Jie; Li, Gang

2014-01-15

Surface stress-based biosensors, as one kind of label-free biosensors, have attracted lots of attention in the process of information gathering and measurement for the biological, chemical and medical application with the development of technology and society. This kind of biosensors offers many advantages such as short response time (less than milliseconds) and a typical sensitivity at nanogram, picoliter, femtojoule and attomolar level. Furthermore, it simplifies sample preparation and testing procedures. In this work, progress made towards the use of surface stress-based biosensors for achieving better performance is critically reviewed, including our recent achievement, the optimally circular membrane-based biosensors and biosensor array. The further scientific and technological challenges in this field are also summarized. Critical remark and future steps towards the ultimate surface stress-based biosensors are addressed. Copyright © 2013 Elsevier B.V. All rights reserved.

Enzymatic biofuel cell-based self-powered biosensing of protein kinase activity and inhibition via thiophosphorylation-mediated interface engineering.

PubMed

Gu, Chengcheng; Gai, Panpan; Han, Lei; Yu, Wen; Liu, Qingyun; Li, Feng

2018-05-24

We developed a facile and ultrasensitive enzymatic biofuel cell (EBFC)-based self-powered biosensor of protein kinase A (PKA) activity and inhibition via thiophosphorylation-mediated interface engineering. The detection limit was down to 0.00022 U mL-1 (S/N = 3). In addition, the PKA activities from MCF-7 and A549 cell lysates were analyzed and achieved reliable results.

Selective detection of hypertoxic organophosphates pesticides via PDMS composite based acetylcholinesterase-inhibition biosensor.

PubMed

Zhao, Wei; Ge, Pei-Yu; Xu, Jing-Juan; Chen, Hong-Yuan

2009-09-01

We report on a pair of highly sensitive amperometric biosensors for organophosphate pesticides (OPs) based on assembling acetylcholinesterase (AChE) on poly(dimethylsiloxane) (PDMS)-poly(diallydimethylemmonium) (PDDA)/gold nanoparticles (AuNPs) composite film. Two AChE immobilization strategies are proposed based on the composite film with hydrophobic and hydrophilic surface tailored by oxygen plasma. The twin biosensors show interesting different electrochemical performances. The hydrophobic surface based PDMS-PDDAN AuNPs/choline oxidase (ChO)/AChE biosensor (biosensor-1) shows excellent stability and unique selectivity to hypertoxic organophosphate. At optimal conditions, this biosensor-1 could measure 5.0 x 10(-10) g/L paraoxon and 1.0 x 10(-9) g/L parathion. As for the hydrophilic surface based biosensor (biosensor-2), it shows no selectivity but can be commonly used for the detection of most OPs. Based on the structure of AChE, it is assumed that via the hydrophobic interaction between enzyme molecules and hydrophobic surface, the enzyme active sites surrounded by hydrophobic amino acids face toward the surface and get better protection from OPs. This assumption may explain the different performances of the twin biosensors and especially the unique selectivity of biosensor-1 to hypertoxic OPs. Real sample detection was performed and the omethoate residue on Cottomrose Hibiscus leaves was detected with biosensor-1.

Photonic crystal waveguide-based biosensor for detection of diseases

NASA Astrophysics Data System (ADS)

Chopra, Harshita; Kaler, Rajinder S.; Painam, Balveer

2016-07-01

A biosensor is a device that is used to detect the analytes or molecules of a sample by means of a binding mechanism. A two-dimensional photonic crystal waveguide-based biosensor is designed with a diamond-shaped ring resonator and two waveguides: a bus waveguide and a drop waveguide. The sensing mechanism is based on change in refractive index of the analytes, leading to a shift in the peak resonant wavelength. This mechanism can be used in the field of biomedical treatment where different body fluids such as blood, tears, saliva, or urine can be used as the analyte in which different components of the fluid can be detected. It can also be used to differentiate between the cell lines of a normal and an unhealthy human being. Average value of quality factor for this device comes out to be 1082.2063. For different analytes used, the device exhibits enhanced sensitivity and, hence, it is useful for the detection of diseases.

Rab-NANOPS: FRET biosensors for Rab membrane nanoclustering and prenylation detection in mammalian cells.

PubMed

Najumudeen, Arafath Kaja; Guzmán, Camilo; Posada, Itziar M D; Abankwa, Daniel

2015-01-01

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.

Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.

PubMed

Steinfeld, Robert; Fuhrmann, Jens C; Gärtner, Jutta

2006-09-01

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Capacitive Biosensors and Molecularly Imprinted Electrodes.

PubMed

Ertürk, Gizem; Mattiasson, Bo

2017-02-17

Capacitive biosensors belong to the group of affinity biosensors that operate by registering direct binding between the sensor surface and the target molecule. This type of biosensors measures the changes in dielectric properties and/or thickness of the dielectric layer at the electrolyte/electrode interface. Capacitive biosensors have so far been successfully used for detection of proteins, nucleotides, heavy metals, saccharides, small organic molecules and microbial cells. In recent years, the microcontact imprinting method has been used to create very sensitive and selective biorecognition cavities on surfaces of capacitive electrodes. This chapter summarizes the principle and different applications of capacitive biosensors with an emphasis on microcontact imprinting method with its recent capacitive biosensor applications.

Development of biosensors based on the one-dimensional semiconductor nanomaterials.

PubMed

Yan, Shancheng; Shi, Yi; Xiao, Zhongdang; Zhou, Minmin; Yan, Wenfu; Shen, Haoliang; Hu, Dong

2012-09-01

Biosensors are becoming increasingly important due to their applications in biological and chemical analyses, food safety industry, biomedical diagnostics, clinical detection, and environmental monitoring. Recent years, nanostructured semiconductor materials have been used to fabricate biosensors owing to their biocompatibility, low toxicity, high electron mobility, and easy fabrication. In the present study, we focus on recent various biosensors based on the one-dimensional semiconductor nanomaterials such as electrochemical biosensor, field-effect transistors biosensor, and label-free optical biosensor. In particular, the development of the electrochemical biosensor is discussed detailedly.

Simple and rapid fabrication of disposable carbon-based electrochemical cells using an electronic craft cutter for sensor and biosensor applications.

PubMed

Afonso, André S; Uliana, Carolina V; Martucci, Diego H; Faria, Ronaldo C

2016-01-01

This work describes the construction of an all-plastic disposable carbon-based electrochemical cell (DCell) using a simple procedure based on the use of a home cutter printer for prototyping and laminating. The cutter printer and adhesive vinyl films were used to produce three electrodes in an electrochemical cell layout, and a laminating process was then used to define the geometric area and insulate the electrodes. The DCell showed excellent performance in several applications including the determination of toxic metals in water samples, the immobilization of DNA and the detection of Salmonella. An unmodified DCell was applied for Pb and Cd detection in the range of 100-300 ng mL(-1) with a limit of detection of 50 and 39 ng mL(-1) for Cd and Pb, respectively. DNA was successfully immobilized on a DCell and used for studies of interaction between bisphenol A and DNA. The square wave voltammetry of a DNA modified DCell presented a guanine oxidation current 2.5 times greater after exposure of the electrode to bisphenol A and no current variation for the adenine moiety indicating that bisphenol A showed a preference for DNA interaction sites. A magneto-immunoassay was developed using a DCell for Salmonella detection in milk samples. The system presented a linear range from 100 to 700 cells mL(-1) with a limit of detection of 100 cells mL(-1) and good recovery values between 93% and 101% in milk samples, with no interference from Escherichia coli. Using the proposed method, hundreds of DCells can be assembled in less than two hours, at a material cost of less than US $0.02 per cell. The all-plastic disposable electrochemical cell developed was successfully applied as an electrochemical sensor and biosensor. The feasibility of the developed all-plastic disposable electrochemical cell was demonstrated in applications as both sensor and biosensor. Copyright © 2015 Elsevier B.V. All rights reserved.

Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

PubMed

Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

2017-11-02

The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

Bio-analytical applications of microbial fuel cell-based biosensors for onsite water quality monitoring.

PubMed

ElMekawy, A; Hegab, H M; Pant, D; Saint, C P

2018-01-01

Globally, sustainable provision of high-quality safe water is a major challenge of the 21st century. Various chemical and biological monitoring analytics are presently utilized to guarantee the availability of high-quality water. However, these techniques still face some challenges including high costs, complex design and onsite and online limitations. The recent technology of using microbial fuel cell (MFC)-based biosensors holds outstanding potential for the rapid and real-time monitoring of water source quality. MFCs have the advantages of simplicity in design and efficiency for onsite sensing. Even though some sensing applications of MFCs were previously studied, e.g. biochemical oxygen demand sensor, recently numerous research groups around the world have presented new practical applications of this technique, which combine multidisciplinary scientific knowledge in materials science, microbiology and electrochemistry fields. This review presents the most updated research on the utilization of MFCs as potential biosensors for monitoring water quality and considers the range of potentially toxic analytes that have so far been detected using this methodology. The advantages of MFCs over established technology are also considered as well as future work required to establish their routine use. © 2017 The Society for Applied Microbiology.

Naringenin-responsive riboswitch-based fluorescent biosensor module for Escherichia coli co-cultures.

PubMed

Xiu, Yu; Jang, Sungho; Jones, J Andrew; Zill, Nicholas A; Linhardt, Robert J; Yuan, Qipeng; Jung, Gyoo Yeol; Koffas, Mattheos A G

2017-10-01

The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA Technical Reports Server (NTRS)

Tieze, Sofia Massaro; Santa Maria, Sergio R.; Liddell, Lauren C.; Bhattacharya, Sharmila

2017-01-01

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

A novel biosensor for p-nitrophenol based on an aerobic anode microbial fuel cell.

PubMed

Chen, Zhengjun; Niu, Yongyan; Zhao, Shuai; Khan, Aman; Ling, Zhenmin; Chen, Yong; Liu, Pu; Li, Xiangkai

2016-11-15

P-nitrophenol is one of the most common contaminants in chemical industrial wastewater, and in situ real-time monitoring of PNP cannot be achieved by conventional analytical techniques. Here, a two-chamber microbial fuel cell with an aerobic anode chamber was tested as a biosensor for in situ real-time monitoring of PNP. Pseudomonas monteilii LZU-3, which was used as the biological recognition element, can form a biofilm on the anode electrode using PNP as a sole substrate. The optimal operation parameters of the biosensor were as follows: external resistance 1000Ω, pH 7.8, temperature 30°C, and maximum PNP concentration 50mgL(-1). Under these conditions, the maximum voltages showed a linear relationship with PNP concentrations ranging from 15±5 to 44±4.5mgL(-1). Furthermore, we developed a novel portable device for in situ real-time monitoring of PNP. When the device was applied to measure PNP in wastewater containing various additional aromatic compounds and metal ions, the performance of the biosensor was not affected and the correlation between the maximum voltages and the PNP concentrations ranging from 9±4mgL(-1) to 36 ± 5mgL(-1) was conserved. The results demonstrated that the MFC biosensor provides a rapid and cost-efficient analytical method for real-time monitoring of toxic and recalcitrant pollutants in environmental samples. Copyright © 2016 Elsevier B.V. All rights reserved.

A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

PubMed

Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

2015-12-15

The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sensitive photoelectrochemical biosensor for kinase activity detection and inhibition based on the surface defect recognition and multiple signal amplification of metal-organic frameworks.

PubMed

Wang, Zonghua; Yan, Zhiyong; Wang, Feng; Cai, Jibao; Guo, Lei; Su, Jiakun; Liu, Yang

2017-11-15

A turn-on photoelectrochemical (PEC) biosensor based on the surface defect recognition and multiple signal amplification of metal-organic frameworks (MOFs) was proposed for highly sensitive protein kinase activity analysis and inhibitor evaluation. In this strategy, based on the phosphorylation reaction in the presence of protein kinase A (PKA), the Zr-based metal-organic frameworks (UiO-66) accommodated with [Ru(bpy) 3 ] 2+ photoactive dyes in the pores were linked to the phosphorylated kemptide modified TiO 2 /ITO electrode through the chelation between the Zr 4+ defects on the surface of UiO-66 and the phosphate groups in kemptide. Under visible light irradiation, the excited electrons from [Ru(bpy) 3 ] 2+ adsorbed in the pores of UiO-66 injected into the TiO 2 conduction band to generate photocurrent, which could be utilized for protein kinase activities detection. The large surface area and high porosities of UiO-66 facilitated a large number of [Ru(bpy) 3 ] 2+ that increased the photocurrent significantly, and afforded a highly sensitive PEC analysis of kinase activity. The detection limit of the as-proposed PEC biosensor was 0.0049UmL -1 (S/N!=!3). The biosensor was also applied for quantitative kinase inhibitor evaluation and PKA activities detection in MCF-7 cell lysates. The developed visible-light PEC biosensor provides a simple detection procedure and a cost-effective manner for PKA activity assays, and shows great potential in clinical diagnosis and drug discoveries. Copyright © 2017 Elsevier B.V. All rights reserved.

Classification of biosensor time series using dynamic time warping: applications in screening cancer cells with characteristic biomarkers.

PubMed

Rai, Shesh N; Trainor, Patrick J; Khosravi, Farhad; Kloecker, Goetz; Panchapakesan, Balaji

2016-01-01

The development of biosensors that produce time series data will facilitate improvements in biomedical diagnostics and in personalized medicine. The time series produced by these devices often contains characteristic features arising from biochemical interactions between the sample and the sensor. To use such characteristic features for determining sample class, similarity-based classifiers can be utilized. However, the construction of such classifiers is complicated by the variability in the time domains of such series that renders the traditional distance metrics such as Euclidean distance ineffective in distinguishing between biological variance and time domain variance. The dynamic time warping (DTW) algorithm is a sequence alignment algorithm that can be used to align two or more series to facilitate quantifying similarity. In this article, we evaluated the performance of DTW distance-based similarity classifiers for classifying time series that mimics electrical signals produced by nanotube biosensors. Simulation studies demonstrated the positive performance of such classifiers in discriminating between time series containing characteristic features that are obscured by noise in the intensity and time domains. We then applied a DTW distance-based k -nearest neighbors classifier to distinguish the presence/absence of mesenchymal biomarker in cancer cells in buffy coats in a blinded test. Using a train-test approach, we find that the classifier had high sensitivity (90.9%) and specificity (81.8%) in differentiating between EpCAM-positive MCF7 cells spiked in buffy coats and those in plain buffy coats.

Electrochemical and AFM Characterization of G-Quadruplex Electrochemical Biosensors and Applications

PubMed Central

2018-01-01

Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited. PMID:29666699

Impedance biosensor based on interdigitated electrode array for detection of E.coli O157:H7 in food products

NASA Astrophysics Data System (ADS)

Ghosh Dastider, Shibajyoti; Barizuddin, Syed; Dweik, Majed; Almasri, Mahmoud F.

2012-05-01

An impedance biosensor was designed, fabricated and tested for detection of viable Escherichia coli O157:H7 in food samples. This device consists of interdigitated microelectrode array (IDEA) fabricated using thin layer of sputtered gold, embedded under a polydimethylsiloxane (PDMS) microchannel. The array of electrodes is designed to detect viable EColi in different food products. The active surface area of the detection array was modified using goat anti-E.coli polyclonal IgG antibody. Contaminated food samples were tested by infusing the supernatant containing bacteria over the IDEA's, through the microchannel. Antibody-antigen binding on the electrodes results in impedance change. Four serial concentrations of E.coli contaminated food samples (3x102 CFUmL-1 to 3x105 CFUmL-1) were tested. The biosensor successfully detected the E.coli samples, with the lower detection limit being 3x103 CFUmL-1 (up to 3cells/μl). Comparing the test results with an IDEA impedance biosensor without microchannel (published elsewhere) indicates that this biosensor have two order of magnitude times higher sensitivity. The proposed biosensor provides qualitative and quantitative detection, and potentially could be used for detection of other type of bacteria by immobilizing the specific type of antibody.

An SU-8 liquid cell for surface acoustic wave biosensors

NASA Astrophysics Data System (ADS)

Francis, Laurent A.; Friedt, Jean-Michel; Bartic, Carmen; Campitelli, Andrew

2004-08-01

One significant challenge facing biosensor development is packaging. For surface acoustic wave based biosensors, packaging influences the general sensing performance. The acoustic wave is generated and received thanks to interdigital transducers and the separation between the transducers defines the sensing area. Liquids used in biosensing experiments lead to an attenuation of the acoustic signal while in contact with the transducers. We have developed a liquid cell based on photodefinable epoxy SU-8 that prevents the presence of liquid on the transducers, has a small disturbance effect on the propagation of the acoustic wave, does not interfere with the biochemical sensing event, and leads to an integrated sensor system with reproducible properties. The liquid cell is achieved in two steps. In a first step, the SU-8 is precisely patterned around the transducers to define 120 μm thick walls. In a second step and after the dicing of the sensors, a glass capping is placed manually and glued on top of the SU-8 walls. This design approach is an improvement compared to the more classical solution consisting of a pre-molded cell that must be pressed against the device in order to avoid leaks, with negative consequences on the reproducibility of the experimental results. We demonstrate the effectiveness of our approach by protein adsorption monitoring. The packaging materials do not interfere with the biomolecules and have a high chemical resistance. For future developments, wafer level bonding of the quartz capping onto the SU-8 walls is envisioned.

Development of biosensors and their application in metabolic engineering.

PubMed

Zhang, Jie; Jensen, Michael K; Keasling, Jay D

2015-10-01

In a sustainable bioeconomy, many commodities and high value chemicals, including pharmaceuticals, will be manufactured using microbial cell factories from renewable feedstocks. These cell factories can be efficiently generated by constructing libraries of diversified genomes followed by screening for the desired phenotypes. However, methods available for microbial genome diversification far exceed our ability to screen and select for those variants with optimal performance. Genetically encoded biosensors have shown the potential to address this gap, given their ability to respond to small molecule binding and ease of implementation with high-throughput analysis. Here we describe recent progress in biosensor development and their applications in a metabolic engineering context. We also highlight examples of how biosensors can be integrated with synthetic circuits to exert feedback regulation on the metabolism for improved performance of cell factories. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cholinesterase-based biosensors.

PubMed

Štěpánková, Šárka; Vorčáková, Katarína

2016-01-01

Recently, cholinesterase-based biosensors are widely used for assaying anticholinergic compounds. Primarily biosensors based on enzyme inhibition are useful analytical tools for fast screening of inhibitors, such as organophosphates and carbamates. The present review is aimed at compilation of the most important facts about cholinesterase based biosensors, types of physico-chemical transduction, immobilization strategies and practical applications.

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

PubMed

Wu, Lei; Yang, Shu-Hou; Xiong, Hao; Yang, Jia-Qian; Guo, Jun; Yang, Wen-Chao; Yang, Guang-Fu

2017-03-21

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction.

PubMed

Wang, Dingzhong; Tang, Wei; Wu, Xiaojie; Wang, Xinyi; Chen, Gengjia; Chen, Qiang; Li, Na; Liu, Feng

2012-08-21

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.

Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging.

PubMed

Li, Kai; Liu, Bin

2014-09-21

Polymer encapsulated organic nanoparticles have recently attracted increasing attention in the biomedical field because of their unique optical properties, easy fabrication and outstanding performance as imaging and therapeutic agents. Of particular importance is the polymer encapsulated nanoparticles containing conjugated polymers (CP) or fluorogens with aggregation induced emission (AIE) characteristics as the core, which have shown significant advantages in terms of tunable brightness, superb photo- and physical stability, good biocompatibility, potential biodegradability and facile surface functionalization. In this review, we summarize the latest advances in the development of polymer encapsulated CP and AIE fluorogen nanoparticles, including preparation methods, material design and matrix selection, nanoparticle fabrication and surface functionalization for fluorescence and photoacoustic imaging. We also discuss their specific applications in cell labeling, targeted in vitro and in vivo imaging, blood vessel imaging, cell tracing, inflammation monitoring and molecular imaging. We specially focus on strategies to fine-tune the nanoparticle property (e.g. size and fluorescence quantum yield) through precise engineering of the organic cores and careful selection of polymer matrices. The review also highlights the merits and limitations of these nanoparticles as well as strategies used to overcome the limitations. The challenges and perspectives for the future development of polymer encapsulated organic nanoparticles are also discussed.

Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

PubMed

Zhang, Hui; Pi, Fengmei; Shu, Dan; Vieweger, Mario; Guo, Peixuan

2015-01-01

RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

PubMed

Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

2017-03-31

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Specificity Bio-identification of CNT-Based Transistor

NASA Astrophysics Data System (ADS)

Wang, Sheng-Yu; Wu, Hue-Min

2017-12-01

In this research, we report a simple and general approach to π-π stacking functionalization of the sidewalls of CNTs by 1-pyrenebutanoic acid, succinimidyl ester (PSE), and subsequent immobilization of insulin-like growth factor 1 receptor (IGF1R) onto SWNTs with a high degree of control and specificity. The selection of PSE provides visualization and characterization of individual CNTs based on its strong luminescence. In addition, we designed a simple and efficient electrode with a staggered pattern to increase the effect of electrophoresis by using electric field for the macroscopic alignment of CNTs to complete a field-effect device for CNT-based biosensors. Scanning Electron Microscopy (SEM) was used to investigate the morphology of the biosensors. The results of four-point probe method demonstrated high selectivity and sensitivity of detection. The functionalization of SWNTs was investigated by Fourier transform infrared spectroscopy (FTIR). Experimental results imply that specific binding between IGF1R and its specific mAb results in a dramatic change in electrical current of CNT-based devices, and suggest that the devices are very promising biosensor candidates to detect circulating cancer cells.

Method for detection of a few pathogenic bacteria and determination of live versus dead cells

NASA Astrophysics Data System (ADS)

Horikawa, Shin; Chen, I.-Hsuan; Du, Songtao; Liu, Yuzhe; Wikle, Howard C.; Suh, Sang-Jin; Barbaree, James M.; Chin, Bryan A.

2016-05-01

This paper presents a method for detection of a few pathogenic bacteria and determination of live versus dead cells. The method combines wireless phage-coated magnetoelastic (ME) biosensors and a surface-scanning dectector, enabling real-time monitoring of the growth of specific bacteria in a nutrient broth. The ME biosensor used in this investigation is composed of a strip-shaped ME resonator upon which an engineered bacteriophage is coated to capture a pathogen of interest. E2 phage with high binding affinity for Salmonella Typhimurium was used as a model study. The specificity of E2 phage has been reported to be 1 in 105 background bacteria. The phage-coated ME biosensors were first exposed to a low-concentration Salmonella suspension to capture roughly 300 cells on the sensor surface. When the growth of Salmonella in the broth occurs, the mass of the biosensor increases, which results in a decrease in the biosensor's resonant frequency. Monitoring of this mass- induced resonant frequency change allows for real-time detection of the presence of Salmonella. Detection of a few bacteria is also possible by growing them to a sufficient number. The surface-scanning detector was used to measure resonant frequency changes of 25 biosensors sequentially in an automated manner as a function of time. This methodology offers direct, real-time detection, quantification, and viability determination of specific bacteria. The rate of the sensor's resonant frequency change was found to be largely dependent on the number of initially bound cells and the efficiency of cell growth.

Genetically encoded pH sensor for tracking surface proteins through endocytosis.

PubMed

Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D; Watkins, Simon C; Waggoner, Alan S; Bruchez, Marcel P

2012-05-14

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorogenic Behaviour of the Hetero-Diels-Alder Ligation of 5-Alkoxyoxazoles with Maleimides and their Applications.

PubMed

Renault, Kévin; Jouanno, Laurie-Anne; Lizzul-Jurse, Antoine; Renard, Pierre-Yves; Sabot, Cyrille

2016-12-19

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels-Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion.

PubMed

Li, Xiaokang; Soler, Maria; Szydzik, Crispin; Khoshmanesh, Khashayar; Schmidt, Julien; Coukos, George; Mitchell, Arnan; Altug, Hatice

2018-06-01

Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

PubMed Central

Shcherbakova, Daria M.; Baloban, Mikhail; Emelyanov, Alexander V.; Brenowitz, Michael; Guo, Peng; Verkhusha, Vladislav V.

2016-01-01

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. PMID:27539380

A graphene-based physiometer array for the analysis of single biological cells

NASA Astrophysics Data System (ADS)

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-10-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer - a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene - in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.

Development and characterization of a whole-cell bioluminescent sensor for bioavailable middle-chain alkanes in contaminated groundwater samples.

PubMed Central

Sticher, P; Jaspers, M C; Stemmler, K; Harms, H; Zehnder, A J; van der Meer, J R

1997-01-01

A microbial whole-cell biosensor was developed, and its potential to measure water-dissolved concentrations of middle-chain-length alkanes and some related compounds by bioluminescence was characterized. The biosensor strain Escherichia coli DH5 alpha(pGEc74, pJAMA7) carried the regulatory gene alkS from Pseudomonas oleovorans and a transcriptional fusion of PalkB from the same strain with the promoterless luciferase luxAB genes from Vibrio harveyi on two separately introduced plasmids. In standardized assays, the biosensor cells were readily inducible with octane, a typical inducer of the alk system. Light emission after induction periods of more than 15 min correlated well with octane concentration. In well-defined aqueous samples, there was a linear relationship between light output and octane concentrations between 24 and 100 nM. The biosensor responded to middle-chain-length alkanes but not to alicyclic or aromatic compounds. In order to test its applicability for analyzing environmentally relevant samples, the biosensor was used to detect the bioavailable concentration of alkanes in heating oil-contaminated groundwater samples. By the extrapolation of calibrated light output data to low octane concentrations with a hyperbolic function, a total inducer concentration of about 3 nM in octane equivalents was estimated. The whole-cell biosensor tended to underestimate the alkane concentration in the groundwater samples by about 25%, possibly because of the presence of unknown inhibitors. This was corrected for by spiking the samples with a known amount of an octane standard. Biosensor measurements of alkane concentrations were further verified by comparing them with the results of chemical analyses. PMID:9327569

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

PubMed

Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Nano-particle enhanced impedimetric biosensor for detedtion of foodborne pathogens

NASA Astrophysics Data System (ADS)

Kim, G.; Om, A. S.; Mun, J. H.

2007-03-01

Recent outbreaks of foodborne illness have been increased the need for rapid and sensitive methods for detection of these pathogens. Conventional methods for pathogens detection and identification involve prolonged multiple enrichment steps. Even though some immunological rapid assays are available, these assays still need enrichment steps result in delayed detection. Biosensors have shown great potential for rapid detection of foodborne pathogens. They are capable of direct monitoring the antigen-antibody reactions in real time. Among the biosensors, impedimetric biosensors have been widely adapted as an analysis tool for the study of various biological binding reactions because of their high sensitivity and reagentless operation. In this study a nanoparticle-enhanced impedimetric biosensor for Salmonella enteritidis detection was developed which detected impedance changes caused by the attachment of the cells to the anti-Salmonella antibodies immobilized on interdigitated gold electrodes. Successive immobilization of neutravidin followed by anti-Salmonella antibodies was performed to the sensing area to create a biological detection surface. To enhance the impedance responses generated by antigen-antibody reactions, anti-Salmonella antibody conjugated nanoparticles were introduced on the sensing area. Using a portable impedance analyzer, the impedance across the interdigital electrodes was measured after the series of antigen-antibody bindings. Bacteria cells present in solution attached to capture antibodies and became tethered to the sensor surface. Attached bacteria cells changed the dielectric constant of the media between the electrodes thereby causing a change in measured impedance. Optimum input frequency was determined by analyzing frequency characteristics of the biosensor over ranges of applied frequencies from 10 Hz to 400 Hz. At 100 Hz of input frequency, the biosensor was most sensitive to the changes of the bacteria concentration and this frequency was used for the detection experiments. The biosensor was able to detect 106 CFU/mL in phosphate buffered saline (PBS) with a detection time of 3 minutes. Additional use of nanoparticles significantly enhanced the detection performance. By using the nanoparticles the biosensor could detect 104 CFU/mL of Salmonella enteritidis in PBS and 105 CFU/mL of cells in milk.

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

PubMed

Perkins, Lydia A; Fisher, Gregory W; Naganbabu, Matharishwan; Schmidt, Brigitte F; Mun, Frederick; Bruchez, Marcel P

2018-03-05

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

Biosensors for Real-Time Monitoring of Radiation-Induced Biologic Effects in Space

NASA Technical Reports Server (NTRS)

Baker, James R.; Balogh, Lajos; Majoros, Istvan; Keszler, Balazs; Myc, Andrzej; Kukowska-Latallo, Jolanta; Norris, Theodore; delaiglesia, Felix; Beeson, Nicholas W. (Compiler)

2002-01-01

This work seeks to develop cellular biosensors based on dendritic polymers. Nanoscale polymer structures less than 20 nm in diameter will be used as the basis of the biosensors. The structures will be designed to target into specific cells of an astronaut and be able to monitor health issues such as exposure to radiation. Multiple components can be assembled on the polymers including target directors, analytical devices (such as molecular probes), and reporting agents. The reporting will be accomplished through fluorescence signal monitoring, with the use of multispectral analysis for signal interpretation. These nanosensors could facilitate the success and increase the safety of extended space flight. The design and assembly of these devices has been pioneered at the Center for Biologic Nanotechnology in the University of Michigan. This period, synthesis of the test-bed biosensors continued. Studies were performed on the candidate fluorescent dyes to determine which might be suitable for the biosensor under development. Development continued on producing an artificial capillary bed as a tool for the use in the production of the fluorescence signal monitor. Work was also done on the in vitro multispectral analysis system, which uses the robotic microscope.

Development of an acoustic wave based biosensor for vapor phase detection of small molecules

NASA Astrophysics Data System (ADS)

Stubbs, Desmond

For centuries scientific ingenuity and innovation have been influenced by Mother Nature's perfect design. One of her more elusive designs is that of the sensory olfactory system, an array of highly sensitive receptors responsible for chemical vapor recognition. In the animal kingdom this ability is magnified among canines where ppt (parts per trillion) sensitivity values have been reported. Today, detection dogs are considered an essential part of the US drug and explosives detection schemes. However, growing concerns about their susceptibility to extraneous odors have inspired the development of highly sensitive analytical detection tools or biosensors known as "electronic noses". In general, biosensors are distinguished from chemical sensors in that they use an entity of biological origin (e.g. antibody, cell, enzyme) immobilized onto a surface as the chemically-sensitive film on the device. The colloquial view is that the term "biosensors" refers to devices which detect the presence of entities of biological origin, such as proteins or single-stranded DNA and that this detection must take place in a liquid. Our biosensor utilizes biomolecules, specifically IgG monoclonal antibodies, to achieve molecular recognition of relatively small molecules in the vapor phase.

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

PubMed

Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

PubMed Central

Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains. PMID:24465669

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach.

PubMed

Pei, Zhichao; Saint-Guirons, Julien; Käck, Camilla; Ingemarsson, Björn; Aastrup, Teodor

2012-05-15

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

PubMed

Jia, Kun; Ionescu, Rodica Elena

2016-01-01

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

SPECIES-SPECIFIC DETECTION OF THREE HUMAN-PATHOGENIC MICROSPORIDIAL SPECIES FROM THE GENUS ENCEPHALITOZOON VIA FLUOROGENIC 5' NUCLEASE PCR ASSAYS

EPA Science Inventory

This describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, quantitative, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cunicli and E. intestinalis. The assays utilize species-specific primer sets and a g...

TanA: a fluorogenic probe for thiaminase activity

USGS Publications Warehouse

Zhu, Wanjun; Zajicek, James L.; Tillitt, Donald E.; Glass, Timothy E.

2013-01-01

A fluorogenic thiamine analogue is presented as a fluorescent probe for thiaminase activity. The emission of the fluorophore is quenched by photoinduced electron transfer (PET) to the N-substituted pyridinium portion of the probe. Action of the enzyme releases the free pyridine group causing a substantial increase in fluorescence.

A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates

ERIC Educational Resources Information Center

Johnson, R. Jeremy; Hoops, Geoffrey C.; Savas, Christopher J.; Kartje, Zachary; Lavis, Luke D.

2015-01-01

Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein…

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

PubMed

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Impedance spectroscopy assisted by magnetic nanoparticles as a potential biosensor principle for breast cancer cells in suspension.

PubMed

Silva, Jesús G; Cárdenas, Rey A; Quiróz, Alan R; Sánchez, Virginia; Lozano, Lucila M; Pérez, Nadia M; López, Jaime; Villanueva, Cleva; González, César A

2014-06-01

Breast cancer (BC) is the leading cause of cancer death in women worldwide, with a higher mortality reported in undeveloped countries. Ideal adjuvant therapeutic strategies require the continuous monitoring of patients by regular blood tests to detect circulating cancer cells, in order to determine whether additional treatment is necessary to prevent cancer dissemination. This circumstance requires a non-complex design of tumor cell biosensor in whole blood with feasibility for use in poor regions. In this work we have evaluated an inexpensive and simple technique of relative bioimpedance measurement, assisted by magnetic nanoparticles, as a potential biosensor of BC cells in suspension. Measurements represent the relative impedance changes caused by the magnetic holding of an interphase of tumor cells versus a homogenous condition in the frequency range of 10-100 kHz. The results indicate that use of a magnet to separate tumor cells in suspension, coupled to magnetic nanoparticles, is a feasible technique to fix an interphase of tumor cells in close proximity to gold electrodes. Relative impedance changes were shown to have potential value as a biosensor method for BC cells in whole blood, at frequencies around 20 kHz. Additional studies are warranted with respect to electrode design and sensitivity at micro-scale levels, according to the proposed technique.

A novel biosensor selective for organoarsenicals.

PubMed

Chen, Jian; Zhu, Yong-Guan; Rosen, Barry P

2012-10-01

Organoarsenicals used as herbicides and growth promoters for farm animals are degraded to inorganic arsenic. Available bacterial whole-cell biosensors detect only inorganic arsenic. We report a biosensor selective for the trivalent organoarsenicals methylarsenite and phenylarsenite over inorganic arsenite. This sensor may be useful for detecting degradation of arsenic-containing herbicides and growth promoters.

Recent advances in electrochemical biosensors based on graphene two-dimensional nanomaterials.

PubMed

Song, Yang; Luo, Yanan; Zhu, Chengzhou; Li, He; Du, Dan; Lin, Yuehe

2016-02-15

Graphene as a star among two-dimensional nanomaterials has attracted tremendous research interest in the field of electrochemistry due to their intrinsic properties, including the electronic, optical, and mechanical properties associated with their planar structure. The marriage of graphene and electrochemical biosensors has created many ingenious biosensing strategies for applications in the areas of clinical diagnosis and food safety. This review provides a comprehensive overview of the recent advances in the development of graphene based electrochemical biosensors. Special attention is paid to graphene-based enzyme biosensors, immunosensors, and DNA biosensors. Future perspectives on high-performance graphene-based electrochemical biosensors are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

A special issue on reviews in biomedical applications of nanomaterials, tissue engineering, stem cells, bioimaging, and toxicity.

PubMed

Nalwa, Hari Singh

2014-10-01

This second special issue of the Journal of Biomedical Nanotechnology in a series contains another 30 state-of-the-art reviews focused on the biomedical applications of nanomaterials, biosensors, bone tissue engineering, MRI and bioimaging, single-cell detection, stem cells, endothelial progenitor cells, toxicity and biosafety of nanodrugs, nanoparticle-based new therapeutic approaches for cancer, hepatic and cardiovascular disease.

Device considerations for development of conductance-based biosensors

PubMed Central

Lee, Kangho; Nair, Pradeep R.; Scott, Adina; Alam, Muhammad A.; Janes, David B.

2009-01-01

Design and fabrication of electronic biosensors based on field-effect-transistor (FET) devices require understanding of interactions between semiconductor surfaces and organic biomolecules. From this perspective, we review practical considerations for electronic biosensors with emphasis on molecular passivation effects on FET device characteristics upon immobilization of organic molecules and an electrostatic model for FET-based biosensors. PMID:24753627

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics.

PubMed

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t 1/2  = 620 ms at [GSH] = 1 mM), as well as appropriate K d values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

NASA Astrophysics Data System (ADS)

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Disposable electrochemiluminescent biosensor for lactate determination in saliva.

PubMed

Ballesta Claver, J; Valencia Mirón, M C; Capitán-Vallvey, L F

2009-07-01

An electrochemiluminescence-based disposable biosensor for lactate is characterized. The lactate recognition system is based on lactate oxidase (LOx) and the transduction system consists of luminol. All the needed reagents, luminol, LOx, BSA, electrolyte and buffer have been immobilized by a Methocel membrane placed on the working electrode of the screen-printed electrochemical cell. The measurement of the electrochemiluminescence (ECL) is made possible via a photocounting head when 50 microl of sample is placed into the screen-printed cell with a circular container containing the disposable sensing membrane. The compositions of the membrane and reaction conditions have been optimized to obtain adequate sensitivity. The disposable biosensor responds to lactate after 20 s when two 1 s pulses at 0.5 V are applied to obtain the analytical parameter, the ECL initial rate. The linearized double logarithmic dependence for lactate shows a dynamic range from 10(-5) to 5 x 10(-4) M with a detection limit of 5 x 10(-6) M and a sensor-to-sensor repeatability, as relative standard deviation, RSD, of 3.30% at the medium level of the range. The ECL disposable biosensor was applied to the analysis of lactate in human saliva as an alternative procedure for obtaining the lactate level in a non-invasive way. Interferences coming from components of saliva were studied and eliminated in a simple way that was easy to handle. The procedure was validated for use in human saliva, comparing the results against an enzymatic reference procedure. The proposed method is quick, inexpensive, selective and sensitive and uses conventional ECL instrumentation.

Electrochemical DNA biosensor based on grafting-to mode of terminal deoxynucleoside transferase-mediated extension.

PubMed

Chen, Jinyuan; Liu, Zhoujie; Peng, Huaping; Zheng, Yanjie; Lin, Zhen; Liu, Ailin; Chen, Wei; Lin, Xinhua

2017-12-15

Previously reported electrochemical DNA biosensors based on in-situ polymerization approach reveal that terminal deoxynucleoside transferase (TdTase) has good amplifying performance and promising application in the design of electrochemical DNA biosensor. However, this method, in which the background is significantly affected by the amount of TdTase, suffers from being easy to produce false positive result and poor stability. Herein, we firstly present a novel electrochemical DNA biosensor based on grafting-to mode of TdTase-mediated extension, in which DNA targets are polymerized in homogeneous solution and then hybridized with DNA probes on BSA-based DNA carrier platform. It is surprising to find that the background in the grafting-to mode of TdTase-based electrochemical DNA biosensor have little interference from the employed TdTase. Most importantly, the proposed electrochemical DNA biosensor shows greatly improved detection performance over the in-situ polymerization approach-based electrochemical DNA biosensor. Copyright © 2017 Elsevier B.V. All rights reserved.

Insights into the HyPer biosensor as molecular tool for monitoring cellular antioxidant capacity.

PubMed

Hernández, Helen; Parra, Alejandra; Tobar, Nicolas; Molina, Jessica; Kallens, Violeta; Hidalgo, Miltha; Varela, Diego; Martínez, Jorge; Porras, Omar

2018-06-01

Aerobic metabolism brings inexorably the production of reactive oxygen species (ROS), which are counterbalanced by intrinsic antioxidant defenses avoiding deleterious intracellular effects. Redox balance is the resultant of metabolic functioning under environmental inputs (i.e. diet, pollution) and the activity of intrinsic antioxidant machinery. Monitoring of intracellular hydrogen peroxide has been successfully achieved by redox biosensor advent; however, to track the intrinsic disulfide bond reduction capacity represents a fundamental piece to understand better how redox homeostasis is maintained in living cells. In the present work, we compared the informative value of steady-state measurements and the kinetics of HyPer, a H 2 O 2 -sensitive fluorescent biosensor, targeted at the cytosol, mitochondrion and endoplasmic reticulum. From this set of data, biosensor signal recovery from an oxidized state raised as a suitable parameter to discriminate reducing capacity of a close environment. Biosensor recovery was pH-independent, condition demonstrated by experiments on pH-clamped cells, and sensitive to pharmacological perturbations of enzymatic disulfide reduction. Also, ten human cell lines were characterized according their H 2 O 2 -pulse responses, including their capacity to reduce disulfide bonds evaluated in terms of their migratory capacity. Finally, cellular migration experiments were conducted to study whether migratory efficiency was associated with the disulfide reduction activity. The migration efficiency of each cell type correlates with the rate of signal recovery measured from the oxidized biosensor. In addition, HyPer-expressing cells treated with N-acetyl-cysteine had accelerated recovery rates and major migratory capacities, both reversible effects upon treatment removal. Our data demonstrate that the HyPer signal recovery offers a novel methodological tool to track the cellular impact of redox active biomolecules. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Biosensors for monitoring the isothermal breakdown kinetics of peanut oil heated at 180°C. Comparison with results obtained for extra virgin olive oil.

PubMed

Tomassetti, M; Vecchio, S; Campanella, L; Dragone, R

2013-10-15

The present research was devoted to studying the kinetics of the artificial rancidification of peanut oil (PO) when a sample of this oil was isothermally heated at 180°C in an air stream. The formation of radical species due to heating was evaluated using a radical index whose value was determined using a biosensor method based on a superoxide dismutase (SOD), while the increasing toxicity was monitored using a suitable toxicity measuring probe based on the Clark electrode and immobilized yeast cells. An extra virgin olive oil was isothermally rancidified under the same experimental conditions and the corresponding data were used for the purpose of comparison. Both the so-called "model-fitting" and the classical kinetic methods were applied to the isothermal process biosensor data in order to obtain the kinetic constant rate value at 180°C. Copyright © 2012 Elsevier Ltd. All rights reserved.

Utilizing hyaluronic acid as a versatile platform for fluorescence resonance energy transfer-based glucose sensing.

PubMed

Ge, Minghao; Bai, Pengli; Chen, Mingli; Tian, Jingjing; Hu, Jun; Zhi, Xu; Yin, Huancai; Yin, Jian

2018-03-01

Here, we utilized the ultrasonic emulsification technique to generate hyaluronic acid microspheres incorporating a fluorescence-based glucose biosensor. We synthesized a novel lanthanide ion luminophore based on Eu 3+ . Eu sulfosuccinimidyl dextran (Eu-dextran) and Alexa Fluor 647 sulfosuccinimidyl-ConA (Alexa Fluor 647-ConA) were encapsulated in hyaluronic acid hydrogel to generate microspheres. Glucose sensing was carried out using a fluorescence resonance energy transfer (FRET)-based assay principle. A proportional fluorescence intensity increase was found within a 0.5-10-mM glucose concentration range. The glucose-sensing strategy showed an excellent tolerance for potential interferents. Meanwhile, the fluorescent signal of hyaluronic acid microspheres was very stable after testing for 72 h in glucose solution. Overall, hyaluronic acid microspheres encapsulating sensing biomolecules offer a stable and biocompatible biosensor for a variety of applications including cell culture systems, tissue engineering, detection of blood glucose, etc. Graphical abstract We report an ingenious biosensor encapsulated in hyaluronic acid microspheres for monitoring of glucose. Glucose sensing is carried out using a fluorescence resonance energy transfer-based assay principle with a novel lanthanide ions luminophore. The glucose detection system has excellent biocompatibility and stability for monitoring of glucose.

Simulation of a broadband nano-biosensor based on an onion-like quantum dot-quantum well structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Absalan, H; SalmanOgli, A; Rostami, R

The fluorescence resonance energy transfer is studied between modified quantum-dots and quantum-wells used as a donor and an acceptor. Because of the unique properties of quantum dots, including diverse surface modification flexibility, bio-compatibility, high quantum yields and wide absorption, their use as nano-biosensors and bio-markers used in diagnosis of cancer is suggested. The fluorescence resonance energy transfer is simulated in a quantum dot-quantum well system, where the energy can flow from donor to acceptor. If the energy transfer can be either turned on or off by a specific interaction, such as interaction with any dyes, a molecular binding event ormore » a cleavage reaction, a sensor can be designed (under assumption that the healthy cells have a known effect or unyielding effect on output parameters while cancerous cells, due to their pandemic optical properties, can impact the fluorescence resonance energy transfer parameters). The developed nano-biosensor can operate in a wide range of wavelengths (310 - 760 nm). (laser applications in biology and medicine)« less

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

PubMed

Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

2015-11-01

Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

PubMed

Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

2017-01-18

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

Highly Selective and Sensitive Self-Powered Glucose Sensor Based on Capacitor Circuit.

PubMed

Slaughter, Gymama; Kulkarni, Tanmay

2017-05-03

Enzymatic glucose biosensors are being developed to incorporate nanoscale materials with the biological recognition elements to assist in the rapid and sensitive detection of glucose. Here we present a highly sensitive and selective glucose sensor based on capacitor circuit that is capable of selectively sensing glucose while simultaneously powering a small microelectronic device. Multi-walled carbon nanotubes (MWCNTs) is chemically modified with pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at anode and cathode, respectively, in the biofuel cell arrangement. The input voltage (as low as 0.25 V) from the biofuel cell is converted to a stepped-up power and charged to the capacitor to the voltage of 1.8 V. The frequency of the charge/discharge cycle of the capacitor corresponded to the oxidation of glucose. The biofuel cell structure-based glucose sensor synergizes the advantages of both the glucose biosensor and biofuel cell. In addition, this glucose sensor favored a very high selectivity towards glucose in the presence of competing and non-competing analytes. It exhibited unprecedented sensitivity of 37.66 Hz/mM.cm 2 and a linear range of 1 to 20 mM. This innovative self-powered glucose sensor opens new doors for implementation of biofuel cells and capacitor circuits for medical diagnosis and powering therapeutic devices.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

PubMed Central

Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

2017-01-01

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

ZnO-Based Amperometric Enzyme Biosensors

PubMed Central

Zhao, Zhiwei; Lei, Wei; Zhang, Xiaobing; Wang, Baoping; Jiang, Helong

2010-01-01

Nanostructured ZnO with its unique properties could provide a suitable microenvironment for immobilization of enzymes while retaining their biological activity, and thus lead to an expanded use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance. ZnO-based enzyme electrochemical biosensors are summarized in several tables for an easy overview according to the target biosensing analyte (glucose, hydrogen peroxide, phenol and cholesterol), respectively. Moreover, recent developments in enzyme electrochemical biosensors based on ZnO nanomaterials are reviewed with an emphasis on the fabrications and features of ZnO, approaches for biosensor construction (e.g., modified electrodes and enzyme immobilization) and biosensor performances. PMID:22205864

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

Multienzyme decorated polysaccharide amplified electrogenerated chemiluminescence biosensor for cytosensing and cell surface carbohydrate profiling.

PubMed

Zhang, Ling; Wang, Yangzhong; Tian, Qianqian; Liu, Yang; Li, Jinghong

2017-03-15

A novel ECL biosensor for cytosensing and cell surface carbohydrate expression evaluation was developed, by the integration of the peptide modified interface for highly specific carbohydrate recognition and sodium alginate loaded glucose oxidase as the signal probe with high signal amplification efficiency. A cysteine-terminated peptide self-assembled on the electrode through Au-S bond to construct a functional interface for cell capture, with decent biocompatibility and high affinity for the human breast cancer cell MCF-7. Concanavalin A lectin modified gold nanoparticles specifically recognized the cell surface carbohydrates and were absorbed on the electrode, followed by the immobilization of multiple glucose oxidase conjugated sodium alginate, which could remarkably increase the sensitivity of the biosensor with enhanced catalysis. The as-proposed ECL cytosensor was successfully applied for the detection of the MCF-7 tumor cells, whose glycans on the cell membranes are over-expressed. A low detection limit of 150cellsmL -1 was obtained, with a wide dynamic linear range from 5.0×10 2 to 5.0×10 5 cellsmL -1 . Due to the excellent sensitivity, stability and biocompatibility, the ECL biosensor would be promising in reliable diagnostics of glycan relevant biomarkers for cancer and other diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface engineered biosensors for the early detection of cancer

NASA Astrophysics Data System (ADS)

Islam, Muhymin

Cancer commences in the building block of human body which is cells and in most of the cases remains silent at early stage. Diseases are only expressed at molecular and cellular level at primary stages. Recognition of diseases at this micro and nano level might reduce the mortality rate of cancer significantly. This research work aimed to introduce novel electronic biosensors for for identification of cancer at cellular level. The dissertation study focuses on 1) Label-Free Isolation of Metastatic Tumor Cells Using Filter Based Microfluidic device; 2) Nanotextured Polymer Substrates for Enhanced Cancer Cell Isolation and Cell Growth; 3) Nanotextured Microfluidic Channel for Electrical Profiling and Detection of Tumor Cells from Blood; and 4) Single Biochip for the Detection of Tumor Cells by Electrical Profile and Surface Immobilized Aptamer. Standard silicon processing techniques were followed to fabricate all of the biosensors. Nantoextruing and surface functionalizon were also incorporated to elevate the efficiency of the devices. The first approach aimed to detect cancer cells from blood based on their mechanophysical properties. Cancer cells are larger than blood cells but highly elastic in nature. These cells can squeeze through small microchannels much smaller than their size. The cross sectional area of the microchannels was optimized to isolate tumor cells from blood. Nanotextured polymer substrates, a platform inspired from the natural basement membrane was used to enhance the isolation and growth of tumor cells. Micro reactive ion etching was performed to have better control on features of nantoxtured surfaces and did not require any template. Next, electrical measurement of ionic current was performed across single microchannel to detect tumor cells from blood. Later, nanotexturing enhanced the efficiency of the device by selectively altering the translocation profile of cancer cells. Eventually aptamer functionalized nanotextured polymer surface was integrated with current measurement facilities in a single biochip to discriminate tumor cells from blood with higher efficiency and selectivity. This biochip can be an implemented as a point-of-care device for the early detection of cancer at cellular level.

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution.

PubMed

Shin, Hae Ja

2011-02-01

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.

Ultrasensitive electrochemical detection of tumor cells based on multiple layer CdS quantum dots-functionalized polystyrene microspheres and graphene oxide - polyaniline composite.

PubMed

Wang, Jidong; Wang, Xiaoyu; Tang, Hengshan; Gao, Zehua; He, Shengquan; Li, Jian; Han, Shumin

2018-02-15

In this work, a novel ultrasensitive electrochemical biosensor was developed for the detection of K562 cell by a signal amplification strategy based on multiple layer CdS QDs functionalized polystyrene microspheres(PS) as bioprobe and graphene oxide(GO) -polyaniline(PANI) composite as modified materials of capture electrode. Due to electrostatic force of different charge, CdS QDs were decorated on the surface of PS by PDDA (poly(diallyldimethyl-ammonium chloride)) through a layer-by-layer(LBL) assemble technology, in which the structure of multiple layer CdS QDs increased the detection signal intensity. Moreover, GO-PANI composite not only enhanced the electron transfer rate, but also increased tumor cells load ratio. The resulting electrochemical biosensor was used to detect K562 cells with a lower detection limit of 3 cellsmL -1 (S/N = 3) and a wider linear range from 10 to 1.0 × 10 7 cellsmL -1 . This sensor was also used for mannosyl groups on HeLa cells and Hct116 cells, which showed high specificity and sensitivity. This signal amplification strategy would provide a novel approach for detection, diagnosis and treatment for tumor cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Chitosan-induced Au/Ag nanoalloy dispersed in IL and application in fabricating an ultrasensitive glucose biosensor based on luminol-H₂O₂-Cu²⁺/IL chemiluminescence system.

PubMed

Chaichi, M J; Alijanpour, S O

2014-11-01

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated hydrogen peroxide (H₂O₂) was constructed by one covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell. In following, chitosan-induced Au/Ag nanoparticles dispersed in ion liquid (IL) were synthesised and immobilized on it. Herein, chitosan molecules acted as both the reducing and stabilizing agent for the preparation of NPs and also, as a coupling agent GOD and Au/Ag alloy NPs. In addition to catalyze luminol CL reaction, these NPs offered excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose. The used IL in fabrication of biosensor increased its stability. Also, IL alongside Cu(2+) accelerated enzymatic and CL reaction kinetic, and decreased luminol CL reaction optimum pH to 7.5 which would enable sensitive and precision determination of glucose. Under optimum condition, linear response range of glucose was found to be 1.0 × 10(-6)-7.5 × 10(-3)M, and detection limit was 4.0 × 10(-7)M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been applied satisfactory to analysis of glucose in real serum and urine samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor

NASA Astrophysics Data System (ADS)

Parashurama, Natesh; O'Sullivan, Thomas D.; De La Zerda, Adam; El Kalassi, Pascale; Cho, Seongjae; Liu, Hongguang; Teed, Robert; Levy, Hart; Rosenberg, Jarrett; Cheng, Zhen; Levi, Ofer; Harris, James S.; Gambhir, Sanjiv S.

2012-11-01

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor

PubMed Central

Parashurama, Natesh; O’Sullivan, Thomas D.; De La Zerda, Adam; El Kalassi, Pascale; Cho, Seongjae; Liu, Hongguang; Teed, Robert; Levy, Hart; Rosenberg, Jarrett; Cheng, Zhen; Levi, Ofer; Harris, James S.

2012-01-01

Abstract. Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications. PMID:23123976

Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

PubMed Central

Heitzer, A; Malachowsky, K; Thonnard, J E; Bienkowski, P R; White, D C; Sayler, G S

1994-01-01

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures. PMID:8017932

Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids

PubMed Central

Rocchitta, Gaia; Spanu, Angela; Babudieri, Sergio; Latte, Gavinella; Madeddu, Giordano; Galleri, Grazia; Nuvoli, Susanna; Bagella, Paola; Demartis, Maria Ilaria; Fiore, Vito; Manetti, Roberto; Serra, Pier Andrea

2016-01-01

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented. PMID:27249001

On the challenges of detecting whole Staphylococcus aureus cells with biosensors.

PubMed

Templier, V; Roupioz, Y

2017-11-01

Due to the increasing number of nosocomial infections and multidrug-resistant bacterial strains, Staphylococcus aureus is now a major worldwide concern. Rapid detection and characterization of this bacterium has become an important issue for biomedical applications. Biosensors are increasingly appearing as low-cost, easy-to-operate and fast alternatives for rapid detection. In this review, we will introduce the main characteristics of S. aureus and will focus on the interest of biosensors for a faster detection of whole S. aureus cells. In particular, we will review the most promising strategies in the choice of ligand for the design of selective and efficient biosensors. Their specific characteristics as well as their advantages and/or disadvantages will also be commented. © 2017 The Society for Applied Microbiology.

Characterizing the reliability of a bioMEMS-based cantilever sensor

NASA Astrophysics Data System (ADS)

Bhalerao, Kaustubh D.

2004-12-01

The cantilever-based BioMEMS sensor represents one instance from many competing ideas of biosensor technology based on Micro Electro Mechanical Systems. The advancement of BioMEMS from laboratory-scale experiments to applications in the field will require standardization of their components and manufacturing procedures as well as frameworks to evaluate their performance. Reliability, the likelihood with which a system performs its intended task, is a compact mathematical description of its performance. The mathematical and statistical foundation of systems-reliability has been applied to the cantilever-based BioMEMS sensor. The sensor is designed to detect one aspect of human ovarian cancer, namely the over-expression of the folate receptor surface protein (FR-alpha). Even as the application chosen is clinically motivated, the objective of this study was to demonstrate the underlying systems-based methodology used to design, develop and evaluate the sensor. The framework development can be readily extended to other BioMEMS-based devices for disease detection and will have an impact in the rapidly growing $30 bn industry. The Unified Modeling Language (UML) is a systems-based framework for design and development of object-oriented information systems which has potential application for use in systems designed to interact with biological environments. The UML has been used to abstract and describe the application of the biosensor, to identify key components of the biosensor, and the technology needed to link them together in a coherent manner. The use of the framework is also demonstrated in computation of system reliability from first principles as a function of the structure and materials of the biosensor. The outcomes of applying the systems-based framework to the study are the following: (1) Characterizing the cantilever-based MEMS device for disease (cell) detection. (2) Development of a novel chemical interface between the analyte and the sensor that provides a degree of selectivity towards the disease. (3) Demonstrating the performance and measuring the reliability of the biosensor prototype, and (4) Identification of opportunities in technological development in order to further refine the proposed biosensor. Application of the methodology to design develop and evaluate the reliability of BioMEMS devices will be beneficial in the streamlining the growth of the BioMEMS industry, while providing a decision-support tool in comparing and adopting suitable technologies from available competing options.

A Terrestrial Single Chamber Microbial Fuel Cell-based Biosensor for Biochemical Oxygen Demand of Synthetic Rice Washed Wastewater

PubMed Central

Logroño, Washington; Guambo, Alex; Pérez, Mario; Kadier, Abudukeremu; Recalde, Celso

2016-01-01

Microbial fuel cells represent an innovative technology which allow simultaneous waste treatment, electricity production, and environmental monitoring. This study provides a preliminary investigation of the use of terrestrial Single chamber Microbial Fuel Cells (SMFCs) as biosensors. Three cells were created using Andean soil, each one for monitoring a BOD concentration of synthetic washed rice wastewater (SRWW) of 10, 100, and 200 mg/L for SMFC1, SMFC2 and SMFC3, respectively. The results showed transient, exponential, and steady stages in the SMFCs. The maximum open circuit voltage (OCV) peaks were reached during the elapsed time of the transient stages, according to the tested BOD concentrations. A good linearity between OCV and time was observed in the increasing stage. The average OCV in this stage increased independently of the tested concentrations. SMFC1 required less time than SMFC2 to reach the steady stage, suggesting the BOD concentration is an influencing factor in SMFCs, and SMFC3 did not reach it. The OCV ratios were between 40.6–58.8 mV and 18.2–32.9 mV for SMFC1 and SMFC2. The reproducibility of the SMFCs was observed in four and three cycles for SMFC1 and SMFC2, respectively. The presented SMFCs had a good response and reproducibility as biosensor devices, and could be an alternative for environmental monitoring. PMID:26784197

ADMET biosensors: up-to-date issues and strategies.

PubMed

Fang, Yan; Offenhaeusser, Andrease

2004-12-01

This insight review introduces the new concepts, theories, technology, instruments, frontier issues, and key strategies of ADMET (absorption, distribution, metabolism, elimination, and toxicity) biosensors, from the fermi to the quantum levels. Information about ADMET, originating from one author's invention, a patented pharmacotherapy for rescuing cardio-cerebral vascular stunning and regulating vascular endothelial growth-factor signaling at the post-genomic level, can be detected by a new generation of ADMET biosensor. This is a single-cell/single-molecule field-effect transistor (FET) hybrid system, where single molecules or single cells are assembled at the FET surface in a high density array manner via complementary metal-oxide-semiconductor (CMOS)-compatible technologies. Within a given nanometer distance, ADMET-mediated oxidation-reduction (redox) potentials, electrochemistry responses, and electron transfer processes can be simultaneously and directly probed by the gates of field-effect transistor arrays. The nanometer details of the functional coupling principles and characterization technologies of DNA single-molecule/single-cell FETs, as well as the design of lab-on-a-chip instruments, are indicated. Four frontier issues and key strategies are elucidated in detail. This can lead to innovative technology for high-throughout screening of labs-on-chips to resolve the pharmaceutical industry's current bottleneck via novel, FET-based drug discovery and single-molecule/single-cell screening methods, which can bring about a pharmaceutical industry revolution in the 21st century.

A Terrestrial Single Chamber Microbial Fuel Cell-based Biosensor for Biochemical Oxygen Demand of Synthetic Rice Washed Wastewater.

PubMed

Logroño, Washington; Guambo, Alex; Pérez, Mario; Kadier, Abudukeremu; Recalde, Celso

2016-01-15

Microbial fuel cells represent an innovative technology which allow simultaneous waste treatment, electricity production, and environmental monitoring. This study provides a preliminary investigation of the use of terrestrial Single chamber Microbial Fuel Cells (SMFCs) as biosensors. Three cells were created using Andean soil, each one for monitoring a BOD concentration of synthetic washed rice wastewater (SRWW) of 10, 100, and 200 mg/L for SMFC1, SMFC2 and SMFC3, respectively. The results showed transient, exponential, and steady stages in the SMFCs. The maximum open circuit voltage (OCV) peaks were reached during the elapsed time of the transient stages, according to the tested BOD concentrations. A good linearity between OCV and time was observed in the increasing stage. The average OCV in this stage increased independently of the tested concentrations. SMFC1 required less time than SMFC2 to reach the steady stage, suggesting the BOD concentration is an influencing factor in SMFCs, and SMFC3 did not reach it. The OCV ratios were between 40.6-58.8 mV and 18.2-32.9 mV for SMFC1 and SMFC2. The reproducibility of the SMFCs was observed in four and three cycles for SMFC1 and SMFC2, respectively. The presented SMFCs had a good response and reproducibility as biosensor devices, and could be an alternative for environmental monitoring.

Escherichia coli biosensors for environmental, food industry and biological warfare agent detection

NASA Astrophysics Data System (ADS)

Allil, R. C. S. B.; Werneck, M. M.; da Silva-Neto, J. L.; Miguel, M. A. L.; Rodrigues, D. M. C.; Wandermur, G. L.; Rambauske, D. C.

2013-06-01

This work has the objective to research and develop a plastic optical fiber biosensor based taper and mPOF LPG techniques to detect Escherichia coli by measurements of index of refraction. Generally, cell detection is crucial in microbiological analysis of clinical, food, water or environmental samples. However, methods current employed are time consuming, taking at least 72 hours in order to produce reliable responses as they depend on sample collection and cell culture in controlled conditions. The delay in obtaining the results of the analysis can result in contamination of a great number of consumers. Plastic Optical Fiber (POF) biosensors consist in a viable alternative for rapid and inexpensive scheme for cells detection. A study the sensitivity of these sensors for microbiological detection, fiber Tapers and Long Period Grating (LPG) both in poly-methyl-methacrylate (PMMA) were realized as possible candidates to take part of a biosensor system to detect Escherichia coli in water samples. In this work we adopted the immunocapture technique, which consists of quantifying bacteria in a liquid sample, attract-ing and fixing the bacteria on the surface of the polymer optical fiber, by the antigen-antibody reaction. The results were obtained by optical setup that consists in a side of the fiber a LED coupled to a photodetector through a POF with the taper in the middle of it. On the other side of the POF a photodetector receives this light producting a photocurrent. The output voltage is fed into the microcontroller A/D input port and its output data is sent via USB to a LabView software running in a microcomputer. The results showed the possibility of the POF in biosensor application capable to detect E. coli for environmental and food industry and for detecting and identifying biological-warfare agents using a very rapid response sensor, applicable to field detection prototypes.

Ion-Sensitive Field-Effect Transistor for Biological Sensing

PubMed Central

Lee, Chang-Soo; Kim, Sang Kyu; Kim, Moonil

2009-01-01

In recent years there has been great progress in applying FET-type biosensors for highly sensitive biological detection. Among them, the ISFET (ion-sensitive field-effect transistor) is one of the most intriguing approaches in electrical biosensing technology. Here, we review some of the main advances in this field over the past few years, explore its application prospects, and discuss the main issues, approaches, and challenges, with the aim of stimulating a broader interest in developing ISFET-based biosensors and extending their applications for reliable and sensitive analysis of various biomolecules such as DNA, proteins, enzymes, and cells. PMID:22423205

Biosensor method and system based on feature vector extraction

DOEpatents

Greenbaum, Elias; Rodriguez, Jr., Miguel; Qi, Hairong; Wang, Xiaoling

2013-07-02

A system for biosensor-based detection of toxins includes providing at least one time-dependent control signal generated by a biosensor in a gas or liquid medium, and obtaining a time-dependent biosensor signal from the biosensor in the gas or liquid medium to be monitored or analyzed for the presence of one or more toxins selected from chemical, biological or radiological agents. The time-dependent biosensor signal is processed to obtain a plurality of feature vectors using at least one of amplitude statistics and a time-frequency analysis. At least one parameter relating to toxicity of the gas or liquid medium is then determined from the feature vectors based on reference to the control signal.

Microfluidic biosensing systems. Part I. Development and optimisation of enzymatic chemiluminescent micro-biosensors based on silicon microchips.

PubMed

Davidsson, Richard; Genin, Frédéric; Bengtsson, Martin; Laurell, Thomas; Emnéus, Jenny

2004-10-01

Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (micro-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells. Part I of this study focuses on the development and optimisation of the micro-biosensors in a microfluidic sequential injection analysis (microSIA) system. Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips. The hydrogen peroxide produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol (PIP). All steps in the microSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled. The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor. It was found that all estimated single factors and the highest order of interaction were significant. The optimum was found at 250 microM luminol and 150 microM PIP at a flow rate of 18 microl min(-1), the latter as a compromise between signal intensity and analysis time. Using the optimised system settings one sample was processed within 5 min. Two different immobilisation chemistries were investigated for both micro-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation. GOX-HRP micro-biosensors responded linear in a log-log format within the range 10-1000 microM glucose. Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive. The AOX-HRP micro-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive. Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.

An open-pattern droplet-in-oil planar array for single cell analysis based on sequential inkjet printing technology.

PubMed

Wang, Chenyu; Liu, Wenwen; Tan, Manqing; Sun, Hongbo; Yu, Yude

2017-07-01

Cellular heterogeneity represents a fundamental principle of cell biology for which a readily available single-cell research tool is urgently required. Here, we present a novel method combining cell-sized well arrays with sequential inkjet printing. Briefly, K562 cells with phosphate buffer saline buffer were captured at high efficiency (74.5%) in a cell-sized well as a "primary droplet" and sealed using fluorinated oil. Then, piezoelectric inkjet printing technology was adapted to precisely inject the cell lysis buffer and the fluorogenic substrate, fluorescein-di-β-D-galactopyranoside, as a "secondary droplet" to penetrate the sealing oil and fuse with the "primary droplet." We thereby successfully measured the intracellular β-galactosidase activity of K562 cells at the single-cell level. Our method allows, for the first time, the ability to simultaneously accommodate the high occupancy rate of single cells and sequential addition of reagents while retaining an open structure. We believe that the feasibility and flexibility of our method will enhance its use as a universal single-cell research tool as well as accelerate the adoption of inkjet printing in the study of cellular heterogeneity.

A novel biosensor array with a wheel-like pattern for glucose, lactate and choline based on electrochemiluminescence imaging.

PubMed

Zhou, Zhenyu; Xu, Linru; Wu, Suozhu; Su, Bin

2014-10-07

Electrochemiluminescence (ECL) imaging provides a superior approach to achieve array detection because of its ability for ultrasensitive multiplex analysis. In this paper, we reported a novel ECL imaging biosensor array modified with an enzyme/carbon nanotubes/chitosan composite film for the determination of glucose, choline and lactate. The biosensor array was constructed by integrating a patterned indium tin oxide (ITO) glass plate with six perforated poly(dimethylsiloxane) (PDMS) covers. ECL is generated by the electrochemical reaction between luminol and hydrogen peroxide that is produced by the enzyme catalysed oxidation of different substrates with molecular oxygen, and ECL images were captured by a charge-coupled device (CCD) camera. The separated electrochemical micro-cells enabled simultaneous assay of six samples at different concentrations. From the established calibration curves, the detection limits were 14 μM for glucose, 40 μM for lactate and 97 μM for choline, respectively. Moreover, multicomponent assays and cross reactivity were also studied, both of which were satisfied for the analysis. This biosensing platform based on ECL imaging shows many distinct advantages, including miniaturization, low cost, and multi-functionalization. We believe that this novel ECL imaging biosensor platform will have potential applications in clinical diagnostics, medicine and food inspection.

AuNP-RF sensor: An innovative application of RF technology for sensing pathogens electrically in liquids (SPEL) within the food supply chain.

PubMed

Matta, Leann Lerie; Karuppuswami, Saranraj; Chahal, Premjeet; Alocilja, Evangelyn C

2018-07-15

Rapid detection techniques of pathogenic bacteria in the liquid food supply chain are of significant research interest due to their pivotal role in preventing foodborne outbreaks, and in maintaining high standards of public health and safety. Milk and dairy products are of particular interest due to their widespread consumption across the globe. In this paper, a biosensor for detecting pathogenic bacteria in milk using dextrin-capped gold nanoparticles (d-AuNP) as labels decoded at microwave frequencies is presented. The SPEL (sensing pathogens electrically in liquids) biosensor consists of a 3D printed vial and uses an RF reader and an RFID (radio-frequency identification) compatible Split Ring Resonator (SRR) based tag. The SPEL biosensor is capable of detecting bacteria at 5 log CFU/mL within 75 min, with the possibility of testing multiple concurrent samples. Detection is based on impedance loading of SRR by d-AuNP bound to pathogenic bacteria. Spectrophotometry, along with carbohydrate-functionalized magnetic nanoparticle (MNP) cell capture, is used to verify the sensitivity of the SPEL biosensor with respect to d-AuNP presence. The proof-of-concept device, along with challenges and opportunities for commercialization, are also outlined. Copyright © 2018. Published by Elsevier B.V.

Azobenzene-caged sulforhodamine dyes: a novel class of ‘turn-on’ reactive probes for hypoxic tumor cell imaging

NASA Astrophysics Data System (ADS)

Chevalier, Arnaud; Piao, Wen; Hanaoka, Kenjiro; Nagano, Tetsuo; Renard, Pierre-Yves; Romieu, Anthony

2015-12-01

New sulforhodamine-based fluorescent ‘turn-on’ probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble ‘azobenzene-caged’ fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH 2 ) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH 2 . This valuable fluorogenic response was obtained for the three ‘smart’ probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH 2 -Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

PubMed

Dündar Yenilmez, Ebru; Kökbaş, Umut; Kartlaşmış, Kezban; Kayrın, Levent; Tuli, Abdullah

2018-01-01

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility.

Environmental Stability of Plasmonic Biosensors Based on Natural versus Artificial Antibody.

PubMed

Luan, Jingyi; Xu, Ting; Cashin, John; Morrissey, Jeremiah J; Kharasch, Evan D; Singamaneni, Srikanth

2018-06-13

Plasmonic biosensors based on the refractive index sensitivity of localized surface plasmon resonance (LSPR) are considered to be highly promising for on-chip and point-of-care biodiagnostics. However, most of the current plasmonic biosensors employ natural antibodies as biorecognition elements, which can easily lose their biorecognition ability upon exposure to environmental stressors (e.g., temperature and humidity). Plasmonic biosensors relying on molecular imprints as recognition elements (artificial antibodies) are hypothesized to be an attractive alternative for applications in resource-limited settings due to their excellent thermal, chemical, and environmental stability. In this work, we provide a comprehensive comparison of the stability of plasmonic biosensors based on natural and artificial antibodies. Although the natural antibody-based plasmonic biosensors exhibit superior sensitivity, their stability (temporal, thermal, and chemical) was found to be vastly inferior to those based on artificial antibodies. Our results convincingly demonstrate that these novel classes of artificial antibody-based plasmonic biosensors are highly attractive for point-of-care and resource-limited conditions where tight control over transport, storage, and handling conditions is not possible.

Self-assembly of glucose oxidase on reduced graphene oxide-magnetic nanoparticles nanocomposite-based direct electrochemistry for reagentless glucose biosensor.

PubMed

Pakapongpan, Saithip; Poo-Arporn, Rungtiva P

2017-07-01

A novel approach of the immobilization of a highly selective and stable glucose biosensor based on direct electrochemistry was fabricated by a self-assembly of glucose oxidase (GOD) on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) modified on a magnetic screen-printed electrode (MSPE). The RGO-Fe 3 O 4 nanocomposite has remarkable enhancement in large surface areas, is favorable environment for enzyme immobilization, facilitates electron transfer between enzymes and electrode surfaces and possesses superparamagnetism property. The morphology and electrochemical properties of RGO-Fe 3 O 4 /GOD were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, cyclic voltammetry (CV) and amperometry. The modified electrode was a fast, direct electron transfer with an apparent electron transfer rate constant (k s ) of 13.78s -1 . The proposed biosensor showed fast amperometric response (3s) to glucose with a wide linear range from 0.05 to 1mM, a low detection limit of 0.1μM at a signal to noise ratio of 3 (S/N=3) and good sensitivity (5.9μA/mM). The resulting biosensor has high stability, good reproducibility, excellent selectivity and successfully applied detection potential at -0.45V. This mediatorless glucose sensing used the advantages of covalent bonding and self-assembly as a new approach for immobilizing enzymes without any binder. It would be worth noting that it opens a new avenue for fabricating excellent electrochemical biosensors. This is a new approach that reporting the immobilization of glucose oxidase on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) by electrostatic interaction and modified screen printed electrode. We propose the reagentless with fabrication method without binder and adhesive agents for immobilized enzyme. Fe 3 O 4 NPs increasing surface area to enhance the immobilization and prevent the leaching of enzymes at electrode surfaces by magnetic stickers which is improve the stability of the biosensor. Based on this synthesis technique, it is a good new strategy and simple used to fabrication of third-generation glucose biosensor and this nanocomposite could be used as a platform for disposable biosensor and biofuel cell applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

PubMed

Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

2016-01-15

Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Biosensor for remote monitoring of airborne toxins

NASA Astrophysics Data System (ADS)

Knopf, George K.; Bassi, Amarjeet S.; Singh, Shikha; Macleod, Roslyn

1999-12-01

The rapid detection of toxic contaminants released into the air by chemical processing facilities is a high priority for many manufacturers. This paper describes a novel biosensor for the remote monitoring of toxic sites. The proposed biosensor is a measurement system that employs immobilized luminescent Vibrio fisheri bacteria to detect airborne contaminants. The presence of toxic chemicals will lead to a detectable decrease in the intensity of light produced by the bacteria. Both cellular and environmental factors control the bioluminescence of these bacteria. Important design factors are the appropriate cell growth media, environmental toxicity, oxygen and cell concentrations. The luminescent bacteria are immobilized on polyvinyl alcohol (PVA) gels and placed inside a specially constructed, miniature flow cell which houses a transducer, power source, and transmitter to convert the light signal information into radio frequencies that are picked up by a receiver at a remote location. The biosensor prototype is designed to function either as a single unit mounted on an exploratory robot or numerous units spatially distributed throughout a contaminated environment for remote sensing applications.

A chromogenic and fluorogenic rhodol-based chemosensor for hydrazine detection and its application in live cell bioimaging

NASA Astrophysics Data System (ADS)

Tiensomjitr, Khomsan; Noorat, Rattha; Chomngam, Sinchai; Wechakorn, Kanokorn; Prabpai, Samran; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

2018-04-01

A rhodol-based fluorescent probe has been developed as a selective hydrazine chemosensor using levulinate as a recognition site. The rhodol levulinate probe (RL) demonstrated high selectivity and sensitivity toward hydrazine among other molecules. The chromogenic response of RL solution to hydrazine from colorless to pink could be readily observed by the naked eye, while strong fluorescence emission could be monitored upon excitation at 525 nm. The detection process occurred via a ring-opening process of the spirolactone initiated by hydrazinolysis, triggering the fluorescence emission with a 53-fold enhancement. The probe rapidly reacted with hydrazine in aqueous medium with the detection limit of 26 nM (0.83 ppb), lower than the threshold limit value (TLV) of 10 ppb suggested by the U.S. Environmental Protection Agency. Furthermore, RL-impregnated paper strips could detect hydrazine vapor. For biological applicability of RL, its membrane-permeable property led to bioimaging of hydrazine in live HepG2 cells by confocal fluorescence microscopy.

Amperometric biosensor based on a single antibody of dual function for rapid detection of Streptococcus agalactiae.

PubMed

Vásquez, Gersson; Rey, Alba; Rivera, Camilo; Iregui, Carlos; Orozco, Jahir

2017-01-15

Pathogenic bacteria are responsible for several diseases in humans and in a variety of hosts. Detection of pathogenic bacteria is imperative to avoid and/or fight their potential harmful effects. This work reports on the first amperometric biosensor for the rapid detection of Streptococcus agalactiae (S. agalactiae). The biosensor relies on a single biotinylated antibody that immobilizes the bacteria on a screen-printed carbon electrode while is further linked to a streptavidin-conjugated HRP reporter. The biotinylated antibody provides selectivity to the biosensor whereas serves as an anchoring point to the reporter for further amplification of the electrochemical signal. The resultant immunosensor is simple, responds rapidly, and allows for the selective and highly sensitive quantification of S. agalactiae cells in a concentration range of 10 1 -10 7 CFUml -1 , with a detection limit of 10CFUml -1 . The approach not only enables a rapid detection and quantification of S. agalactiae in environmental samples but also opens up new opportunities for the simple fabrication of electrochemical immunosensors for different target pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of Pt-Au-Graphene-Carbon Nanotube Composite for Fuel Cells and Biosensors Applications

DTIC Science & Technology

2011-02-11

1 Project Title:- Development of Pt-Au- Graphene -Carbon nanotube composites for fuel cells and biosensors applications Objectives:- This...project addresses the architectures needed for the processing of Pt-Au- graphene -carbon nanotube (Pt-Au/f-G/f-CNT) nanocomposites and aims at the...cells:- Graphene and nitrogen doped graphene as catalyst support materials:- Graphene and nitrogen doped graphene have been used as a catalyst

Molecular Modulation of Inhibitors of Apoptosis as a Novel Approach for Radiosensitization of Human Prostate Cancer

DTIC Science & Technology

2006-11-01

6 well plate at the concentration of 2X105/ml, then exposed by SH130 (10 uM) with or without the pan-caspase inhibitor zVAD (2.5 uM) ( Biovision ...treated with SH- 130 and radiation. DU-145 cell were treated as described in Figure 7. Cells were lysed by the lysis buffer ( Biovision ) as indicated...Total extracted proteins were determined and normalized, and then reacted with fluorogenic substrates ( Biovision , DEVD-AFC and LEHD- AFC for Caspase

Spiroguanidine rhodamines as fluorogenic probes for lysophosphatidic acid

PubMed Central

Wang, Lei; Sibrian-Vazquez, Martha; Escobedo, Jorge O.; Wang, Jialu; Moore, Richard G.

2015-01-01

Direct determination of total lysophosphatidic acid (LPA) was accomplished using newly developed spiroguanidines derived from rhodamine B as universal fluorogenic probes. Optimum conditions for the quantitative analysis of total LPA were investigated. The linear range for the determination of total LPA is up to 5 μM with a limit of detection of 0.512 μM. PMID:25516957

Nanoscale hybrid systems based on carbon nanotubes for biological sensing and control

PubMed Central

Cho, Youngtak; Shin, Narae; Kim, Daesan; Park, Jae Yeol

2017-01-01

This paper provides a concise review on the recent development of nanoscale hybrid systems based on carbon nanotubes (CNTs) for biological sensing and control. CNT-based hybrid systems have been intensively studied for versatile applications of biological interfaces such as sensing, cell therapy and tissue regeneration. Recent advances in nanobiotechnology not only enable the fabrication of highly sensitive biosensors at nanoscale but also allow the applications in the controls of cell growth and differentiation. This review describes the fabrication methods of such CNT-based hybrid systems and their applications in biosensing and cell controls. PMID:28188158

A Green Microbial Fuel Cell-Based Biosensor for In Situ Chromium (VI) Measurement in Electroplating Wastewater.

PubMed

Wu, Li-Chun; Tsai, Teh-Hua; Liu, Man-Hai; Kuo, Jui-Ling; Chang, Yung-Chu; Chung, Ying-Chien

2017-10-27

The extensive use of Cr(VI) in many industries and the disposal of Cr(VI)-containing wastes have resulted in Cr(VI)-induced environmental contamination. Cr(VI) compounds are associated with increased cancer risks; hence, the detection of toxic Cr(VI) compounds is crucial. Various methods have been developed for Cr(VI) measurement, but they are often conducted offsite and cannot provide real-time toxicity monitoring. A microbial fuel cell (MFC) is an eco-friendly and self-sustaining device that has great potential as a biosensor for in situ Cr(VI) measurement, especially for wastewater generated from different electroplating units. In this study, Exiguobacterium aestuarii YC211, a facultatively anaerobic, Cr(VI)-reducing, salt-tolerant, and exoelectrogenic bacterium, was isolated and inoculated into an MFC to evaluate its feasibility as a Cr(VI) biosensor. The Cr(VI) removal efficiency of E. aestuarii YC211 was not affected by the surrounding environment (pH 5-9, 20-35 °C, coexisting ions, and salinity of 0-15 g/L). The maximum power density of the MFC biosensor was 98.3 ± 1.5 mW/m² at 1500 Ω. A good linear relationship ( r ² = 0.997) was observed between the Cr(VI) concentration (2.5-60 mg/L) and the voltage output. The developed MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in the actual electroplating wastewater that is generated from different electroplating units within 30 min with low deviations (-6.1% to 2.2%). After treating the actual electroplating wastewater with the MFC, the predominant family in the biofilm was found to be Bacillaceae (95.3%) and was further identified as the originally inoculated E. aestuarii YC211 by next generation sequencing (NGS). Thus, the MFC biosensor can measure Cr(VI) concentrations in situ in the effluents from different electroplating units, and it can potentially help in preventing the violation of effluent regulations.

A Green Microbial Fuel Cell-Based Biosensor for In Situ Chromium (VI) Measurement in Electroplating Wastewater

PubMed Central

Wu, Li-Chun; Tsai, Teh-Hua; Liu, Man-Hai; Kuo, Jui-Ling; Chang, Yung-Chu

2017-01-01

The extensive use of Cr(VI) in many industries and the disposal of Cr(VI)-containing wastes have resulted in Cr(VI)-induced environmental contamination. Cr(VI) compounds are associated with increased cancer risks; hence, the detection of toxic Cr(VI) compounds is crucial. Various methods have been developed for Cr(VI) measurement, but they are often conducted offsite and cannot provide real-time toxicity monitoring. A microbial fuel cell (MFC) is an eco-friendly and self-sustaining device that has great potential as a biosensor for in situ Cr(VI) measurement, especially for wastewater generated from different electroplating units. In this study, Exiguobacterium aestuarii YC211, a facultatively anaerobic, Cr(VI)-reducing, salt-tolerant, and exoelectrogenic bacterium, was isolated and inoculated into an MFC to evaluate its feasibility as a Cr(VI) biosensor. The Cr(VI) removal efficiency of E. aestuarii YC211 was not affected by the surrounding environment (pH 5–9, 20–35 °C, coexisting ions, and salinity of 0–15 g/L). The maximum power density of the MFC biosensor was 98.3 ± 1.5 mW/m2 at 1500 Ω. A good linear relationship (r2 = 0.997) was observed between the Cr(VI) concentration (2.5–60 mg/L) and the voltage output. The developed MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in the actual electroplating wastewater that is generated from different electroplating units within 30 min with low deviations (−6.1% to 2.2%). After treating the actual electroplating wastewater with the MFC, the predominant family in the biofilm was found to be Bacillaceae (95.3%) and was further identified as the originally inoculated E. aestuarii YC211 by next generation sequencing (NGS). Thus, the MFC biosensor can measure Cr(VI) concentrations in situ in the effluents from different electroplating units, and it can potentially help in preventing the violation of effluent regulations. PMID:29076985

Self-assembly of an upconverting nanocomplex and its application to turn-on detection of metalloproteinase-9 in living cells

NASA Astrophysics Data System (ADS)

Nguyen, Phuong-Diem; Thanh Cong, Vu; Baek, Changyoon; Min, Junhong

2016-10-01

Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml-1. Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.

Membrane-based electrochemical nanobiosensor for Escherichia coli detection and analysis of cells viability.

PubMed

Cheng, Ming Soon; Lau, Suk Hiang; Chow, Vincent T; Toh, Chee-Seng

2011-08-01

A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL(-1) (R(2) = 0.999) over a wide linear working range of 10 to 10(6) cfu mL(-1). It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells' enzyme activity using the same redox probe in the analysis solution, in the presence of glucose.

A 3D paper-based enzymatic fuel cell for self-powered, low-cost glucose monitoring.

PubMed

Fischer, Christopher; Fraiwan, Arwa; Choi, Seokheun

2016-05-15

In this work, we demonstrate a novel low-cost, self-powered paper-based biosensor for glucose monitoring. The device operating mechanism is based on a glucose/oxygen enzymatic fuel cell using an electrochemical energy conversion as a transducing element for glucose monitoring. The self-powered glucose biosensor features (i) a 3D origami paper-based structure for easy system integration onto paper, (ii) an air-cathode on paper for low-cost production and easy operation, and (iii) a screen printed chitosan/glucose oxidase anode for stable current generation as an analytical signal for glucose monitoring. The sensor showed a linear range of output current at 1-5mM glucose (R(2)=0.996) with a sensitivity of 0.02 µA mM(-1). The advantages offered by such a device, including a low cost, lack of external power sources/sophisticated external transducers, and the capacity to rapidly generate reliable results, are well suited for the clinical and social settings of the developing world. Copyright © 2015 Elsevier B.V. All rights reserved.

Advances in nano-scaled biosensors for biomedical applications.

PubMed

Wang, Jianling; Chen, Guihua; Jiang, Hui; Li, Zhiyong; Wang, Xuemei

2013-08-21

Recently, a growing amount of attention has been focused on the utility of biosensors for biomedical applications. Combined with nanomaterials and nanostructures, nano-scaled biosensors are installed for biomedical applications, such as pathogenic bacteria monitoring, virus recognition, disease biomarker detection, among others. These nano-biosensors offer a number of advantages and in many respects are ideally suited to biomedical applications, which could be made as extremely flexible devices, allowing biomedical analysis with speediness, excellent selectivity and high sensitivity. This minireview discusses the literature published in the latest years on the advances in biomedical applications of nano-scaled biosensors for disease bio-marking and detection, especially in bio-imaging and the diagnosis of pathological cells and viruses, monitoring pathogenic bacteria, thus providing insight into the future prospects of biosensors in relevant clinical applications.

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

PubMed

Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

2017-04-17

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

Highly ordered mesoporous carbons as electrode material for the construction of electrochemical dehydrogenase- and oxidase-based biosensors.

PubMed

Zhou, Ming; Shang, Li; Li, Bingling; Huang, Lijian; Dong, Shaojun

2008-11-15

In this work, the excellent catalytic activity of highly ordered mesoporous carbons (OMCs) to the electrooxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) was described for the construction of electrochemical alcohol dehydrogenase (ADH) and glucose oxidase (GOD)-based biosensors. The high density of edge-plane-like defective sites and high specific surface area of OMCs could be responsible for the electrocatalytic behavior at OMCs modified glassy carbon electrode (OMCs/GE), which induced a substantial decrease in the overpotential of NADH and H(2)O(2) oxidation reaction compared to carbon nanotubes modified glassy carbon electrode (CNTs/GE). Such ability of OMCs permits effective low-potential amperometric biosensing of ethanol and glucose, respectively, at Nafion/ADH-OMCs/GE and Nafion/GOD-OMCs/GE. Especially, as an amperometric glucose biosensor, Nafion/GOD-OMCs/GE showed large determination range (500-15,000 micromoll(-1)), high sensitivity (0.053 nA micromol(-1)), fast (9+/-1s) and stable response (amperometric response retained 90% of the initial activity after 10h stirring of 2 mmoll(-1) glucose solution) to glucose as well as the effective discrimination to the possible interferences, which may make it to readily satisfy the need for the routine clinical diagnosis of diabetes. By comparing the electrochemical performance of OMCs with that of CNTs as electrode material for the construction of ADH- and GOD-biosensors in this work, we reveal that OMCs could be a favorable and promising carbon electrode material for constructing other electrochemical dehydrogenase- and oxidase-based biosensors, which may have wide potential applications in biocatalysis, bioelectronics and biofuel cells.

DNA nanotechnology-enabled biosensors.

PubMed

Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

2016-02-15

Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Biosensor method and system based on feature vector extraction

DOEpatents

Greenbaum, Elias [Knoxville, TN; Rodriguez, Jr., Miguel; Qi, Hairong [Knoxville, TN; Wang, Xiaoling [San Jose, CA

2012-04-17

A method of biosensor-based detection of toxins comprises the steps of providing at least one time-dependent control signal generated by a biosensor in a gas or liquid medium, and obtaining a time-dependent biosensor signal from the biosensor in the gas or liquid medium to be monitored or analyzed for the presence of one or more toxins selected from chemical, biological or radiological agents. The time-dependent biosensor signal is processed to obtain a plurality of feature vectors using at least one of amplitude statistics and a time-frequency analysis. At least one parameter relating to toxicity of the gas or liquid medium is then determined from the feature vectors based on reference to the control signal.

A graphene-based physiometer array for the analysis of single biological cells

PubMed Central

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-01-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer – a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene – in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation. PMID:25359450

Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

NASA Astrophysics Data System (ADS)

Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

2016-10-01

C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1-3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites.

Magneto-impedance based detection of magnetically labeled cancer cells and bio-proteins

NASA Astrophysics Data System (ADS)

Devkota, J.; Howell, M.; Mohapatra, S.; Nhung, T. H.; Mukherjee, P.; Srikanth, H.; Phan, M. H.

2015-03-01

A magnetic biosensor with enhanced sensitivity and immobilized magnetic markers is essential for a reliable analysis of the presence of a biological entity in a fluid. Based on conventional approaches, however, it is quite challenging to create such a sensor. We report on a novel magnetic biosensor using the magneto-impedance (MI) effect of a Co-based amorphous ribbon with a microhole-patterned surface that fulfils these requirements. The sensor probe was fabricated by patterning four microholes, each of diameter 2 μm and depth 2 μm, on the ribbon surface using FIB lithography. The magnetically labeled Luis Lung Carcinoma (LLC) cancer cells and Bovine serum albumin (BSA) proteins were drop-casted on the ribbon surface, and MI was measured over 0.1 - 10 MHz frequency range. As the analytes were trapped into the microholes, their physical motion was minimized and interaction among the magnetic fields was strengthened, thus yielding a more reliable and sensitive detection of the biological entities. The presence of magnetically labeled LLC cells (8.25x105 cells/ml, 10 μl) and BSA proteins (2x1011 particles/ml, 10 μl) were found to result in a ~ 2% change in MI with respect to the reference signal.

Detection of Antibiotics and Evaluation of Antibacterial Activity with Screen-Printed Electrodes

PubMed Central

Titoiu, Ana Maria; Marty, Jean-Louis

2018-01-01

This review provides a brief overview of the fabrication and properties of screen-printed electrodes and details the different opportunities to apply them for the detection of antibiotics, detection of bacteria and antibiotic susceptibility. Among the alternative approaches to costly chromatographic or ELISA methods for antibiotics detection and to lengthy culture methods for bacteria detection, electrochemical biosensors based on screen-printed electrodes present some distinctive advantages. Chemical and (bio)sensors for the detection of antibiotics and assays coupling detection with screen-printed electrodes with immunomagnetic separation are described. With regards to detection of bacteria, the emphasis is placed on applications targeting viable bacterial cells. While the electrochemical sensors and biosensors face many challenges before replacing standard analysis methods, the potential of screen-printed electrodes is increasingly exploited and more applications are anticipated to advance towards commercial analytical tools. PMID:29562637

Research on optical biosensor with up-converting phosphor marker

NASA Astrophysics Data System (ADS)

Zhao, Yongkai; Zhou, Lei; Wang, Jing; Huang, Lihua; Yan, Zhongqiang; Huang, Huijie; Yang, Ruifu; Liu, Lei; Ren, Bingqiang; Wang, Xiangzhao

2006-08-01

An optical biosensor with up-converting phosphor (UCP) marker is developed for the sensitive rapid immunoassay to the specific biomolecule. UCP can emit visible light when excited by infrared light. Through detecting and analyzing the content of UCP particles on the test strip after immunoreaction, the concentration of target analyte in the sample can be obtained. The detection sensitivity to plague IgG is better than 5 ng/ml; to plague FI-Ab is better than 100 pg/ml; to plague Yersinia pestis cell is better than 3*10^(4) CFU/ml. Good linear response characteristics and an excellent correlation (R2>=0.95) have been verified by quantitative detection results. In the practical application, detection results to 167 analytic samples have an excellent consistency with those obtained by reverse hemagglutination test. The up-converting phosphor technology (UPT) based biosensor has stable, reliable, and sensitive performances. It can meet the need of various bioassay applications.

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression.

PubMed

Zager, Valerija; Cemazar, Maja; Hreljac, Irena; Lah, Tamara T; Sersa, Gregor; Filipic, Metka

2010-03-01

Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

Portable microsystem integrates multifunctional dielectrophoresis manipulations and a surface stress biosensor to detect red blood cells for hemolytic anemia.

PubMed

Sang, Shengbo; Feng, Qiliang; Jian, Aoqun; Li, Huiming; Ji, Jianlong; Duan, Qianqian; Zhang, Wendong; Wang, Tao

2016-09-20

Hemolytic anemia intensity has been suggested as a vital factor for the growth of certain clinical complications of sickle cell disease. However, there is no effective and rapid diagnostic method. As a powerful platform for bio-particles testing, biosensors integrated with microfluidics offer great potential for a new generation of portable point of care systems. In this paper, we describe a novel portable microsystem consisting of a multifunctional dielectrophoresis manipulations (MDM) device and a surface stress biosensor to separate and detect red blood cells (RBCs) for diagnosis of hemolytic anemia. The peripheral circuit to power the interdigitated electrode array of the MDM device and the surface stress biosensor test platform were integrated into a portable signal system. The MDM includes a preparing region, a focusing region, and a sorting region. Simulation and experimental results show the RBCs trajectories when they are subjected to the positive DEP force, allowing the successful sorting of living/dead RBCs. Separated RBCs are then transported to the biosensor and the capacitance values resulting from the variation of surface stress were measured. The diagnosis of hemolytic anemia can be realized by detecting RBCs and the portable microsystem provides the assessment to the hemolytic anemia patient.

Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Recent advances in transition-metal dichalcogenides based electrochemical biosensors: A review.

PubMed

Wang, Yi-Han; Huang, Ke-Jing; Wu, Xu

2017-11-15

Layered transition metal dichalcogenides (TMDCs) comprise a category of two-dimensional (2D) materials that offer exciting properties, including large surface area, metallic and semi-conducting electrical capabilities, and intercalatable morphologies. Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. TMDCs nanomaterials have been widely applied in various electrochemical biosensors with high sensitivity and selectivity. The marriage of TMDCs and electrochemical biosensors has created many productive sensing strategies for applications in the areas of clinical diagnosis, environmental monitoring and food safety. In recent years, an increasing number of TMDCs-based electrochemical biosensors are reported, suggesting TMDCs offers new possibilities of improving the performance of electrochemical biosensors. This review summarizes recent advances in electrochemical biosensors based on TMDCs for detection of various inorganic and organic analytes in the last five years, including glucose, proteins, DNA, heavy metal, etc. In addition, we also point out the challenges and future perspectives related to the material design and development of TMDCs-based electrochemical biosensors. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucose biosensor based on the immobilization of glucose oxidase on electrochemically synthesized polypyrrole-poly(vinyl sulphonate) composite film by cross-linking with glutaraldehyde.

PubMed

Colak, Ozlem; Yaşar, Ahmet; Cete, Servet; Arslan, Fatma

2012-10-01

In this study, a novel amperometric glucose biosensor was developed by immobilizing glucose oxidase (GOX) by cross-linking via glutaraldehyde on electrochemically polymerized polypyrrole-poly(vinyl sulphonate) (PPy-PVS) films on the surface of a platinum (Pt) electrode. Electropolymerization of pyrrole and poly(vinyl sulphonate) on the Pt surface was carried out with an electrochemical cell containing pyrrole and poly(vinyl sulphonate) by cyclic voltammetry between -1.0 and + 2.0 V (vs.Ag/AgCl) at a scan rate of 50 mV/s upon the Pt electrode. The amperometric determination was based on the electrochemical detection of H(2)O(2) generated in enzymatic reaction of glucose. Determination of glucose was carried out by the oxidation of enzymatically produced H(2)O(2) at 0.4 V vs. Ag/AgCl. The effects of pH and temperature were investigated and optimum parameters were found to be 7.5 and 65°C, respectively. The effect of working potential was investigated and optimum potential was determined to be 0.4 V. The operational stability of the enzyme electrode was also studied. The response of the PPy/PVS-GOX glucose biosensor exhibited good reproducibility with a relative standard deviation (RSD) of 2.48%. The glucose biosensor retained 63% of initial activity after 93 days when stored in 0.1 M phosphate buffer solution of pH 7.5 at 4°C. With the low operating potential, the biosensor demonstrated little interference from the possible interferants.

Electrochemical biosensing of galactose based on carbon materials: graphene versus multi-walled carbon nanotubes.

PubMed

Dalkıran, Berna; Erden, Pınar Esra; Kılıç, Esma

2016-06-01

In this study, two enzyme electrodes based on graphene (GR), Co3O4 nanoparticles and chitosan (CS) or multi-walled carbon nanotubes (MWCNTs), Co3O4 nanoparticles, and CS, were fabricated as novel biosensing platforms for galactose determination, and their performances were compared. Galactose oxidase (GaOx) was immobilized onto the electrode surfaces by crosslinking with glutaraldehyde. Optimum working conditions of the biosensors were investigated and the analytical performance of the biosensors was compared with respect to detection limit, linearity, repeatability, and stability. The MWCNTs-based galactose biosensor provided about 1.6-fold higher sensitivity than its graphene counterpart. Moreover, the linear working range and detection limit of the MWCNTs-based galactose biosensor was superior to the graphene-modified biosensor. The successful application of the purposed biosensors for galactose biosensing in human serum samples was also investigated.

Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

NASA Astrophysics Data System (ADS)

Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

2013-11-01

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

Development of a biosensor for caffeine.

PubMed

Babu, V R Sarath; Patra, S; Karanth, N G; Kumar, M A; Thakur, M S

2007-01-23

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL(-1). With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible. Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30+/-2 degrees C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis.

Biocompatible electrochemiluminescent biosensor for choline based on enzyme/titanate nanotubes/chitosan composite modified electrode.

PubMed

Dai, Hong; Chi, Yuwu; Wu, Xiaoping; Wang, Youmei; Wei, Mingdeng; Chen, Guonan

2010-02-15

A new biocompatible ECL biosensor based on enzyme/titanate nanotubes/chitosan composite film was developed for the determination of analytes in biological samples. In the fabrication of the new ECL biosensor, biocompatible titanate nanotubes (TNTs) and a model enzyme, i.e., choline oxidase (ChOX), were immobilized on a chitosan modified glassy carbon electrode (GCE) via electrostatic adsorption and covalent interaction, respectively. By this ECL biosensor, choline was enzymatically oxidized to hydrogen peroxide and detected by a sensitive luminol ECL system. The use of TNTs not only provided a biocompatible microenvironment for the immobilized enzyme, which resulted in an excellent stability and long lifetime of the ECL biosensor, but also exhibited great enhancement towards luminol ECL and thus led to a significant improvement in sensitivity of ECL biosensor. Satisfactory results were obtained when employing this biosensor in assaying the total choline in milk samples. The work would provide a common platform to develop various sensitive, selective and biocompatible ECL biosensors based on using enzyme/TNTs/CHIT composite films. Copyright 2009 Elsevier B.V. All rights reserved.

Fluorescent Biosensor for Phosphate Determination Based on Immobilized Polyfluorene-Liposomal Nanoparticles Coupled with Alkaline Phosphatase.

PubMed

Kahveci, Zehra; Martínez-Tomé, Maria José; Mallavia, Ricardo; Mateo, C Reyes

2017-01-11

This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.

Comprehensive analysis of chemokine-induced cAMP-inhibitory responses using a real-time luminescent biosensor.

PubMed

Felouzis, Virginia; Hermand, Patricia; de Laissardière, Guy Trambly; Combadière, Christophe; Deterre, Philippe

2016-01-01

Chemokine receptors are members of the G-protein-coupled receptor (GPCR) family coupled to members of the Gi class, whose primary function is to inhibit the cellular adenylate cyclase. We used a cAMP-related and PKA-based luminescent biosensor (GloSensor™ F-22) to monitor the real-time downstream response of chemokine receptors, especially CX3CR1 and CXCR4, after activation with their cognate ligands CX3CL1 and CXCL12. We found that the amplitudes and kinetic profiles of the chemokine responses were conserved in various cell types and were independent of the nature and concentration of the molecules used for cAMP prestimulation, including either the adenylate cyclase activator forskolin or ligands mediating Gs-mediated responses like prostaglandin E2 or beta-adrenergic agonist. We conclude that the cAMP chemokine response is robustly conserved in various inflammatory conditions. Moreover, the cAMP-related luminescent biosensor appears as a valuable tool to analyze the details of Gi-mediated cAMP-inhibitory cellular responses, even in native conditions and could help to decipher their precise role in cell function. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-enzymatic Fluorescent Biosensor for Glucose Sensing Based on ZnO Nanorods

NASA Astrophysics Data System (ADS)

Mai, Hong Hanh; Pham, Van Thanh; Nguyen, Viet Tuyen; Sai, Cong Doanh; Hoang, Chi Hieu; Nguyen, The Binh

2017-06-01

We have developed a non-enzymatic fluorescent biosensor for glucose sensing based on ZnO nanorods. ZnO nanorods of high density, high crystallinity, and good alignment were grown on low-cost industrial copper substrates at low temperature. To grow them directly on the substrates without using a seed layer, we utilized a simple one-step seedless hydrothermal method, which is based on galvanic cell structure. Herein, the glucose-treated ZnO nanorods together with the ultraviolet (UV) irradiation of the sample during the photoluminescent measurement played the role of a catalyst. They decomposed glucose into hydrogen peroxide (H2O2) and gluconic acid, which is similar to the glucose oxidase enzyme (GOx) used in enzymatic sensors. Due to the formation of H2O2, the photoluminescence intensity of the UV emission peak of ZnO nanorods decreased as the glucose concentration increased from 1 mM to 100 mM. In comparison with glucose concentration of a normal human serum, which is in the range of 4.4-6.6 mM, the obtained results show potential of non-enzymatic fluorescent biosensors in medical applications.

REV, A BRET-Based Sensor of ERK Activity

PubMed Central

Xu, Chanjuan; Peter, Marion; Bouquier, Nathalie; Ollendorff, Vincent; Villamil, Ignacio; Liu, Jianfeng; Fagni, Laurent; Perroy, Julie

2013-01-01

Networks of signaling molecules are activated in response to environmental changes. How are these signaling networks dynamically integrated in space and time to process particular information? To tackle this issue, biosensors of single signaling pathways have been engineered. Bioluminescence resonance energy transfer (BRET)-based biosensors have proven to be particularly efficient in that matter due to the high sensitivity of this technology to monitor protein–protein interactions or conformational changes in living cells. Extracellular signal-regulated kinases (ERK) are ubiquitously expressed and involved in many diverse cellular functions that might be encoded by the strength and spatio-temporal pattern of ERK activation. We developed a BRET-based sensor of ERK activity, called Rluc8-ERKsubstrate-Venus (REV). As expected, BRET changes of REV were correlated with ERK phosphorylation, which is required for its kinase activity. In neurons, the nature of the stimuli determines the strength, the location, or the moment of ERK activation, thus highlighting how acute modulation of ERK may encode the nature of initial stimulus to specify the consequences of this activation. This study provides evidence for suitability of REV as a new biosensor to address biological questions. PMID:23908646

Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

PubMed

Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

2016-04-01

Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

Design and application of a lactulose biosensor.

PubMed

Wu, Jieyuan; Jiang, Peixia; Chen, Wei; Xiong, Dandan; Huang, Linglan; Jia, Junying; Chen, Yuanyuan; Jin, Jian-Ming; Tang, Shuang-Yan

2017-04-07

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

Simultaneous detection and determination of mercury (II) and lead (II) ions through the achievement of novel functional nucleic acid-based biosensors.

PubMed

Khoshbin, Zahra; Housaindokht, Mohammad Reza; Verdian, Asma; Bozorgmehr, Mohammad Reza

2018-09-30

The serious threats of mercury (Hg 2+ ) and lead (Pb 2+ ) ions for the public health makes it important to achieve the detection methods of the ions with high affinity and specificity. Metal ions usually coexist in some environment and foodstuff or clinical samples. Therefore, it is very necessary to develop a fast and simple method for simultaneous monitoring the amount of metal ions, especially when Hg 2+ and Pb 2+ coexist. DNAzyme-based biosensors and aptasensors have been highly regarded for this purpose as two main groups of the functional nucleic acid (FNA)-based biosensors. In this review, we summarize the recent achievements of functional nucleic acid-based biosensors for the simultaneous detection of Hg 2+ and Pb 2+ ions in two main optical and electrochemical groups. The tremendous interest in utilizing the various nanomaterials is also highlighted in the fabrication of the FNA-based biosensors. Finally, some results are presented based on the advantages and disadvantages of the studied FNA-based biosensors to compare their validation. Copyright © 2018 Elsevier B.V. All rights reserved.

Biomimetic Trehalose Biosensor Using Gustatory Receptor (Gr5a) Expressed in Drosophila Cells and Ion-Sensitive Field-Effect Transistor

NASA Astrophysics Data System (ADS)

Lau, Hui-Chong; Bae, Tae-Eon; Jang, Hyun-June; Kwon, Jae-Young; Cho, Won-Ju; Lim, Jeong-Ok

2013-04-01

The development of potential applications of biosensors using the sensory systems of vertebrates and invertebrates has progressed rapidly, especially in clinical diagnosis. The biosensor developed here involves the use of Drosophila cells expressing the gustatory receptor Gr5a and an ion-sensitive field-effect transistor (ISFET) sensor device. Gustatory receptor Gr5a is expressed abundantly in gustatory neurons and acts as a primary marker for tastants, especially sugar, in Drosophila. As a result, it could potentially serve as a good candidate for potential biomarkers of diseases in which the current knowledge of the cause and treatment is limited. The developed ISFET was based on the outstanding electrical characteristics of the metal-oxide-semiconductor field-effect transistor (MOSFET) with a subthreshold swing of 85 mV/dec, low leakage current of <10-12 and high on/off current ratio of 7.3×106. The SiO2 sensing membrane with a pH sensitivity of 34.9 mV/pH and drift rate 1.17 mV/h was sufficient for biosensing applications. In addition, the sensor device also showed significant compatibility with the Drosophila cells expressing Gr5a and their response to sugar, particularly trehalose. Moreover, the interactions between the transfected Drosophila cells and trehalose were consistent and reliable. This suggests that the developed ISFET sensor device could have potential use in the future as a screening device in diagnosis.

In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity.

PubMed

Krause, Jason; Frost, Carminita L

2018-04-01

Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

PubMed

Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro

2018-02-01

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.

Current Trends in Nanomaterial-Based Amperometric Biosensors

PubMed Central

Hayat, Akhtar; Catanante, Gaëlle; Marty, Jean Louis

2014-01-01

The last decade has witnessed an intensive research effort in the field of electrochemical sensors, with a particular focus on the design of amperometric biosensors for diverse analytical applications. In this context, nanomaterial integration in the construction of amperometric biosensors may constitute one of the most exciting approaches. The attractive properties of nanomaterials have paved the way for the design of a wide variety of biosensors based on various electrochemical detection methods to enhance the analytical characteristics. However, most of these nanostructured materials are not explored in the design of amperometric biosensors. This review aims to provide insight into the diverse properties of nanomaterials that can be possibly explored in the construction of amperometric biosensors. PMID:25494347

Function-based Biosensor for Hazardous Waste Toxin Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

James J Hickman

There is a need for new types of toxicity sensors in the DOE and other agencies that are based on biological function as the toxins encountered during decontamination or waste remediation may be previously unknown or their effects subtle. Many times the contents of the environmental waste, especially the minor components, have not been fully identified and characterized. New sensors of this type could target unknown toxins that cause death as well as intermediate levels of toxicity that impair function or cause long term impairment that may eventually lead to death. The primary question posed in this grant was tomore » create an electronically coupled neuronal cellular circuit to be used as sensor elements for a hybrid non-biological/biological toxin sensor system. A sensor based on the electrical signals transmitted between two mammalian neurons would allow the marriage of advances in solid state electronics with a functioning biological system to develop a new type of biosensor. Sensors of this type would be a unique addition to the field of sensor technology but would also be complementary to existing sensor technology that depends on knowledge of what is to be detected beforehand. We integrated physics, electronics, surface chemistry, biotechnology, and fundamental neuroscience in the development of this biosensor. Methods were developed to create artificial surfaces that enabled the patterning of discrete cells, and networks of cells, in culture; the networks were then aligned with transducers. The transducers were designed to measure electromagnetic fields (EMF) at low field strength. We have achieved all of the primary goals of the project. We can now pattern neurons routinely in our labs as well as align them with transducers. We have also shown the signals between neurons can be modulated by different biochemicals. In addition, we have made another significant advance where we have repeated the patterning results with adult hippocampal cells. Finally, we demonstrated that patterned cardiac cells on microelectrode arrays could act as sensors as well.« less

Microbial Fuel Cell Inoculated with Ochrobactrum Tritici KCC210 for Chromium (VI) Measurement in Electroplating Wastewater

NASA Astrophysics Data System (ADS)

Kuo, Jongtar; Kuo, Juiling; Cheng, Chiuyu; Chung, Yingchien

2018-01-01

Many methods/techniques have been developed for Cr(VI) measurement, but they are often conducted offsite or/and cannot provide real-time for Cr(VI) monitoring. A microbial fuel cell (MFC) is a self-sustaining device and has great potential as a biosensor for in situ Cr(VI) measurement, especially for wastewater generated from different electroplating units. In this study, Ochrobactrum tritici KCC210, a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic bacterium, was isolated and inoculated into the MFC to evaluate its feasibility as a Cr(VI) biosensor. The results indicated that O. tritici KCC210 exhibited high adaptability to pH, and temperature under anaerobic conditions. The maximum power density of the MFC biosensor was 17.5±0.9 mW/m2 at 2,000 Ω. A good linear relationship was observed between the Cr(VI) concentration (10-80 mg/L) and voltage output. The stable performance of the MFC biosensor indicated its potential as a reliable biosensor system. Moreover, the developed MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in the actual electroplating wastewater generated from different electroplating units within 15 min with low deviations (-1.8% to 7.8%) in comparison with the values determined using standard method. Thus, the MFC biosensor can measure Cr(VI) concentrations in situ in the effluents and has potential as an early warning detection device.

In Vitro Evaluation of Fluorescence Glucose Biosensor Response

PubMed Central

Aloraefy, Mamdouh; Pfefer, T. Joshua; Ramella-Roman, Jessica C.; Sapsford, Kim E.

2014-01-01

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor. PMID:25006996

In vitro evaluation of fluorescence glucose biosensor response.

PubMed

Aloraefy, Mamdouh; Pfefer, T Joshua; Ramella-Roman, Jessica C; Sapsford, Kim E

2014-07-08

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

Role of Aspergillus niger acrA in Arsenic Resistance and Its Use as the Basis for an Arsenic Biosensor

PubMed Central

Choe, Se-In; Gravelat, Fabrice N.; Al Abdallah, Qusai; Lee, Mark J.; Gibbs, Bernard F.

2012-01-01

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter). PMID:22467499

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Label-free nano-biosensing on the road to tuberculosis detection.

PubMed

Golichenari, Behrouz; Velonia, Kelly; Nosrati, Rahim; Nezami, Alireza; Farokhi-Fard, Aref; Abnous, Khalil; Behravan, Javad; Tsatsakis, Aristidis M

2018-08-15

Tuberculosis, an ailment caused by the bacterium Mycobacterium tuberculosis (Mtb) complex, is one of the catastrophic transmittable diseases that affect human. Reports published by WHO indicate that in 2017 about 6.3 million people progressed to TB and 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. Common diagnostics like the traditional PPD test and antibody-assisted assays suffer the lack of sensitivity, long processing time and cumbersome post-test proceedings. These shortcomings restrict their use and encourage innovations in TB diagnostics. In recent years, the biosensor concept opened up new horizons in sensitive and fast detection of the disease, reducing the interval time between sampling and diagnostic result. Among new diagnostics, label-free nano-biosensors are highly promising for sensitive and accessible detection of tuberculosis. Various specific label-free nano-biosensors have been recently reported detecting the whole cell of M. tuberculosis, mycobacterial proteins and IFN-γ as crucial markers in early diagnosis of TB. This article provides a focused overview on nanomaterial-based label-free biosensors for tuberculosis detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Recombinant antibodies and their use in biosensors.

PubMed

Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

2012-04-01

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

PubMed

Kucherenko, I S; Kucherenko, D Yu; Soldatkin, O O; Lagarde, F; Dzyadevych, S V; Soldatkin, A P

2016-04-01

The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

New CNT/poly(brilliant green) and CNT/poly(3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors.

PubMed

Barsan, Madalina M; Pifferi, Valentina; Falciola, Luigi; Brett, Christopher M A

2016-07-13

A combination of the electroactive polymer poly(brilliant green) (PBG) or conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) with carbon nanotubes to obtain CNT/PBG and CNT/PEDOT modified carbon film electrodes (CFE) has been investigated as a new biosensor platform, incorporating the enzymes glucose oxidase (GOx) as test enzyme, alcohol oxidase (AlcOx) or alcohol dehydrogenase (AlcDH). The sensing parameters were optimized for all biosensors based on CNT/PBG/CFE, CNT/PEDOT/CFE platforms. Under optimized conditions, both GOx biosensors exhibited very similar sensitivities, while in the case of AlcOx and AlcDH biosensors, AlcOx/CNT/PBG/CFE was found to give a higher sensitivity and lower detection limit. The influence of dissolved O2 on oxidase-biosensor performance was investigated and was shown to be different for each enzyme. Comparisons were made with similar reported biosensors, showing the advantages of the new biosensors, and excellent selectivity against potential interferents was successfully demonstrated. Finally, alcohol biosensors were successfully used for the determination of ethanol in alcoholic beverages. Copyright © 2016 Elsevier B.V. All rights reserved.

Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

PubMed

Natarajan, A; Molnar, P; Sieverdes, K; Jamshidi, A; Hickman, J J

2006-04-01

The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods.

Papers Based Electrochemical Biosensors: From Test Strips to Paper-Based Microfluidics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Bingwen; Du, Dan; Hua, Xin

2014-05-08

Papers based biosensors such as lateral flow test strips and paper-based microfluidic devices (or paperfluidics) are inexpensive, rapid, flexible, and easy-to-use analytical tools. An apparent trend in their detection is to interpret sensing results from qualitative assessment to quantitative determination. Electrochemical detection plays an important role in quantification. This review focuses on electrochemical (EC) detection enabled biosensors. The first part provides detailed examples in paper test strips. The second part gives an overview of paperfluidics engaging EC detections. The outlook and recommendation of future directions of EC enabled biosensors are discussed in the end.

Test/QA Plan for Verification of Coliform Detection Technologies for Drinking Water

EPA Science Inventory

The coliform detection technologies to be tested use chromatogenic and fluorogenic growth media to detect coliforms and E. coli based on the enzymatic activity of these organisms. The systems consist of single-use sample containers that contain pre-measured reagents and can be u...

QUANTITATIVE MEASUREMENT OF STACHYBOTRYS CHARTARUM CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS WITH THE TAQMAN TM FLUOROGENIC PROBE SYSTEM

EPA Science Inventory

The occurence of Stachybotrys chartarum in indoor environments has been associated with a number of human health concerns, including fatal pulmonary haemosiderosis in infants. Currently used culture-based and microscopic methods of fungal species identification are poorly suited ...

Amperometric biosensor system for simultaneous determination of adenosine-5'-triphosphate and glucose.

PubMed

Kucherenko, Ivan S; Didukh, Daria Yu; Soldatkin, Oleksandr O; Soldatkin, Alexei P

2014-06-03

The majority of biosensors for adenosine-5'-triphosphate (ATP) determination are based on cascades of enzymatic reactions; therefore, they are sensitive to glucose or glycerol (depending on the enzymatic system) as well as to ATP. The presence of unknown concentrations of these substances in the sample greatly complicates the determination of ATP. To overcome this disadvantage of known biosensors, we developed a biosensor system consisting of two biosensors: the first one is based on glucose oxidase and is intended for measuring glucose concentration, and the second one is based on glucose oxidase and hexokinase and is sensitive toward both glucose and ATP. Using glucose concentration measured by the first biosensor, we can analyze the total response to glucose and ATP obtained by the second biosensor. Platinum disc electrodes were used as amperometric transducers. The polyphenilenediamine membrane was deposited onto the surface of platinum electrodes to avoid the response to electroactive substances. The effect of glucose concentration on biosensor determination of ATP was studied. The reproducibility of biosensor responses to glucose and ATP during a day was tested (relative standard deviation, RSD, of responses to glucose was 3-6% and to ATP was 8-12%) as well as storage stability of the biosensors (no decrease of glucose responses and 43% drop of ATP responses during 50 days). The measurements of ATP and glucose in pharmaceutical vials (including mixtures of ATP and glucose) were carried out. It was shown that the developed biosensor system can be used for simultaneous analysis of glucose and ATP concentrations in water solutions.

Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

PubMed

Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

2016-07-18

Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

Solvent-Free Off-On Detection of the Improvised Explosive Triacetone Triperoxide (TATP) with Fluorogenic Materials.

PubMed

Calvo-Gredilla, Patricia; García-Calvo, José; Cuevas, José V; Torroba, Tomás; Pablos, Jesús-Luis; García, Félix C; García, José-Miguel; Zink-Lorre, Nathalie; Font-Sanchis, Enrique; Sastre-Santos, Ángela; Fernández-Lázaro, Fernando

2017-10-09

A fluorogenic perylenediimide-functionalized polyacrylate capable of generating color and fluorescence changes in the presence of triacetone triperoxide TATP), an improvised explosive used in terrorist attacks, under solvent-free, solid-state conditions has been developed. The material works by accumulating volatile TATP until it reaches a threshold; therefore, triggering colorimetric and fluorescent responses. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorogenic PNA probes

PubMed Central

2018-01-01

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years. PMID:29507634

Nanomaterial-Based Electrochemical Biosensors and Bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Guodong; Mao, Xun; Gurung, Anant

2010-08-31

This book chapter summarizes the recent advance in nanomaterials for electrochemical biosensors and bioassays. Biofunctionalization of nanomaterials for biosensors fabrication and their biomedical applications are discussed.

Novel fluorogenic probe for fluoride ion based on the fluoride-induced cleavage of tert-butyldimethylsilyl ether

NASA Astrophysics Data System (ADS)

Yang, Xiao-Feng

2007-06-01

A highly sensitive and selective fluorogenic probe for fluoride ion, 4-methylumbelliferyl tert-butyldimethylsilyl ether (4-MUTBS), was designed and synthesized. 4-MUTBS was a weakly fluorescent compound and was synthesized via the one-step reaction of 4-MU with tert-butyldimethylsilyl chloride. Upon incubation with fluoride ion in acetone-water solution (7:3, v/v), the Si-O bond of 4-MUTBS was cleaved and highly fluorescent 4-methylumbelliferone (4-MU) was released, hence leading to the fluorescence increase of the reaction solution. The fluorescence increase is linearly with fluoride concentration in the range 50-8000 nmol l -1 with a detection limit of 19 nmol l -1 (3 σ). Because of the high affinity of silicon toward fluoride ion, the proposed probe shows excellent selectivity toward fluoride ion over other anions. The method has been successfully applied to the fluoride determination in toothpaste and tap water samples.

Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.

PubMed

Grimm, Jonathan B; Klein, Teresa; Kopek, Benjamin G; Shtengel, Gleb; Hess, Harald F; Sauer, Markus; Lavis, Luke D

2016-01-26

The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

PubMed Central

Grimm, Jonathan B.; Klein, Teresa; Kopek, Benjamin G.; Shtengel, Gleb; Hess, Harald F.; Sauer, Markus

2015-01-01

Abstract The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules. PMID:26661345

Chromatographic determination of aliphatic aldehydes in human serum after pre-column derivatization using 2,2'-furil, a novel fluorogenic reagent.

PubMed

Fathy Bakr Ali, Marwa; Kishikawa, Naoya; Ohyama, Kaname; Abdel-Mageed Mohamed, Horria; Mohamed Abdel-Wadood, Hanaa; Mohamed Mohamed, Ashraf; Kuroda, Naotaka

2013-07-26

A novel, highly sensitive and selective fluorimetric liquid chromatographic method for simultaneous determination of medium chain aliphatic aldehydes was developed. The method was based on the derivatization of aliphatic aldehydes with 1,2-di(2-furyl)-1,2-ethanedione (2,2'-furil), a novel fluorogenic reagent, to form highly fluorescent difurylimidazole derivatives. The fluorescence derivatives were separated in less than 20min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (80:20, v/v%). The detection limits were from 0.19 to 0.50nM (1-10fmol/injection) at a signal-to-noise ratio (S/N) of 3. This method was successfully applied for monitoring of aliphatic aldehydes in healthy human sera by a simple pretreatment procedure without interferences from serum constituents. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantifying time-varying cellular secretions with local linear models.

PubMed

Byers, Jeff M; Christodoulides, Joseph A; Delehanty, James B; Raghu, Deepa; Raphael, Marc P

2017-07-01

Extracellular protein concentrations and gradients initiate a wide range of cellular responses, such as cell motility, growth, proliferation and death. Understanding inter-cellular communication requires spatio-temporal knowledge of these secreted factors and their causal relationship with cell phenotype. Techniques which can detect cellular secretions in real time are becoming more common but generalizable data analysis methodologies which can quantify concentration from these measurements are still lacking. Here we introduce a probabilistic approach in which local-linear models and the law of mass action are applied to obtain time-varying secreted concentrations from affinity-based biosensor data. We first highlight the general features of this approach using simulated data which contains both static and time-varying concentration profiles. Next we apply the technique to determine concentration of secreted antibodies from 9E10 hybridoma cells as detected using nanoplasmonic biosensors. A broad range of time-dependent concentrations was observed: from steady-state secretions of 230 pM near the cell surface to large transients which reached as high as 56 nM over several minutes and then dissipated.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution.

PubMed

Jha, Ramesh K; Bingen, Jeremy M; Johnson, Christopher W; Kern, Theresa L; Khanna, Payal; Trettel, Daniel S; Strauss, Charlie E M; Beckham, Gregg T; Dale, Taraka

2018-06-01

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli- based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Ramesh K.; Bingen, Jeremy M.; Johnson, Christopher W.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. In this study, we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain formore » the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.« less

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

DOE PAGES

Jha, Ramesh K.; Bingen, Jeremy M.; Johnson, Christopher W.; ...

2018-06-01

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. In this study, we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain formore » the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.« less

Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors.

PubMed

Mannan, Ahmad A; Liu, Di; Zhang, Fuzhong; Oyarzún, Diego A

2017-10-20

Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.

Precise mass determination of single cell with cantilever-based microbiosensor system.

PubMed

Łabędź, Bogdan; Wańczyk, Aleksandra; Rajfur, Zenon

2017-01-01

Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

Nanomaterials-based enzyme electrochemical biosensors operating through inhibition for biosensing applications.

PubMed

Kurbanoglu, Sevinc; Ozkan, Sibel A; Merkoçi, Arben

2017-03-15

In recent years great progress has been made in applying nanomaterials to design novel biosensors. Use of nanomaterials offers to biosensing platforms exceptional optical, electronic and magnetic properties. Nanomaterials can increase the surface of the transducing area of the sensors that in turn bring an increase in catalytic behaviors. They have large surface-to-volume ratio, controlled morphology and structure that also favor miniaturization, an interesting advantage when the sample volume is a critical issue. Biosensors have great potential for achieving detect-to-protect devices: devices that can be used in detections of pollutants and other treating compounds/analytes (drugs) protecting citizens' life. After a long term focused scientific and financial efforts/supports biosensors are expected now to fulfill their promise such as being able to perform sampling and analysis of complex samples with interest for clinical or environment fields. Among all types of biosensors, enzymatic biosensors, the most explored biosensing devices, have an interesting property, the inherent inhibition phenomena given the enzyme-substrate complex formation. The exploration of such phenomena is making remarkably important their application as research and applied tools in diagnostics. Different inhibition biosensor systems based on nanomaterials modification has been proposed and applied. The role of nanomaterials in inhibition-based biosensors for the analyses of different groups of drugs as well as contaminants such as pesticides, phenolic compounds and others, are discussed in this review. This deep analysis of inhibition-based biosensors that employ nanomaterials will serve researchers as a guideline for further improvements and approaching of these devices to real sample applications so as to reach society needs and such biosensor market demands. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

PubMed Central

Oliinyk, Olena S.; Chernov, Konstantin G.

2017-01-01

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs. PMID:28771184

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

PubMed

Dzyadevych, Sergei V; Soldatkin, Alexey P; Korpan, Yaroslav I; Arkhypova, Valentyna N; El'skaya, Anna V; Chovelon, Jean-Marc; Martelet, Claude; Jaffrezic-Renault, Nicole

2003-10-01

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

PubMed

Castro, Liliana R V; Guiot, Elvire; Polito, Marina; Paupardin-Tritsch, Daniéle; Vincent, Pierre

2014-02-01

Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amperometric Glucose Biosensor Based on Effective Self-Assembly Technology for Preparation of Poly(allylamine hydrochloride)/Au Nanoparticles Multilayers.

PubMed

Ye, Yuhang; Xie, Hangqing; Shao, Xiaobao; Wei, Yuan; Liu, Yuhong; Zhao, Wenbo; Xia, Xinyi

2016-03-01

Novel nanomaterials and nanotechnology for use in bioassay applications represent a rapidly advancing field. This study developed a novel method to fabricate the glucose biosensor with good gold nanoparticles (AuNPs) fixed efficiency based on effective self-assembly technology for preparation of multilayers composed of poly(allylamine hydrochloride) (PAH) and AuNPs. The electrochemical properties of the biosensor based on (AuNPs/PAH)n/AuNPs/glucose oxide (GOD) with different multilayers were systematically investigated. Among the resulting glucose biosensors, electrochemical properties of the biosensor with three times self-assembly processes ((AuNPs/PAH)3/AuNPs/GOD) is best. The GOD biosensor exhibited a fast amperometric response (5 s) to glucose, a good linear current-time relation over a wide range of glucose concentrations from 0.05 to 162 mM, and a low detection limit of 0.029 mM. The GOD biosensor modified with (AuNPs/PAH)n layers will have essential significance and practical application in future owing to the simple method of fabrication and good performance.

An Amperometric Biosensor Utilizing a Ferrocene-Mediated Horseradish Peroxidase Reaction for the Determination of Capsaicin (Chili Hotness)

PubMed Central

Mohammad, Rosmawani; Ahmad, Musa; Heng, Lee Yook

2013-01-01

Chili hotness is very much dependent on the concentration of capsaicin present in the chili fruit. A new biosensor based on a horseradish peroxidase enzyme-capsaicin reaction mediated by ferrocene has been successfully developed for the amperometric determination of chili hotness. The amperometric biosensor is fabricated based on a single-step immobilization of both ferrocene and horseradish peroxidase in a photocurable hydrogel membrane, poly(2-hydroxyethyl methacrylate). With mediation by ferrocene, the biosensor could measure capsaicin concentrations at a potential 0.22 V (vs. Ag/AgCl), which prevented potential interference from other electroactive species in the sample. Thus a good selectivity towards capsaicin was demonstrated. The linear response range of the biosensor towards capsaicin was from 2.5–99.0 μM with detection limit of 1.94 μM. A good relative standard deviation (RSD) for reproducibility of 6.4%–9.9% was obtained. The capsaicin biosensor demonstrated long-term stability for up to seven months. The performance of the biosensor has been validated using a standard method for the analysis of capsaicin based on HPLC. PMID:23921830

[Physiopathology of cAMP/PKA signaling in neurons].

PubMed

Castro, Liliana; Yapo, Cedric; Vincent, Pierre

2016-01-01

Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, synaptic transmission, regulation of excitability or long term changes in the nucleus. Genetically-encoded optical biosensors for cAMP or PKA considerably improved our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progresses made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the subcellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus and axon. Combining this imaging approach with pharmacology or genetical models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly help understand the mechanism of action of current drugs as well as help in devising novel therapeutic strategies for neuropsychiatric diseases. © Société de Biologie, 2017.

Effects of Operating Parameters on Measurements of Biochemical Oxygen Demand Using a Mediatorless Microbial Fuel Cell Biosensor.

PubMed

Hsieh, Min-Chi; Cheng, Chiu-Yu; Liu, Man-Hai; Chung, Ying-Chien

2015-12-28

The conventional Biochemical Oxygen Demand (BOD) method takes five days to analyze samples. A microbial fuel cell (MFC) may be an alternate tool for rapid BOD determination in water. However, a MFC biosensor for continuous BOD measurements of water samples is still unavailable. In this study, a MFC biosensor inoculated with known mixed cultures was used to determine the BOD concentration. Effects of important parameters on establishing a calibration curve between the BOD concentration and output signal from the MFC were evaluated. The results indicate monosaccharides were good fuel, and methionine, phenylalanine, and ethanol were poor fuels for electricity generation by the MFC. Ions in the influent did not significantly affect the MFC performance. CN(-) in the influent could alleviate the effect of antagonistic electron acceptors on the MFC performance. The regression equation for BOD concentration and current density of the biosensor was y = 0.0145x + 0.3317. It was adopted to measure accurately and continuously the BOD concentration in actual water samples at an acceptable error margin. These results clearly show the developed MFC biosensor has great potential as an alternative BOD sensing device for online measurements of wastewater BOD.

A heating-superfusion platform technology for the investigation of protein function in single cells.

PubMed

Xu, Shijun; Ainla, Alar; Jardemark, Kent; Jesorka, Aldo; Jeffries, Gavin D M

2015-01-06

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

A Multi-Walled Carbon Nanotube-based Biosensor for Monitoring Microcystin-LR in Sources of Drinking Water Supplies

EPA Science Inventory

A multi-walled carbon nanotube-based electrochemical biosensor is developed for monitoring microcystin-LR (MC-LR), a toxic cyanobacterial toxin, in sources of drinking water supplies. The biosensor electrodes are fabricated using dense, mm-long multi-walled CNT (MWCNT) arrays gro...

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

PubMed Central

2016-01-01

Rapid progress in disease biomarker discovery has increased the need for robust detection technologies. In the past several years, the designs of many immunoaffinity reagents have focused on lowering costs and improving specificity while also promoting stability. Antibody fragments (scFvs) have long been displayed on the surface of yeast and phage libraries for selection; however, the stable production of such fragments presents challenges that hamper their widespread use in diagnostics. Membrane and cell wall proteins similarly suffer from stability problems when solubilized from their native environment. Recently, cell envelope compositions that maintain membrane proteins in native or native-like lipid environment to improve their stability have been developed. This cell envelope composition approach has now been adapted toward stabilizing antibody fragments by retaining their native cell wall environment. A new class of immunoaffinity reagents has been developed that maintains antibody fragment attachment to yeast cell wall. Herein, we review recent strategies that incorporate cell wall fragments with functional scFvs, which are designed for easy production while maintaining specificity and stability when in use with simple detection platforms. These cell wall based antibody fragments are globular in structure, and heterogeneous in size, with fragments ranging from tens to hundreds of nanometers in size. These fragments appear to retain activity once immobilized onto biosensor surfaces for the specific and sensitive detection of pathogen antigens. They can be quickly and economically generated from a yeast display library and stored lyophilized, at room temperature, for up to a year with little effect on stability. This new format of scFvs provides stability, in a simple and low-cost manner toward the use of scFvs in biosensor applications. The production and “panning” of such antibody cell wall composites are also extremely facile, enabling the rapid adoption of stable and inexpensive affinity reagents for emerging infectious threats. PMID:27762541

Early-stage detection of VE-cadherin during endothelial differentiation of human mesenchymal stem cells using SPR biosensor.

PubMed

Fathi, Farzaneh; Rezabakhsh, Aysa; Rahbarghazi, Reza; Rashidi, Mohammad-Reza

2017-10-15

Surface plasmon resonance (SPR) biosensors are most commonly applied for real-time dynamic analysis and measurement of interactions in bio-molecular studies and cell-surface analysis without the need for labeling processes. Up to present, SPR application in stem cell biology and biomedical sciences was underused. Herein, a very simple and sensitive method was developed to evaluate human mesenchymal stem cells trans-differentiation to endothelial lineage of over a period of 14 days based on VE-cadherin biomarker. The SPR signals increased with the increase of the amount of VE-cadherin expression on the cell surface during cell differentiation process. The method was able to detect ≈27 cells permm 2 . No significant effect was observed on the cell viability during the cell attachment to the surface of immune-reactive biochips and during the SPR analysis. Using this highly sensitive SPR method, it was possible to sense the early stage of endothelial differentiation on day 3 in label-free form, whereas flow cytometry and fluorescent microscopy methods were found unable to detect the cell differentiation at the same time. Therefore, the proposed method can rapidly and accurately detect cell differentiation in live cells and label-free manner without any need of cell breakage and has great potential for both diagnostic and experimental approaches. Copyright © 2017. Published by Elsevier B.V.

Label-free electrical detection using carbon nanotube-based biosensors.

PubMed

Maehashi, Kenzo; Matsumoto, Kazuhiko

2009-01-01

Label-free detections of biomolecules have attracted great attention in a lot of life science fields such as genomics, clinical diagnosis and practical pharmacy. In this article, we reviewed amperometric and potentiometric biosensors based on carbon nanotubes (CNTs). In amperometric detections, CNT-modified electrodes were used as working electrodes to significantly enhance electroactive surface area. In contrast, the potentiometric biosensors were based on aptamer-modified CNT field-effect transistors (CNTFETs). Since aptamers are artificial oligonucleotides and thus are smaller than the Debye length, proteins can be detected with high sensitivity. In this review, we discussed on the technology, characteristics and developments for commercialization in label-free CNT-based biosensors.

The Electrophysiological Biosensor for Batch-Measurement of Cell Signals

NASA Astrophysics Data System (ADS)

Suzuki, Kengo; Tanabe, Masato; Ezaki, Takahiro; Konishi, Satoshi; Oka, Hiroaki; Ozaki, Nobuhiko

This paper presents the development of electrophysiological biosensor. The developed sensor allows a batch-measurement by detecting all signals from a large number of cells together. The developed sensor employs the same measurement principle as the patch-clamp technique. A single cell is sucked and clamped in a micro hole with detecting electrode. Detecting electrodes in arrayed micro holes are connected together for the batch-measurement of signals a large number of cell signals. Furthermore, an array of sensors for batch-measurement is designed to improve measurement-throughput to satisfy requirements for the drug screening application.

Electrochemical Sunset Yellow Biosensor Based on Photocured Polyacrylamide Membrane for Food Dye Monitoring

PubMed Central

Rozi, Normazida; Ahmad, Amalina; Yook Heng, Lee; Shyuan, Loh Kee; Hanifah, Sharina Abu

2018-01-01

An enzyme-based electrochemical biosensor was investigated for the analysis of Sunset Yellow synthetic food dye. A glassy carbon electrode was coated with a poly(acrylamide-co-ethyl methacrylate) membrane to immobilize laccase using a single-step photopolymerization procedure. Poly(acrylamide-co-ethyl methacrylate) membrane was demonstrated to have acceptable water absorption and suitable for biosensor application. Sunset Yellow biosensor exhibited a linear response range from 0.08 to 10.00 µM with a detection limit of 0.02 µM. This biosensor was successfully used to determine Sunset Yellow in soft drinks with recoveries of 99.0–101.6%. The method was validated using high-performance liquid chromatography, indicating the biosensor can be as a promising alternative method for Sunset Yellow detection. PMID:29301262

A Self-Immobilizing and Fluorogenic Probe for β-Lactamase Detection.

PubMed

Mao, Wuyu; Xia, Lingying; Wang, Yaqun; Xie, Hexin

2016-12-19

The spread of antibiotic resistance in pathogenic bacteria has become one of the major concerns to public health. Improved monitoring of drug resistance is of high importance for infectious disease control. One of the major mechanisms for bacteria to overcome treatment of antibiotics is the production of β-lactamases, which are enzymes that hydrolyze the β-lactam ring of the antibiotic. In this study, we have developed a self-immobilizing and fluorogenic probe for the detection of β-lactamase activity. This fluorogenic reagent, upon activation by β-lactamases, turns on a fluorescence signal and, more importantly, generates a covalent linkage to the target enzymes or the nearby proteins. The covalent labeling of enzymes was confirmed by SDS-PAGE analysis and MALDI-TOF mass spectrometry. The utility of this structurally simple probe was further confirmed by the fluorescent labeling of a range of β-lactamase-expressing bacteria. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.