Emerging applications of label-free optical biosensors

NASA Astrophysics Data System (ADS)

Zanchetta, Giuliano; Lanfranco, Roberta; Giavazzi, Fabio; Bellini, Tommaso; Buscaglia, Marco

2017-01-01

Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

NASA Astrophysics Data System (ADS)

Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

2006-01-01

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

Label-free virus detection using silicon photonic microring resonators

PubMed Central

McClellan, Melinda S.; Domier, Leslie L; Bailey, Ryan C.

2013-01-01

Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications. PMID:22138465

Label-free virus detection using silicon photonic microring resonators.

PubMed

McClellan, Melinda S; Domier, Leslie L; Bailey, Ryan C

2012-01-15

Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications. Copyright © 2011 Elsevier B.V. All rights reserved.

Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating

NASA Astrophysics Data System (ADS)

Yasui, Takao; Ogawa, Kensuke; Kaji, Noritada; Nilsson, Mats; Ajiri, Taiga; Tokeshi, Manabu; Horiike, Yasuhiro; Baba, Yoshinobu

2016-08-01

Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

Dynamic and label-free high-throughput detection of biomolecular interactions based on phase-shift interferometry

NASA Astrophysics Data System (ADS)

Li, Qiang; Huang, Guoliang; Gan, Wupeng; Chen, Shengyi

2009-08-01

Biomolecular interactions can be detected by many established technologies such as fluorescence imaging, surface plasmon resonance (SPR)[1-4], interferometry and radioactive labeling of the analyte. In this study, we have designed and constructed a label-free, real-time sensing platform and its operating imaging instrument that detects interactions using optical phase differences from the accumulation of biological material on solid substrates. This system allows us to monitor biomolecular interactions in real time and quantify concentration changes during micro-mixing processes by measuring the changes of the optical path length (OPD). This simple interferometric technology monitors the optical phase difference resulting from accumulated biomolecular mass. A label-free protein chip that forms a 4×4 probe array was designed and fabricated using a commercial microarray robot spotter on solid substrates. Two positive control probe lines of BSA (Bovine Serum Albumin) and two experimental human IgG and goat IgG was used. The binding of multiple protein targets was performed and continuously detected by using this label-free and real-time sensing platform.

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.

Label-Free Direct Detection of miRNAs with Poly-Silicon Nanowire Biosensors

PubMed Central

Gong, Changguo; Qi, Jiming; Xiao, Han; Jiang, Bin; Zhao, Yulan

2015-01-01

Background The diagnostic and prognostic value of microRNAs (miRNAs) in a variety of diseases is promising. The novel silicon nanowire (SiNW) biosensors have advantages in molecular detection because of their high sensitivity and fast response. In this study, poly-crystalline silicon nanowire field-effect transistor (poly-SiNW FET) device was developed to achieve specific and ultrasensitive detection of miRNAs without labeling and amplification. Methods The poly-SiNW FET was fabricated by a top–down Complementary Metal Oxide Semiconductor (CMOS) wafer fabrication based technique. Single strand DNA (ssDNA) probe was bind to the surface of the poly-SiNW device which was silanated and aldehyde-modified. By comparing the difference of resistance value before and after ssDNA and miRNA hybridization, poly-SiNW device can be used to detect standard and real miRNA samples. Results Poly-SiNW device with different structures (different line width and different pitch) was applied to detect standard Let-7b sample with a detection limitation of 1 fM. One-base mismatched sequence could be distinguished meanwhile. Furthermore, these poly-SiNW arrays can detect snRNA U6 in total RNA samples extracted from HepG2 cells with a detection limitation of 0.2 μg/mL. In general, structures with pitch showed better results than those without pitch in detection of both Let-7b and snRNA U6. Moreover, structures with smaller pitch showed better detection efficacy. Conclusion Our findings suggest that poly-SiNW arrays could detect standard and real miRNA sample without labeling or amplification. Poly-SiNW biosensor device is promising for miRNA detection. PMID:26709827

Plasmonic biosensor for label-free G-quadruplexes detection

NASA Astrophysics Data System (ADS)

Qiu, Suyan; Zhao, Fusheng; Santos, Greggy M.; Shih, Wei-Chuan

2016-03-01

G-quadruplex, readily formed by the G-rich sequence, potentially distributes in over 40 % of all human genes, such as the telomeric DNA with the G-rich sequence found at the end of the chromosome. The G-quadruplex structure is supposed to possess a diverse set of critical functions in the mammalian genome for transcriptional regulation, DNA replication and genome stability. However, most of the currently available methods for G-quadruplex identification are restricted to fluorescence techniques susceptible to poor sensitivity. It is essential to propose methods with higher sensitivity to specifically recognize the G-quadruplexes. In this study, we demonstrate a label-free plasmonic biosensor for G-quadruplex detection by relying on the advantages of nanoporous gold (NPG) disks that provide high-density plasmonic hot spots, suitable for molecular recognition capability without the requirement for labeling processes.

H2O2-sensitive quantum dots for the label-free detection of glucose.

PubMed

Hu, Mei; Tian, Jing; Lu, Hao-Ting; Weng, Li-Xing; Wang, Lian-Hui

2010-08-15

A novel label-free detection system based on CdTe/CdS quantum dots (QDs) was designed for the direct measurement of glucose. Herein we demonstrated that the photoluminescence (PL) of CdTe/CdS QDs was sensitive to hydrogen peroxide (H(2)O(2)). With d-glucose as a substrate, H(2)O(2) that intensively quenched the QDs PL can be produced via the catalysis of glucose oxidase (GOx). Experimental results showed that the decrease of the QDs PL was proportional to the concentration of glucose within the range of 1.8 microM to 1mM with the detection limit of 1.8 microM under the optimized experimental conditions. In addition, the QD-based label-free glucose sensing platform was adapted to 96-well plates for fluorescent assay, enhancing the capabilities and conveniences of this detection platform. An excellent response to the concentrations of glucose was found within the range of 2-30 mM. Glucose in blood and urine samples was effectively detected via this strategy. The comparison with commercialized glucose meter indicated that this proposed glucose assay system is not only simple, sensitive, but also reliable and suitable for practical application. The high sensitivity, versatility, portability, high-throughput and low cost of this glucose sensor implied its potential in point-of-care clinical diagnose of diabetes and other fields. Copyright 2010 Elsevier B.V. All rights reserved.

Ionic liquid-capped graphene quantum dots as label-free fluorescent probe for direct detection of ferricyanide.

PubMed

Sun, Xue; Qian, Yuting; Jiao, Yajie; Liu, Jiyang; Xi, Fengna; Dong, Xiaoping

2017-04-01

Despite complex molecular and atomic doping, efficient post-functionalization strategies for graphene quantum dots (GQDs) are of key importance to control the physicochemical properties and broaden the practical applications. With ionic liquid as specific modification agents, herein, the preparation of ionic liquid-capped GQDs (IL-GQDs) and its application as label-free fluorescent probe for direct detection of anion were reported. Hydroxyl-functionalized GQDs that could be easily gram-scale synthesized and possessed single-crystalline were chosen as the model GQDs. Also, the most commonly used ionic liquids, water-soluble 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIMBF 4 ) was chosen as the model IL. Under the ultrasonic treatment, BMIMBF 4 easily composited with GQDs to form IL-GQDs. The synthesized IL-GQDs were characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and fluorescence (FL) spectrum. After successful combination with IL, the excitation-independent photoluminescence behavior of GQDs presented almost no change, whereas, the anion responsiveness of IL-GQDs drastically improved, which afforded the IL-GQDs a sensitive response to Fe(CN) 6 3- . Based on the strong fluorescence quench, a facile and sensitive detection of Fe(CN) 6 3- was achieved. A wide linear range of 1.0×10 -7 to 2.5×10 -3 moll -1 with a low detection limit of 40 nmol l -1 was obtained. As the composition and properties of IL and GQDs could be easily tuned by varying the structure, ionic liquids-capped GQDs might present promising potential for their applications in sensing and catalysis. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Label-free SERS in biological and biomedical applications: Recent progress, current challenges and opportunities

NASA Astrophysics Data System (ADS)

Zheng, Xiao-Shan; Jahn, Izabella Jolan; Weber, Karina; Cialla-May, Dana; Popp, Jürgen

2018-05-01

To achieve an insightful look within biomolecular processes on the cellular level, the development of diseases as well as the reliable detection of metabolites and pathogens, a modern analytical tool is needed that is highly sensitive, molecular-specific and exhibits fast detection. Surface-enhanced Raman spectroscopy (SERS) is known to meet these requirements and, within this review article, the recent progress of label-free SERS in biological and biomedical applications is summarized and discussed. This includes the detection of biomolecules such as metabolites, nucleic acids and proteins. Further, the characterization and identification of microorganisms has been achieved by label-free SERS-based approaches. Eukaryotic cells can be characterized by SERS in order to gain information about the outer cell wall or to detect intracellular molecules and metabolites. The potential of SERS for medically relevant detection schemes is emphasized by the label-free detection of tissue, the investigation of body fluids as well as applications for therapeutic and illicit drug monitoring. The review article is concluded with an evaluation of the recent progress and current challenges in order to highlight the direction of label-free SERS in the future.

On the Determination of Uncertainty and Limit of Detection in Label-Free Biosensors.

PubMed

Lavín, Álvaro; Vicente, Jesús de; Holgado, Miguel; Laguna, María F; Casquel, Rafael; Santamaría, Beatriz; Maigler, María Victoria; Hernández, Ana L; Ramírez, Yolanda

2018-06-26

A significant amount of noteworthy articles reviewing different label-free biosensors are being published in the last years. Most of the times, the comparison among the different biosensors is limited by the procedure used of calculating the limit of detection and the measurement uncertainty. This article clarifies and establishes a simple procedure to determine the calibration function and the uncertainty of the concentration measured at any point of the measuring interval of a generic label-free biosensor. The value of the limit of detection arises naturally from this model as the limit at which uncertainty tends when the concentration tends to zero. The need to provide additional information, such as the measurement interval and its linearity, among others, on the analytical systems and biosensor in addition to the detection limit is pointed out. Finally, the model is applied to curves that are typically obtained in immunoassays and a discussion is made on the application validity of the model and its limitations.

Less label, more free: approaches in label-free quantitative mass spectrometry.

PubMed

Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A

2011-02-01

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A label-free immunoassay for Flavivirus detection by the Reflective Phantom Interface technology.

PubMed

Tagliabue, Giovanni; Faoro, Valentina; Rizzo, Serena; Sblattero, Daniele; Saccani, Andrea; Riccio, Gabriele; Bellini, Tommaso; Salina, Matteo; Buscaglia, Marco; Marcello, Alessandro

2017-10-28

Flaviviruses are widespread and cause clinically relevant arboviral diseases that impact locally and as imported travel-related infections. Direct detection of viraemia is limited, being typically undetectable at onset of symptoms. Therefore, diagnosis is primarily based on serology, which is complicated by high cross-reactivity across different species. The overlapping geographical distribution of the vectors in areas with a weak healthcare system, the increase of international travel and the similarity of symptoms highlight the need for rapid and reliable multi-parametric diagnostic tests in point-of-care formats. To this end we developed a bi-parametric serological microarray using recombinant NS1 proteins from Tick-borne encephalitis virus and West Nile virus coupled to a low-cost, label-free detection device based on the Reflective Phantom Interface (RPI) principle. Specific sequential detection of antibodies in solution demonstrates the feasibility of the approach for the surveillance and diagnosis of Flaviviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

NASA Astrophysics Data System (ADS)

Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

2016-10-01

Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

Multiplex surface plasmon resonance imaging platform for label-free detection of foodborne pathogens

USDA-ARS?s Scientific Manuscript database

Salmonellae are among the leading causes of foodborne outbreaks in the United States, and more rapid and efficient detection methods are needed. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multiple targets simultaneous...

Surface plasmon resonance imaging for label-free detection of foodborne pathogens and toxins

USDA-ARS?s Scientific Manuscript database

More rapid and efficient detection methods for foodborne pathogenic bacteria and toxins are needed to address the long assay time and limitations in multiplex capacity. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multi...

Label-free imaging and spectroscopy for early detection of cervical cancer.

PubMed

Jing, Yueyue; Wang, Yulan; Wang, Xinyi; Song, Chuan; Ma, Jiong; Xie, Yonghui; Fei, Yiyan; Zhang, Qinghua; Mi, Lan

2018-05-01

The label-free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label-free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free SnO2 nanowire FET biosensor for protein detection

NASA Astrophysics Data System (ADS)

Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

2017-06-01

Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

"Peak tracking chip" for label-free optical detection of bio-molecular interaction and bulk sensing.

PubMed

Bougot-Robin, Kristelle; Li, Shunbo; Zhang, Yinghua; Hsing, I-Ming; Benisty, Henri; Wen, Weijia

2012-10-21

A novel imaging method for bulk refractive index sensing or label-free bio-molecular interaction sensing is presented. This method is based on specially designed "Peak tracking chip" (PTC) involving "tracks" of adjacent resonant waveguide gratings (RWG) "micropads" with slowly evolving resonance position. Using a simple camera the spatial information robustly retrieves the diffraction efficiency, which in turn transduces either the refractive index of the liquids on the tracks or the effective thickness of an immobilized biological layer. Our intrinsically multiplex chip combines tunability and versatility advantages of dielectric guided wave biochips without the need of costly hyperspectral instrumentation. The current success of surface plasmon imaging techniques suggests that our chip proposal could leverage an untapped potential to routinely extend such techniques in a convenient and sturdy optical configuration toward, for instance for large analytes detection. PTC design and fabrication are discussed with challenging process to control micropads properties by varying their period (step of 2 nm) or their duty cycle through the groove width (steps of 4 nm). Through monochromatic imaging of our PTC, we present experimental demonstration of bulk index sensing on the range [1.33-1.47] and of surface biomolecule detection of molecular weight 30 kDa in aqueous solution using different surface densities. A sensitivity of the order of 10(-5) RIU for bulk detection and a sensitivity of the order of ∼10 pg mm(-2) for label-free surface detection are expected, therefore opening a large range of application of our chip based imaging technique. Exploiting and chip design, we expect as well our chip to open new direction for multispectral studies through imaging.

Label-free optical detection of action potential in mammalian neurons (Conference Presentation)

NASA Astrophysics Data System (ADS)

Batabyal, Subrata; Satpathy, Sarmishtha; Bui, Loan; Kim, Young-Tae; Mohanty, Samarendra K.; Davé, Digant P.

2017-02-01

Electrophysiology techniques are the gold standard in neuroscience for studying functionality of a single neuron to a complex neuronal network. However, electrophysiology techniques are not flawless, they are invasive nature, procedures are cumbersome to implement with limited capability of being used as a high-throughput recording system. Also, long term studies of neuronal functionality with aid of electrophysiology is not feasible. Non-invasive stimulation and detection of neuronal electrical activity has been a long standing goal in neuroscience. Introduction of optogenetics has ushered in the era of non-invasive optical stimulation of neurons, which is revolutionizing neuroscience research. Optical detection of neuronal activity that is comparable to electro-physiology is still elusive. A number of optical techniques have been reported recording of neuronal electrical activity but none is capable of reliably measuring action potential spikes that is comparable to electro-physiology. Optical detection of action potential with voltage sensitive fluorescent reporters are potential alternatives to electrophysiology techniques. The heavily rely on secondary reporters, which are often toxic in nature with background fluorescence, with slow response and low SNR making them far from ideal. The detection of one shot (without averaging)-single action potential in a true label-free way has been elusive so far. In this report, we demonstrate the optical detection of single neuronal spike in a cultured mammalian neuronal network without using any exogenous labels. To the best of our knowledge, this is the first demonstration of label free optical detection of single action potentials in a mammalian neuronal network, which was achieved using a high-speed phase sensitive interferometer. We have carried out stimulation and inhibition of neuronal firing using Glutamate and Tetrodotoxin respectively to demonstrate the different outcome (stimulation and inhibition) revealed in

Label-free SERS detection of Salmonella Typhimurium on DNA aptamer modified AgNR substrates

USDA-ARS?s Scientific Manuscript database

A straightforward label-free method based on aptamer binding and surface enhanced Raman specstroscopy (SERS) has been developed for the detection of Salmonella Typhimurium, an important foodborne pathogen that causes gastroenteritis in both humans and animals. Surface of the SERS-active silver nanor...

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor.

PubMed

Shalev, Gil; Rosenwaks, Yossi; Levy, Ilan

2012-01-15

We present experimental results in order to establish a correlation between pH sensitivity of immunologically modified nano-scaled field-effect transistor (NS-ImmunoFET) with their sensing capacity for label-free detection. The NS-ImmunoFETs are fabricated from silicon-on-insulator (SOI) wafers and are fully-depleted with thickness of ~20 nm. The data shows that higher sensitivity to pH entails enhanced sensitivity to analyte detection. This suggests that the mechanism of analyte detection as pure electrostatic perturbation induced by antibody-analyte interaction is over simplified. The fundamental assumption, in existing models for field-effect sensing mechanism assumes that the analyte molecules do not directly interact with the surface but rather stand 'deep' in the solution and away from the dielectric surface. Recent studies clearly provide contradicting evidence demonstrating that antibodies lie down flat on the surface. These observations led us to propose that the proteins that cover the gate area intimately interact with active sites on the surface thus forming a network of interacting sites. Since sensitivity to pH is directly correlated with the amount of amphoteric sites, we witness a direct correlation between sensitivity to pH and analyte detection. The highest and lowest threshold voltage shift for a label-free and specific detection of 6.5 nM IgG were 40 mV and 2.3 mV for NS-ImmunoFETs with pH sensitivity of 35 mV/decade and 15 mV/decade, respectively. Finally, physical modeling of the NS-ImmunoFET is presented and charge of a single IgG protein at pH 6 is calculated. The obtained value is consistent with charge of IgG protein cited in literature. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a label-free immunosensor system for detecting plasma cortisol levels in fish.

PubMed

Wu, Haiyun; Ohnuki, Hitoshi; Hibi, Kyoko; Ren, Huifeng; Endo, Hideaki

2016-02-01

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen-antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from -0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.

Label-free image-based detection of drug resistance with optofluidic time-stretch microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hirofumi; Lei, Cheng; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

Acquired drug resistance is a fundamental predicament in cancer therapy. Early detection of drug-resistant cancer cells during or after treatment is expected to benefit patients from unnecessary drug administration and thus play a significant role in the development of a therapeutic strategy. However, the development of an effective method of detecting drug-resistant cancer cells is still in its infancy due to their complex mechanism in drug resistance. To address this problem, we propose and experimentally demonstrate label-free image-based drug resistance detection with optofluidic time-stretch microscopy using leukemia cells (K562 and K562/ADM). By adding adriamycin (ADM) to both K562 and K562/ADM (ADM-resistant K562 cells) cells, both types of cells express unique morphological changes, which are subsequently captured by an optofluidic time-stretch microscope. These unique morphological changes are extracted as image features and are subjected to supervised machine learning for cell classification. We hereby have successfully differentiated K562 and K562/ADM solely with label-free images, which suggests that our technique is capable of detecting drug-resistant cancer cells. Our optofluidic time-stretch microscope consists of a time-stretch microscope with a high spatial resolution of 780 nm at a 1D frame rate of 75 MHz and a microfluidic device that focuses and orders cells. We compare various machine learning algorithms as well as various concentrations of ADM for cell classification. Owing to its unprecedented versatility of using label-free image and its independency from specific molecules, our technique holds great promise for detecting drug resistance of cancer cells for which its underlying mechanism is still unknown or chemical probes are still unavailable.

Label-free amino acid detection based on nanocomposites of graphene oxide hybridized with gold nanoparticles.

PubMed

Zhang, Qian; Zhang, Diming; Lu, Yanli; Xu, Gang; Yao, Yao; Li, Shuang; Liu, Qingjun

2016-03-15

Nanocomposites of graphene oxide and gold nanoparticles (GO/GNPs) were synthesized for label-free detections of amino acids. Interactions between the composites and amino acids were investigated by both naked-eye observation and optical absorption spectroscopy. The GO/GNPs composites displayed apparent color changes and absorption spectra changes in presences of amino acids including glutamate, aspartate, and cysteine. The interaction mechanisms of the composites and amino acids were discussed and explored with sulfhydryl groups and non-α-carboxylic groups on the amino acids. Sensing properties of the composites were tested, while pure gold particles were used as the control. The results suggested that the GO/GNPs composites had better linearity and stability in dose-dependent responses to the amino acids than those of the particles, especially in detections for acidic amino acids. Therefore, the nanocomposites platform can provide a convenient and efficient approach for label-free optical detections of important molecules such as amino acids. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free aptamer-based sensor for specific detection of malathion residues by surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Nie, Yonghui; Teng, Yuanjie; Li, Pan; Liu, Wenhan; Shi, Qianwei; Zhang, Yuchao

2018-02-01

A novel label-free aptamer surface-enhanced Raman scattering (SERS) sensor for trace malathion residue detection was proposed. In this process, the binding of malathion molecule with aptamer is identified directly. The silver nanoparticles modified with positively charged spermine served as enhancing and capture reagents for the negatively charged aptamer. Then, the silver nanoparticles modified by aptamer were used to specifically capture the malathion. The SERS background spectra of spermine, aptamer, and malathion were recorded and distinguished with the spectrum of malathion-aptamer. To enhance the characteristic peak signal of malathion captured by the aptamer, the aggregate reagents (NaCl, KCl, MgCl2) were compared and selected. The selectivity of this method was verified in the mixed-pesticide standard solution, which included malathion, phosmet, chlorpyrifos-methyl, and fethion. Results show that malathion can be specifically identified when the mixed-pesticide interferences existed. The standard curve was established, presenting a good linear range of 5 × 10- 7 to 1 × 10- 5 mol·L- 1. The spiked experiments for tap water show good recoveries from 87.4% to 110.5% with a relative standard deviation of less than 4.22%. Therefore, the proposed label-free aptamer SERS sensor is convenient, specifically detects trace malathion residues, and can be applied for qualitative and quantitative analysis of other pesticides.

Label-free electrical detection of DNA hybridization using carbon nanotubes and graphene

PubMed Central

Fu, Dongliang; Li, Lain-Jong

2010-01-01

The interface between biosystems and nanomaterials is emerging for detection of various biomolecules and subtle cellular activities. In particular, the development of cost-effective and sequence-selective DNA detection is urgent for the diagnosis of genetic or pathogenic diseases. Graphene-based nanocarbon materials, such as carbon nanotubes and thin graphene layers, have been employed as biosensors because they are biocompatible, extraordinarily sensitive, and promising for large-area detection. Electrical and label-free detection of DNA can be achieved by monitoring the conductance change of devices fabricated from these carbon materials. Here, the recent advances in this research area are briefly reviewed. The key issues and perspectives of future development are also discussed. PMID:22110861

Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

NASA Astrophysics Data System (ADS)

Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Cocaine Detection in Complex Biological Fluids.

PubMed

Rauf, Sana; Zhang, Ling; Ali, Asghar; Liu, Yang; Li, Jinghong

2017-02-24

Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

A compact and portable optofluidic device for detection of liquid properties and label-free sensing

NASA Astrophysics Data System (ADS)

Lahoz, F.; Martín, I. R.; Walo, D.; Gil-Rostra, J.; Yubero, F.; Gonzalez-Elipe, A. R.

2017-06-01

Optofluidic lasers have been widely investigated over the last few years mainly because they can be easily integrated in sensor devices. However, high power pulse lasers are required as excitation sources, which, in practice, limit the portability of the system. Trying to overcome some of these limitations, in this paper we propose the combined use of a small CW laser with a Fabry-Perot optofluidic planar microcavity showing high sensitivity and versatility for detection of liquid properties and label-free sensing. Firstly, a fluorescein solution in ethanol is used to demonstrate the high performances of the FP microcavity as a temperature sensor both in the laser (high pump power above laser threshold) and in the fluorescence (low pump power) regimes. A shift in the wavelength of the resonant cavity modes is used to detect changes in the temperature and our results show that high sensitivities could be already obtained using cheap and portable CW diode lasers. In the second part of the paper, the demonstration of this portable device for label-free sensing is illustrated under low CW pumping. The wavelength positions of the optofluidic resonant modes are used to detect glucose concentrations in water solutions using a protein labelled with a fluorescent dye as the active medium.

Label-free in vitro prostate cancer cell detection via photonic-crystal biosensor

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Ding, XiaoFei; Sagredo, Ismael; Bustamante, Gilbert; Sun, Lu-Zhe; Ye, Jing Yong

2018-02-01

Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA's low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-β1. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.

Label-free probing of genes by time-domain terahertz sensing.

PubMed

Haring Bolivar, P; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Büttner, R

2002-11-07

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a freespace analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Label-free detection of 3-nitro-l-tyrosine with nickel-doped graphene localized surface plasmon resonance biosensor.

PubMed

Ng, Siu Pang; Qiu, Guangyu; Ding, Ning; Lu, Xiaoqing; Wu, Chi-Man Lawrence

2017-03-15

3-nitro-l-tyrosine (3-NT) is believed to be a biomarker of neurodegenerative diseases and metal doped graphene possess exceptionally high binding energy of 3-NT with metal-nitro chemisorption. Here we report a novel label-free detection scheme of 3-NT via nickel-doped graphene (NDG) as the functionalized receptor on our phase detecting localized surface plasmon resonance (LSPR) biosensor. When compared with reported 3-NT immunoassay with enzyme-linked immunosorbent assay (ELISA), our NDG-LSPR platform offers two advantages i.e. 1) label-free and 2) capture of 3-NT by direct chemisorption. Our limit of detection for 3-NT in PBS was found to be 0.13pg/ml and the linear dynamic range of response was from 0.5pg/ml to 1ng/ml, i.e. four orders of magnitude. The specificity of our NDG receptor to 3-NT was also verified with l-tyrosine of equivalent concentrations in PBS and diluted human serum, for which the NDG receptor shows negligible responses. In addition, the adsorption of 3-NT and l-tyrosine to the NDG receptor were also investigated by atomic force microscopy and further verified by surface enhanced Raman spectroscopy. Therefore, our NDG-LSPR biosensor competes favorably against ELISA and we believe it should be an attractive and economical solution to early diagnostic of 3-NT related disorders for clinical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

PubMed

Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

2017-10-03

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

Label-free carbon particulates detection in bio (medical) settings (Conference Presentation)

NASA Astrophysics Data System (ADS)

Steuwe, Christian; Bové, Hannelore; vandeVen, Martin J.; Ameloot, Marcel; Roeffaers, Maarten B. J.

2017-02-01

The adverse health effects of particulate matter exposure are a generally accepted concern. Dramatic statistical figures suggest that fine dust is a main environmental risk in Europe and can be held accountable for hundreds of thousands of deaths per year [1]. Locating and tracking these nanometer sized particles, however, is not straight forward: In epidemiological and toxicology research only measurements based on labels [2] such as radionuclide markers have been applied. In this paper we present a direct, label-free optical contrast mechanism to detect carbon nanoparticles immersed in aqueous environments [3]. The virtue of this technique is its ability to perform in body fluids such as urine but also in cells and tissues. The mechanism is based on white light (WL) generation upon illumination with femtosecond pulsed near-infrared and is therefore non-incandescence related. We demonstrate the technique in various biological settings with dry and suspended carbon black particles (CB), a widely used model compound for soot [4]. Our approach allows for the unequivocal localization of CB alongside of common fluorophores and markers and can be performed on multiphoton laser-scanning microscopy platforms, a system commonly available in research laboratories. [1] European Environment Agency (2015). Press release. [2] Kong et al. Int. J. Mol. Sci. 2013, 14, (11), 22529-22543 [3] Bové and Steuwe et al. Nano letters, 2016, (16) , pages 3173-3178 [4] Arnal et al. Combust. Sci. Technol. 2012, 184, (7-8), 1191-1206.

Low-cost label-free electrical detection of artificial DNA nanostructures using solution-processed oxide thin-film transistors.

PubMed

Kim, Si Joon; Jung, Joohye; Lee, Keun Woo; Yoon, Doo Hyun; Jung, Tae Soo; Dugasani, Sreekantha Reddy; Park, Sung Ha; Kim, Hyun Jae

2013-11-13

A high-sensitivity, label-free method for detecting deoxyribonucleic acid (DNA) using solution-processed oxide thin-film transistors (TFTs) was developed. Double-crossover (DX) DNA nanostructures with different concentrations of divalent Cu ion (Cu(2+)) were immobilized on an In-Ga-Zn-O (IGZO) back-channel surface, which changed the electrical performance of the IGZO TFTs. The detection mechanism of the IGZO TFT-based DNA biosensor is attributed to electron trapping and electrostatic interactions caused by negatively charged phosphate groups on the DNA backbone. Furthermore, Cu(2+) in DX DNA nanostructures generates a current path when a gate bias is applied. The direct effect on the electrical response implies that solution-processed IGZO TFTs could be used to realize low-cost and high-sensitivity DNA biosensors.

Label-free direct surface-enhanced Raman scattering (SERS) of nucleic acids (Conference Presentation)

NASA Astrophysics Data System (ADS)

Guerrini, Luca; Morla-Folch, Judit; Gisbert-Quilis, Patricia; Xie, Hainan; Alvarez-Puebla, Ramon

2016-03-01

Recently, plasmonic-based biosensing has experienced an unprecedented level of attention, with a particular focus on the nucleic acid detection, offering efficient solutions to engineer simple, fast, highly sensitive sensing platforms while overcoming important limitations of PCR and microarray techniques. In the broad field of plasmonics, surface-enhanced Raman scattering (SERS) spectroscopy has arisen as a powerful analytical tool for detection and structural characterization of biomolecules. Today applications of SERS to nucleic acid analysis largely rely on indirect strategies, which have been demonstrated very effective for pure sensing purposes but completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS of nucleic acid shows an outstanding potential in terms of chemical-specific information which, however, remained largely unexpressed mainly because of the inherent poor spectral reproducibility and/or limited sensitivity. To address these limitations, we developed a fast and affordable high-throughput screening direct SERS method for gaining detailed genomic information on nucleic acids (DNA and RNA) and for the characterization and quantitative recognition of DNA interactions with exogenous agents. The simple strategy relies on the electrostatic adhesion of DNA/RNA onto positively-charged silver colloids that promotes the nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at picogram level (i.e. the analysis can be performed without the necessity of amplification steps thus providing realistic direct information of the nucleic acid in its native state). We anticipate this method to gain a vast impact and set of applications in different fields, including medical diagnostics, genomic screening, drug discovery, forensic science and even molecular electronics.

Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy.

PubMed

Ortiz-Aguayo, Dionisia; Del Valle, Manel

2018-01-26

This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN)₆] 3- /[Fe(CN)₆] 4- as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM -1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis.

Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy

PubMed Central

2018-01-01

This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN)6]3−/[Fe(CN)6]4− as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM−1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis. PMID:29373502

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A.

PubMed

Radi, Abd-Elgawad; Muñoz-Berbel, Xavier; Lates, Vasilica; Marty, Jean-Louis

2009-03-15

A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (DeltaR(et)) values was linearly proportional to the concentration of OTA in the range of 1-20ngmL(-1), with a detection limit of 0.5ngmL(-1).

High-contrast grating resonators for label-free detection of disease biomarkers

PubMed Central

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-01-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

High-contrast grating resonators for label-free detection of disease biomarkers

NASA Astrophysics Data System (ADS)

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-06-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke

PubMed Central

Liu, Huanling; Jablonska, Anna; Li, Yuguo; Cao, Suyi; Liu, Dexiang; Chen, Hanwei; Van Zijl, Peter CM; Bulte, Jeff W.M.; Janowski, Miroslaw; Walczak, Piotr; Liu, Guanshu

2016-01-01

ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion. PMID:27446492

Integrated planar terahertz resonators for femtomolar sensitivity label-free detection of DNA hybridization.

PubMed

Nagel, Michael; Bolivar, Peter Haring; Brucherseifer, Martin; Kurz, Heinrich; Bosserhoff, Anja; Büttner, Reinhard

2002-04-01

A promising label-free approach for the analysis of genetic material by means of detecting the hybridization of polynucleotides with electromagnetic waves at terahertz (THz) frequencies is presented. Using an integrated waveguide approach, incorporating resonant THz structures as sample carriers and transducers for the analysis of the DNA molecules, we achieve a sensitivity down to femtomolar levels. The approach is demonstrated with time-domain ultrafast techniques based on femtosecond laser pulses for generating and electro-optically detecting broadband THz signals, although the principle can certainly be transferred to other THz technologies.

Ultra-high frequency piezoelectric aptasensor for the label-free detection of cocaine.

PubMed

Neves, Miguel A D; Blaszykowski, Christophe; Bokhari, Sumra; Thompson, Michael

2015-10-15

This paper describes a label-free and real-time piezoelectric aptasensor for the detection of cocaine. The acoustic wave sensing platform is a quartz substrate functionalized with an adlayer of S-(11-trichlorosilyl-undecanyl)-benzenethiosulfonate (BTS) cross-linker onto which the anti-cocaine MN4 DNA aptamer is next immobilized. Preparation of the sensor surface was monitored using X-ray photoelectron spectroscopy (XPS), while the binding of cocaine to surface-attached MN4 was evaluated using the electromagnetic piezoelectric acoustic sensor (EMPAS). The MN4 aptamer, unlike other cocaine aptamer variants, has its secondary structure preformed in the unbound state with only tertiary structure changes occurring during target binding. It is postulated that the highly sensitive EMPAS detected the binding of cocaine through target mass loading coupled to aptamer tertiary structure folding. The sensor achieved an apparent Kd of 45 ± 12 µM, and a limit of detection of 0.9 µM. Repeated regenerability of the sensor platform was also demonstrated. This work constitutes the first application of EMPAS technology in the field of aptasensors. Furthermore, it is so far one of the very few examples of a bulk acoustic wave aptasensor that is able to directly detect the binding interaction between an aptamer and a small molecule in a facile one-step protocol without the use of a complex assay or signal amplification step. Copyright © 2015 Elsevier B.V. All rights reserved.

A Label-Free, Redox Biosensor for Detection of Disease Biomarkers

NASA Astrophysics Data System (ADS)

Archibald, Michelle M.; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.

2014-03-01

Technologies to detect early stage cancer would provide significant benefit to cancer disease patients. Clinical measurement of biomarkers offers the promise of a noninvasive and cost effective screening for early stage detection. We have developed a novel 3-dimensional ``nanocavity'' array for the detection of human cancer biomarkers in serum and other fluids. This all-electronic diagnostic sensor is based on a nanoscale coaxial array architecture that we have modified to enable molecular-level detection and identification. Each individual sensor in the array is a vertically-oriented coaxial capacitor, whose dielectric impedance is measurably changed when target molecules enter the coax annulus. We are designing a nanocoaxial biosensor based on electronic response to antibody recognition of a specific disease biomarker (e . g . CA-125 for early-stage ovarian cancer) on biofunctionalized metal surfaces within the nanocoax structure, thereby providing an all-electronic, ambient temperature, rapid-response, label-free redox biosensor. Our results demonstrate the feasibility of using this nanocoaxial array as an ultrasensitive device to detect a wide range of target proteins, including disease biomarkers. Supported by NIH (National Cancer Institute and the National Institute of Allergy and Infectious Diseases).

Label-free okadaic acid detection using growth of gold nanoparticles in sensor gaps as a conductive tag.

PubMed

Pan, Yuxiang; Wan, Zijian; Zhong, Longjie; Li, Xueqin; Wu, Qi; Wang, Jun; Wang, Ping

2017-06-01

Okadaic acid (OA) is a marine toxin ingested by shellfish. In this work, a simple, sensitive and label-free gap-based electrical competitive bioassay has been developed for this biotoxin detection. The gap-electrical biosensor is constructed by modifying interdigitated microelectrodes with gold nanoparticles (AuNPs) and using the self-catalytic growth of AuNPs as conductive bridges. In this development, the AuNPs growth is realized in the solution of glucose and chloroauric acid, with glucose oxidation used as the catalysis for growth of the AuNPs. The catalytic reaction product H 2 O 2 in turn reduces chloroauric acid to make the AuNPs grow. The conductance signal amplification is directly determined by the growth efficiency of AuNPs and closely related to the catalytic activity of AuNPs upon their interaction with OA molecule and OA aptamer. In the absence of OA molecule, the OA aptamer can absorb onto the surfaces of AuNPs due to electrostatic interaction, and the catalytically active sites of AuNPs are fully blocked. Thus the AuNPs growth would not happen. In contrast, the presence of OA molecule can hinder the interaction of OA aptamer and AuNPs. Then the AuNPs sites are exposed and the catalytic growth induces the conductance signal change. The results demonstrated that developed biosensor was able to specifically respond to OA ranging from 5 ppb to 80 ppb, providing limit of detection of 1 ppb. The strategy is confirmed to be effective for OA detection, which indicates the label-free OA biosensor has great potential to offer promising alternatives to the traditional analytical and immunological methods for OA detection.

LABEL-FREE DETECTION OF Pb2+ USING SPECIFIC DNAZYME AND UNMODIFIED Au NANOPARTICLE PROBE

NASA Astrophysics Data System (ADS)

Li, Chengyong; Zhao, Zike; Liu, Yaoqian; Lv, Lulu; Qi, Bing; Lin, Haixia; He, Lei; Sun, Shengli

A simple and sensitive Pb2+ sensor is developed based on label-free 17E DNAzyme and unmodified Au nanoparticles. On this basis, Pb2+ concentration can be judged according to the color variation of Au nanoparticles. The detection limit is 100nM and linear range is 100nM-16μM. It can serve as a measurement tool for Pb2+ rapid detection, which provides reference for the development of sensors in environmental monitoring and food safety.

The whispering gallery mode biosensor: label-free detection from virus to single protein

NASA Astrophysics Data System (ADS)

Holler, S.; Dantham, V. R.; Keng, D.; Kolchenko, V.; Arnold, S.; Mulroe, Brigid; Paspaley-Grbavac, M.

2014-08-01

The whispering gallery mode (WGM) biosensor is a micro-optical platform capable of sensitive label-free detection of biological particles. Described by the reactive sensing principle (RSP), this analytic formulation quantifies the response of the system to the adsorption of bioparticles. Guided by the RSP, the WGM biosensor enabling from detection of virus (e.g., Human Papillomavirus, HPV) to the ultimate goal of single protein detection. The latter was derived from insights into the RSP, which resulted in the development of a hybrid plasmonic WGM biosensor, which has recently demonstrated detection of individual protein cancer markers. Enhancements from bound gold nanoparticles provide the sensitivity to detect single protein molecules (66 kDa) with good signal-to-noise (S/N > 10), and project that detection of proteins as small as 5 kDa.

Hairpin assembly circuit-based fluorescence cooperative amplification strategy for enzyme-free and label-free detection of small molecule.

PubMed

Feng, Chunjing; Zhu, Jing; Sun, Jiewei; Jiang, Wei; Wang, Lei

2015-10-01

Here, we developed an enzyme-free, label-free, and sensitive fluorescence cooperative amplification strategy based on a hairpin assembly circuit which coupled catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR) for small molecule adenosine. A double-strand DNA probe with aptamer-catalysis strand (Apt-C) and inhibit strand (Inh) was designed for adenosine recognition and signal transduction which was named as Apt-C/Inh. Hairpins H1 and H2 were employed for constructing the CHA, and hairpins H3 and H4 for the HCR. Through the binding of adenosine and the Apt-C, the Inh was released from the Apt-C/Inh. Then the free Apt-C initiated the CHA through successively opening H1 and H2, generating H1/H2 complex and recyclable Apt-C. Next, the released Apt-C entered another CHA cycle, and the H1/H2 complex further initiated the HCR of H3 and H4 which induced the formation of the concatemers of H3/H4 complex. Such a process brought the two ends of hairpins H3 into close proximity, yielding numerous integrated G-quadruplexes which were initially sequestered in the stem and two terminals of H3. Finally, N-methyl mesoporphyrin IX (NMM) was added to generate an enhanced fluorescence signal. In the proposed strategy, driven only by the energy from hybridization, one target could trigger multiple HCR events via CHA-based target-cycle, leading to a remarkable enzyme-free amplification for adenosine. The detection limit could achieve as low as 9.7 × 10(-7) mol L(-1). Furthermore, G-quadruplexes were applied to construct label-free hairpin assembly circuit, which made it more simple and cost-effective. The satisfactory recoveries were obtained when detecting adenosine in spiked human serum and urine samples, demonstrating the feasibility of this detection strategy in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

PubMed

Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-26

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

NASA Astrophysics Data System (ADS)

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

PubMed Central

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-01-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

Label-free electrochemical genosensor based on mesoporous silica thin film.

PubMed

Saadaoui, Maroua; Fernández, Iñigo; Luna, Gema; Díez, Paula; Campuzano, Susana; Raouafi, Noureddine; Sánchez, Alfredo; Pingarrón, José M; Villalonga, Reynaldo

2016-10-01

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

PubMed

Morlay, Alexandra; Roux, Agnès; Templier, Vincent; Piat, Félix; Roupioz, Yoann

2017-01-01

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Shrink-induced graphene sensor for alpha-fetoprotein detection with low-cost self-assembly and label-free assay

NASA Astrophysics Data System (ADS)

Sando, Shota; Zhang, Bo; Cui, Tianhong

2017-12-01

Combination of shrink induced nano-composites technique and layer-by-layer (LbL) self-assembled graphene challenges controlling surface morphology. Adjusting shrink temperature achieves tunability on graphene surface morphology on shape memory polymers, and it promises to be an alternative in fields of high-surface-area conductors and molecular detection. In this study, self-assembled graphene on a shrink polymer substrate exhibits nanowrinkles after heating. Induced nanowrinkles on graphene with different shrink temperature shows distinct surface roughness and wettability. As a result, it becomes more hydrophilic with higher shrink temperatures. The tunable wettability promises to be utilized in, for example, microfluidic devices. The graphene on shrink polymer also exhibits capability of being used in sensing applications for pH and alpha-fetoprotein (AFP) detection with advantages of label free and low cost, due to self-assembly technique, easy functionalization, and antigen-antibody reaction on graphene surface. The detection limit of AFP detection is down to 1 pg/mL, and therefore the sensor also has a significant potential for biosensing as it relies on low-cost self-assembly and label-free assay.

Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

NASA Astrophysics Data System (ADS)

Gao, Shuyan; Jia, Xiaoxia; Chen, Yanli

2013-01-01

Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag+/Ag (0.7996 V) and Hg2+/Hg2 2+ (0.920 V) that makes colorless Hg2+ ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg2+ ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg2+ ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg2+ ions sensing motif shows great promise for sensing Hg2+ ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

Sensitive and label-free detection of miRNA-145 by triplex formation.

PubMed

Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

2016-01-01

The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

Graphene oxide-based electrochemical label-free detection of glycoproteins down to aM level using a lectin biosensor

PubMed Central

Klukova, L.; Filip, J.; Belicky, S.; Vikartovska, A.; Tkac, J.

2017-01-01

A label-free ultrasensitive impedimetric biosensor with lectin immobilised on graphene oxide (GO) for the detection of glycoproteins from 1 aM is shown here. This is the first time a functional lectin biosensor with lectin directly immobilised on a graphene-based interface without any polymer modifier has been described. The study also shows that hydrophilic oxidative debris present on GO has a beneficial effect on the sensitivity of (8.46 ± 0.20)% per decade for the lectin biosensor compared to the sensitivity of (4.52 ± 0.23)% per decade for the lectin biosensor built up from GO with the oxidative debris washed out. PMID:27277703

High-throughput label-free detection of aggregate platelets with optofluidic time-stretch microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Ito, Takuro; Guo, Baoshan; Kobayashi, Hirofumi; Ozeki, Yasuyuki; Yatomi, Yutaka; Goda, Keisuke

2017-02-01

According to WHO, approximately 10 million new cases of thrombotic disorders are diagnosed worldwide every year. In the U.S. and Europe, their related diseases kill more people than those from AIDS, prostate cancer, breast cancer and motor vehicle accidents combined. Although thrombotic disorders, especially arterial ones, mainly result from enhanced platelet aggregability in the vascular system, visual detection of platelet aggregates in vivo is not employed in clinical settings. Here we present a high-throughput label-free platelet aggregate detection method, aiming at the diagnosis and monitoring of thrombotic disorders in clinical settings. With optofluidic time-stretch microscopy with a spatial resolution of 780 nm and an ultrahigh linear scanning rate of 75 MHz, it is capable of detecting aggregated platelets in lysed blood which flows through a hydrodynamic-focusing microfluidic device at a high throughput of 10,000 particles/s. With digital image processing and statistical analysis, we are able to distinguish them from single platelets and other blood cells via morphological features. The detection results are compared with results of fluorescence-based detection (which is slow and inaccurate, but established). Our results indicate that the method holds promise for real-time, low-cost, label-free, and minimally invasive detection of platelet aggregates, which is potentially applicable to detection of platelet aggregates in vivo and to the diagnosis and monitoring of thrombotic disorders in clinical settings. This technique, if introduced clinically, may provide important clinical information in addition to that obtained by conventional techniques for thrombotic disorder diagnosis, including ex vivo platelet aggregation tests.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

PubMed

Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J

2016-09-02

Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

Detecting and identifying DNA via the THz backbone frequency using a metamaterial-based label-free biosensor

NASA Astrophysics Data System (ADS)

Mirzaei, Sahar; Green, Nicolas G.; Rotaru, Mihai; Pu, Suan Hui

2017-02-01

In genetic diagnostics, laboratory-based equipment generally uses analytical techniques requiring complicated and expensive fluorescent labelling of target DNA molecules. Intense research effort into, and commercial development of, Point-of-Care diagnostics and Personalized Healthcare are driving the development of simple, fast and cost-effective detection methods. One potential label-free DNA detection method uses Terahertz (THz) spectroscopy of the natural responses of DNA in metamaterial structures, which are engineered to have properties that are impossible to obtain in natural materials. This paper presents a study of the development of metamaterials based on asymmetric X-shaped resonator inclusions as a functional sensor for DNA. Gold X-shaped resonator structures with dimensions of 90/85 μm were demonstrated to produce trapped mode resonant frequency in the correct range for DNA detection. Realistic substrate materials in the form of 375 μm thick quartz were investigated, demonstrating that the non-transparent nature of the material resulted in the production of standing waves, affecting the system response, as well as requiring a reduction in scale of the resonator of 85%. As a result, the effect of introducing etched windows in the substrate material were investigated, demonstrating that increased window size significantly reduces the effect of the substrate on the system response. The device design showed a good selectivity when RNA samples were introduced to the model, demonstrating the potential for this design of device in the development of sensors capable of performing cheap and simple genetic analysis of DNA, giving label-free detection at high sensitivity.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

PubMed

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Gold/silver/gold trilayer films on nanostructured polycarbonate substrates for direct and label-free nanoplasmonic biosensing.

PubMed

López-Muñoz, Gerardo A; Estévez, M-Carmen; Vázquez-García, Marc; Berenguel-Alonso, Miguel; Alonso-Chamarro, Julián; Homs-Corbera, Antoni; Lechuga, Laura M

2018-05-01

Ultrasmooth gold/silver/gold trilayer nanostructured plasmonic sensors were obtained using commercial Blu-ray optical discs as nanoslits-based flexible polymer substrates. A thin gold film was used as an adhesion and nucleation layer to improve the chemical stability and reduce the surface roughness of the overlying silver film, without increasing ohmic plasmon losses. The structures were physically and optically characterized and compared with nanostructures of single gold layer. Ultrasmooth and chemically stable trilayer nanostructures with a surface roughness <0.5 nm were obtained following a simple and reproducible fabrication process. They showed a figure of merit (FOM) value up to 69.2 RIU -1 which is significantly higher (more than 95%) than the gold monolayer counterpart. Their potential for biosensing was demonstrated by employing the trilayer sensor for the direct and refractometric (label-free) detection of C-reactive protein (CRP) biomarker in undiluted urine achieving a Limit of Detection (LOD) in the pM order. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A label-free amplified fluorescence DNA detection based on isothermal circular strand-displacement polymerization reaction and graphene oxide.

PubMed

Li, Zhen; Zhu, Wenping; Zhang, Jinwen; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2013-07-07

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples.

PubMed

Tan, Feng; Saucedo, Nuvia Maria; Ramnani, Pankaj; Mulchandani, Ashok

2015-08-04

Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody-antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

PubMed Central

Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

2013-01-01

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

A Label-Free Aptasensor for Ochratoxin a Detection Based on the Structure Switch of Aptamer.

PubMed

Liu, Feng; Ding, Ailing; Zheng, Jiushang; Chen, Jiucun; Wang, Bin

2018-06-01

A label-free sensing platform is developed based on switching the structure of aptamer for highly sensitive and selective fluorescence detection of ochratoxin A (OTA). OTA induces the structure of aptamer, transforms into G-quadruplex and produces strong fluorescence in the presence of zinc(II)-protoporphyrin IX probe due to the specific bind to G-quadruplex. The simple method exhibits high sensitivity towards OTA with a detection limit of 0.03 nM and excellent selectivity over other mycotoxins. In addition, the successful detection of OTA in real samples represents a promising application in food safety.

Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

NASA Astrophysics Data System (ADS)

Qi, Yingying; Li, Li; Li, Baoxin

2009-09-01

A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV-vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10 -13 to 4.5 × 10 -6 mol/L. The detection limit (3 σ) of this method has decreased to pico-molar level.

A Label-Free, Quantitative Fecal Hemoglobin Detection Platform for Colorectal Cancer Screening

PubMed Central

Soraya, Gita V.; Nguyen, Thanh C.; Abeyrathne, Chathurika D.; Huynh, Duc H.; Chan, Jianxiong; Nguyen, Phuong D.; Nasr, Babak; Chana, Gursharan; Kwan, Patrick; Skafidas, Efstratios

2017-01-01

The early detection of colorectal cancer is vital for disease management and patient survival. Fecal hemoglobin detection is a widely-adopted method for screening and early diagnosis. Fecal Immunochemical Test (FIT) is favored over the older generation chemical based Fecal Occult Blood Test (FOBT) as it does not require dietary or drug restrictions, and is specific to human blood from the lower digestive tract. To date, no quantitative FIT platforms are available for use in the point-of-care setting. Here, we report proof of principle data of a novel low cost quantitative fecal immunochemical-based biosensor platform that may be further developed into a point-of-care test in low-resource settings. The label-free prototype has a lower limit of detection (LOD) of 10 µg hemoglobin per gram (Hb/g) of feces, comparable to that of conventional laboratory based quantitative FIT diagnostic systems. PMID:28475117

Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

PubMed

Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

2017-03-15

This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb 2+ ) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb 2+ ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb 2+ with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb 2+ monitoring is enabled with promising application potential. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

2014-02-01

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

Label-free signal-on aptasensor for sensitive electrochemical detection of arsenite.

PubMed

Cui, Lin; Wu, Jie; Ju, Huangxian

2016-05-15

A signal-on aptasensor was fabricated for highly sensitive and selective electrochemical detection of arsenite with a label-free Ars-3 aptamer self-assembled on a screen-printed carbon electrode (SPCE) via Au-S bond. The Ars-3 aptamer could adsorb cationic polydiallyldimethylammonium (PDDA) via electrostatic interaction to repel other cationic species. In the presence of arsenite, the change of Ars-3 conformation due to the formation of Ars-3/arsenite complex led to less adsorption of PDDA, and the complex could adsorb more positively charged [Ru(NH3)6](3+) as an electrochemically active indicator on the aptasensor surface, which produced a sensitive "turn-on" response. The target-induced structure switching could be used for sensitive detection of arsenite with a linear range from 0.2 nM to 100 nM and a detection limit down to 0.15 nM. Benefiting from Ars-3 aptamer, the proposed system exhibited excellent specificity against other heavy metal ions. The SPCE-based aptasensor exhibited the advantages of low cost and simple fabrication, providing potential application of arsenite detection in environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor.

PubMed

Chen, Xuejuan; Pan, Yangang; Liu, Huiqing; Bai, Xiaojing; Wang, Nan; Zhang, Bailin

2016-05-15

Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300 cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability. Copyright © 2015 Elsevier B.V. All rights reserved.

Real-time label-free biosensing with integrated planar waveguide ring resonators

NASA Astrophysics Data System (ADS)

Sohlström, Hans; Gylfason, Kristinn B.; Hill, Daniel

2010-05-01

We review the use of planar integrated optical waveguide ring resonators for label free bio-sensing and present recent results from two European biosensor collaborations: SABIO and InTopSens. Planar waveguide ring resonators are attractive for label-free biosensing due to their small footprint, high Q-factors, and compatibility with on-chip optics and microfluidics. This enables integrated sensor arrays for compact labs-on-chip. One application of label-free sensor arrays is for point-of-care medical diagnostics. Bringing such powerful tools to the single medical practitioner is an important step towards personalized medicine, but requires addressing a number of issues: improving limit of detection, managing the influence of temperature, parallelization of the measurement for higher throughput and on-chip referencing, efficient light-coupling strategies to simplify alignment, and packaging of the optical chip and integration with microfluidics. From the SABIO project we report refractive index measurement and label-free biosensing in an 8-channel slotwaveguide ring resonator sensor array, within a compact cartridge with integrated microfluidics. The sensors show a volume sensing detection limit of 5 x 10-6 RIU and a surface sensing detection limit of 0.9 pg/mm2. From the InTopSens project we report early results on silicon-on-insulator racetrack resonators.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more

A novel label-free fluorescence assay for one-step sensitive detection of Hg2+ in environmental drinking water samples

NASA Astrophysics Data System (ADS)

Li, Ya; Liu, Nan; Liu, Hui; Wang, Yu; Hao, Yuwei; Ma, Xinhua; Li, Xiaoli; Huo, Yapeng; Lu, Jiahai; Tang, Shuge; Wang, Caiqin; Zhang, Yinhong; Gao, Zhixian

2017-04-01

A novel label-free fluorescence assay for detection of Hg2+ was developed based on the Hg2+-binding single-stranded DNA (ssDNA) and SYBR Green I (SG I). Differences from other assays, the designed rich-thymine (T) ssDNA probe without fluorescent labelling can be rapidly formed a T-Hg2+-T complex and folded into a stable hairpin structure in the presence of Hg2+ in environmental drinking water samples by facilitating fluorescence increase through intercalating with SG I in one-step. In the assay, the fluorescence signal can be directly obtained without additional incubation within 1 min. The dynamic quantitative working ranges was 5-1000 nM, the determination coefficients were satisfied by optimization of the reaction conditions. The lowest detection limit of Hg2+ was 3 nM which is well below the standard of U.S. Environmental Protection Agency. This method was highly specific for detecting of Hg2+ without being affected by other possible interfering ions from different background compositions of water samples. The recoveries of Hg2+ spiked in these samples were 95.05-103.51%. The proposed method is more viable, low-costing and simple for operation in field detection than the other methods with great potentials, such as emergency disposal, environmental monitoring, surveillance and supporting of ecological risk assessment and management.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

PubMed

Soler, Maria; Estevez, M-Carmen; Moreno, Maria de Lourdes; Cebolla, Angel; Lechuga, Laura M

2016-05-15

Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly Sensitive, Label-Free Detection of 2,4-Dichlorophenoxyacetic Acid Using an Optofluidic Chip.

PubMed

Feng, Xueling; Zhang, Gong; Chin, Lip Ket; Liu, Ai Qun; Liedberg, Bo

2017-07-28

A highly sensitive approach for rapid and label-free detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) using an optofluidic chip is demonstrated. The optofluidic chip is prepared by covalent immobilization of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to an integrated microring resonator. Subsequent detection of 2,4-D carried out in a competitive immunoreaction format enables selective detection of 2,4-D in different types of water samples, including bottled, tap, and lake water, at a limit of detection (LOD) of 4.5 pg/mL and in a quantitative range of 15-10 5 pg/mL. The microring resonator-based optofluidic chip is reusable with ultrahigh sensitivity that offers real-time and on-site detection of low-molecular-weight targets for potential applications in food safety and environmental monitoring.

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.

Carbon Nanotube Nanoelectrode Array as an Electronic Chip for Ultrasensitive Label-free DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6 and ferrocene derivatives. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. BRCA1 related oligonucleotide probes with 18 bp are selectively functionalized at the open ends of the nanotube array and specifically hybridized with oligonucleotide targets incorporated with a polyG tag. The guanine groups are employed as the signal moieties in the electrochemical measurements. R(bpy)(sup 2+, sub 3) mediator is used to further amplify the guanine oxidation signal. The hybridization of sub-attomoles of DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the R(bpy)(sup 2+, sub 3) amplification mechanism. This technique was employed for direct electrochemical detection of label-free PCR amplicon from a healthy donor through specific hybridization with the BRCA1 probe. The detection limit is estimated to be less than 1000 DNA molecules since abundant guanine bases in the PCR amplicon provides a large signal. This system provides a general platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparation, and low-cost operation.

Label-free optical resonant sensors for biochemical applications

NASA Astrophysics Data System (ADS)

Ciminelli, Caterina; Campanella, Clarissa Martina; Dell'Olio, Francesco; Campanella, Carlo Edoardo; Armenise, Mario Nicola

2013-03-01

For a number of years, the scientific community has been paying growing attention to the monitoring and enhancement of public health and the quality of life through the detection of all dangerous agents for the human body, including gases, proteins, virus, and bacterial agents. When these agents are detected through label-free biochemical sensors, the molecules are not modified structurally or functionally by adding fluorescent or radioactive dyes. This work focuses on label-free optical ring resonator-based configurations suited for bio-chemical sensing, highlighting their physical aspects and specific applications. Resonant wavelength shift and the modal splitting occurring when the analyte interacts with microresonant structures are the two major physical aspects analyzed in this paper. Competitive optical platforms proposed in the literature are also illustrated together with their properties and performance.

Femtomolar Detection by Nanocoated Fiber Label-Free Biosensors.

PubMed

Chiavaioli, Francesco; Zubiate, Pablo; Del Villar, Ignacio; Zamarreño, Carlos R; Giannetti, Ambra; Tombelli, Sara; Trono, Cosimo; Arregui, Francisco J; Matias, Ignacio R; Baldini, Francesco

2018-05-25

The advent of optical fiber-based biosensors combined with that of nanotechnologies has provided an opportunity for developing in situ, portable, lightweight, versatile, and high-performance optical sensing platforms. We report on the generation of lossy mode resonances by the deposition of nanometer-thick metal oxide films on optical fibers, which makes it possible to measure precisely and accurately the changes in optical properties of the fiber-surrounding medium with very high sensitivity compared to other technology platforms, such as long period gratings or surface plasmon resonances, the gold standard in label-free and real-time biomolecular interaction analysis. This property, combined with the application of specialty structures such as D-shaped fibers, permits enhancing the light-matter interaction. SEM and TEM imaging together with X-EDS tool have been utilized to characterize the two films used, i.e., indium tin oxide and tin dioxide. Moreover, the experimental transmission spectra obtained after the deposition of the nanocoatings have been numerically corroborated by means of wave propagation methods. With the use of a conventional wavelength interrogation system and ad hoc developed microfluidics, the shift of the lossy mode resonance can be reliably recorded in response to very low analyte concentrations. Repeated experiments confirm a big leap in performance thanks to the capability to detect femtomolar concentrations in human serum, improving the detection limit by 3 orders of magnitude when compared with other fiber-based configurations. The biosensor has been regenerated several times by injecting sodium dodecyl sulfate, which proves the capability of sensor to be reused.

A microfluidic laser scattering sensor for label-free detection of waterborne pathogens

NASA Astrophysics Data System (ADS)

Wei, Huang; Yang, Limei; Li, Feng

2016-10-01

A microfluidic-based multi-angle laser scattering (MALS) sensor capable of acquiring scattering pattern of single particle is demonstrated. The size and relative refractive index (RI) of polystyrene (PS) microspheres were deduced with accuracies of 60 nm and 0.001 by analyzing the scattering patterns. We measured scattering patterns of waterborne parasites i.e., cryptosporidium parvum (c.parvum) and giardia lamblia (g.lamblia), and some other representative species in 1 L water within 1 hour, and the waterborne parasites were identified with accuracy better than 96% by classification of distinctive scattering patterns with a support-vector-machine (SVM) algorithm. The system provides a promising tool for label-free and rapid detection of waterborne parasites.

Label-free in-flow detection of single DNA molecules using glass nanopipettes.

PubMed

Gong, Xiuqing; Patil, Amol V; Ivanov, Aleksandar P; Kong, Qingyuan; Gibb, Thomas; Dogan, Fatma; deMello, Andrew J; Edel, Joshua B

2014-01-07

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

Label-free optical detection of C-reactive protein by nanoimprint lithography-based 2D-photonic crystal film.

PubMed

Endo, Tatsuro; Kajita, Hiroshi; Kawaguchi, Yukio; Kosaka, Terumasa; Himi, Toshiyuki

2016-06-01

The development of high-sensitive, and cost-effective novel biosensors have been strongly desired for future medical diagnostics. To develop novel biosensor, the authors focused on the specific optical characteristics of photonic crystal. In this study, a label-free optical biosensor, polymer-based two-dimensional photonic crystal (2D-PhC) film fabricated using nanoimprint lithography (NIL), was developed for detection of C-reactive protein (CRP) in human serum. The nano-hole array constructed NIL-based 2D-PhC (hole diameter: 230 nm, distance: 230, depth: 200 nm) was fabricated on a cyclo-olefin polymer (COP) film (100 µm) using thermal NIL and required surface modifications to reduce nonspecific adsorption of target proteins. Antigen-antibody reactions on the NIL-based 2D-PhC caused changes to the surrounding refractive index, which was monitored as reflection spectrum changes in the visible region. By using surface modified 2D-PhC, the calculated detection limit for CRP was 12.24 pg/mL at an extremely short reaction time (5 min) without the need for additional labeling procedures and secondary antibody. Furthermore, using the dual-functional random copolymer, CRP could be detected in a pooled blood serum diluted 100× with dramatic reduction of nonspecific adsorption. From these results, the NIL-based 2D-PhC film has great potential for development of an on-site, high-sensitivity, cost-effective, label-free biosensor for medical diagnostics applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

PubMed Central

Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

2013-01-01

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

PubMed

Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

2007-10-30

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets

PubMed Central

Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

2007-01-01

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format. PMID:17951434

A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

PubMed

Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

2015-01-01

A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

NASA Astrophysics Data System (ADS)

Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

2016-03-01

Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

PubMed

Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

2012-03-21

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

Glutathione-Capped Gold Nanoparticles-Based Photoacoustic Sensor for Label-Free Detection of Lead Ions

NASA Astrophysics Data System (ADS)

Shi, R.; Liu, X.-J.; Ying, Y.

2017-07-01

The photoacoustic signal generated by laser-induced nanobubbles (PA-LINB) proved to be a sensitive tool to monitor the aggregation of gold nanoparticles. Here, a simple and label-free photoacoustic method for the rapid detection of Pb2+ in the aqueous phase was developed. Due to the high affinity of Pb2+ ions to glutathione, the presence of Pb2+ led to the aggregation of glutathione-conjugated gold nanoparticles (GSH-GNPs). Hence, by measuring the variation of the PA-LINB signal after the aggregation of GSH-GNPs, Pb2+ can be quantified. A low detection limit for Pb2+ (42 nM) and a wide linear working range ( 42-1000 nM) were achieved. Furthermore, the proposed method showed good selectivity against other metal ions.

Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

NASA Astrophysics Data System (ADS)

Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

2014-12-01

The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification

Biomimetic nanochannels based biosensor for ultrasensitive and label-free detection of nucleic acids.

PubMed

Sun, Zhongyue; Liao, Tangbin; Zhang, Yulin; Shu, Jing; Zhang, Hong; Zhang, Guo-Jun

2016-12-15

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

PubMed

Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan

2015-11-24

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

Microneedle pH Sensor: Direct, Label-Free, Real-Time Detection of Cerebrospinal Fluid and Bladder pH.

PubMed

Mani, Ganesh Kumar; Miyakoda, Kousei; Saito, Asuka; Yasoda, Yutaka; Kajiwara, Kagemasa; Kimura, Minoru; Tsuchiya, Kazuyoshi

2017-07-05

Acid-base homeostasis (body pH) inside the body is precisely controlled by the kidneys and lungs and buffer systems, such that even a minor pH change could severely affect many organs. Blood and urine pH tests are common in day-to-day clinical trials and require little effort for diagnosis. There is always a great demand for in vivo testing to understand more about body metabolism and to provide effective diagnosis and therapy. In this article, we report the simple fabrication of microneedle-based direct, label-free, and real-time pH sensors. The reference and working electrodes were Ag/AgCl thick films and ZnO thin films on tungsten (W) microneedles, respectively. The morphological and structural characteristics of microneedles were carefully investigated through various analytical methods. The developed sensor exhibited a Nernstian response of -46 mV/pH. Different conditions were used to test the sensor to confirm their accuracy and stability, such as various buffer solutions, with respect to time, and we compared the reading with commercial pH electrodes. Besides that, the fabricated microneedle sensor ability is proven by in vivo testing in mouse cerebrospinal fluid (CSF) and bladders. The pH sensor procedure reported here is totally reversible, and results were reproducible after several rounds of testing.

Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

PubMed

Geissler, David; Belder, Detlev

2015-12-01

One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (<300 nm) allows label-free detection of a broader range of analytes but also mandates the use of expensive fused silica glass, which is transparent to UV light. Herein, we report the first application of label-free deep UV fluorescence detection in non-UV transparent full-body polymer microfluidic devices. This was achieved by means of two-photon excitation in the visible range (λex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

NASA Astrophysics Data System (ADS)

Chirvi, Sajal

Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi

Label-free detection of DNA using a light-addressable potentiometric sensor modified with a positively charged polyelectrolyte layer

NASA Astrophysics Data System (ADS)

Wu, Chunsheng; Bronder, Thomas; Poghossian, Arshak; Werner, Carl Frederik; Schöning, Michael J.

2015-03-01

A multi-spot (16 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al-p-Si-SiO2 structure modified with a weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. To achieve a preferentially flat orientation of DNA strands and thus, to reduce the distance between the DNA charge and MLAPS surface, the negatively charged probe single-stranded DNAs (ssDNA) were electrostatically adsorbed onto the positively charged PAH layer using a simple layer-by-layer (LbL) technique. In this way, more DNA charge can be positioned within the Debye length, yielding a higher sensor signal. The surface potential changes in each spot induced due to the surface modification steps (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), non-specific adsorption of mismatched ssDNA) were determined from the shifts of photocurrent-voltage curves along the voltage axis. A high sensor signal of 83 mV was registered after immobilization of probe ssDNA onto the PAH layer. The hybridization signal increases from 5 mV to 32 mV with increasing the concentration of cDNA from 0.1 nM to 5 μM. In contrast, a small signal of 5 mV was recorded in the case of non-specific adsorption of fully mismatched ssDNA (5 μM). The obtained results demonstrate the potential of the MLAPS in combination with the simple and rapid LbL immobilization technique as a promising platform for the future development of multi-spot light-addressable label-free DNA chips with direct electrical readout.A multi-spot (16 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al-p-Si-SiO2 structure modified with a weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization

Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria.

PubMed

Barreiros dos Santos, M; Azevedo, S; Agusil, J P; Prieto-Simón, B; Sporer, C; Torrents, E; Juárez, A; Teixeira, V; Samitier, J

2015-02-01

Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm(− 2) (0.08 pmol cm(− 2)). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL(− 1)) over a large linear working range (10–10(6) CFU mL(− 1)). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly.

PubMed

Parfenov, Vladislav A; Koudan, Elizaveta V; Bulanova, Elena A; Karalkin, Pavel A; Pereira, Frederico DAS; Norkin, Nikita E; Knyazeva, Alisa D; Gryadunova, Anna A; Petrov, Oleg F; Vasiliev, M M; Myasnikov, Maxim; Chernikov, Valery P; Kasyanov, Vladimir A; Marchenkov, Artem Yu; Brakke, Kenneth A; Khesuani, Yusef D; Demirci, Utkan; Mironov, Vladimir A

2018-05-31

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first-time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering. . © 2018 IOP Publishing Ltd.

Label-free detection of biomolecules with Ta2O5-based field effect devices

NASA Astrophysics Data System (ADS)

Branquinho, Rita Maria Mourao Salazar

Field-effect-based devices (FEDs) are becoming a basic structural element in a new generation of micro biosensors. Their numerous advantages such as small size, labelfree response and versatility, together with the possibility of on-chip integration of biosensor arrays with a future prospect of low-cost mass production, make their development highly desirable. The present thesis focuses on the study and optimization of tantalum pentoxide (Ta2O5) deposited by rf magnetron sputtering at room temperature, and their application as sensitive layer in biosensors based on field effect devices (BioFEDs). As such, the influence of several deposition parameters and post-processing annealing temperature and surface plasma treatment on the film¡¦s properties was investigated. Electrolyte-insulator-semiconductor (EIS) field-effect-based sensors comprising the optimized Ta2O5 sensitive layer were applied to the development of BioFEDs. Enzyme functionalized sensors (EnFEDs) were produced for penicillin detection. These sensors were also applied to the label free detection of DNA and the monitoring of its amplification via polymerase chain reaction (PCR), real time PCR (RT-PCR) and loop mediated isothermal amplification (LAMP). Ion sensitive field effect transistors (ISFETs) based on semiconductor oxides comprising the optimized Ta2O5 sensitive layer were also fabricated. EIS sensors comprising Ta2O5 films produced with optimized conditions demonstrated near Nernstian pH sensitivity, 58+/-0.3 mV/pH. These sensors were successfully applied to the label-free detection of penicillin and DNA. Penicillinase functionalized sensors showed a 29+/-7 mV/mM sensitivity towards penicillin detection up to 4 mM penicillin concentration. DNA detection was achieved with 30 mV/mugM sensitivity and DNA amplification monitoring with these sensors showed comparable results to those obtained with standard fluorescence based methods. Semiconductor oxides-based ISFETs with Ta2O5 sensitive layer were

Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer.

PubMed

Liu, Yingxiong; Zhao, Qiang

2017-06-01

Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

A label-free and portable graphene FET aptasensor for children blood lead detection

NASA Astrophysics Data System (ADS)

Wang, Chenyu; Cui, Xinyi; Li, Ying; Li, Hongbo; Huang, Lei; Bi, Jun; Luo, Jun; Ma, Lena Q.; Zhou, Wei; Cao, Yi; Wang, Baigeng; Miao, Feng

2016-02-01

Lead is a cumulative toxicant, which can induce severe health issues, especially in children’s case due to their immature nervous system. While realizing large-scale monitoring of children blood lead remains challenging by utilizing traditional methods, it is highly desirable to search for alternative techniques or novel sensing materials. Here we report a label-free and portable aptasensor based on graphene field effect transistor (FET) for effective children blood lead detection. With standard solutions of different Pb2+ concentrations, we obtained a dose-response curve and a detection limitation below 37.5 ng/L, which is three orders lower than the safe blood lead level (100 μg/L). The devices also showed excellent selectivity over other metal cations such as, Na+, K+, Mg2+, and Ca2+, suggesting the capability of working in a complex sample matrix. We further successfully demonstrated the detection of Pb2+ ions in real blood samples from children by using our aptasensors, and explored their potential applications for quantification. Our results underscore such graphene FET aptasensors for future applications on fast detection of heavy metal ions for health monitoring and disease diagnostics.

Label-free optical biosensing with slot-waveguides.

PubMed

Barrios, Carlos A; Bañuls, María José; González-Pedro, Victoria; Gylfason, Kristinn B; Sánchez, Benito; Griol, Amadeu; Maquieira, A; Sohlström, H; Holgado, M; Casquel, R

2008-04-01

We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

Engineered biomarkers for leprosy diagnosis using labeled and label-free analysis.

PubMed

de Santana, Juliana F; da Silva, Mariângela R B; Picheth, Guilherme F; Yamanaka, Isabel B; Fogaça, Rafaela L; Thomaz-Soccol, Vanete; Machado-de-Avila, Ricardo A; Chávez-Olórtegui, Carlos; Sierakowski, Maria Rita; de Freitas, Rilton Alves; Alvarenga, Larissa M; de Moura, Juliana

2018-09-01

The biotechnological evolution towards the development of antigens to detect leprosy has been progressing. However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interaction still remains a challenge. The complexity of clinical manifestations requires innovative approaches to improve the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant sequence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to provide better exposure to antibodies; iv. amplifying the signal using biotin-streptavidin detection system in an ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132 patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis exhibits great potential. Copyright © 2018 Elsevier B.V. All rights reserved.

Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

PubMed

Li, Wei; Hou, Ting; Wu, Min; Li, Feng

2016-01-01

MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Copyright © 2015 Elsevier B.V. All rights reserved.

78 FR 47154 - Food Labeling; Gluten-Free Labeling of Foods

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-05

...The Food and Drug Administration (FDA or we) is issuing a final rule to define the term ``gluten-free'' for voluntary use in the labeling of foods. The final rule defines the term ``gluten-free'' to mean that the food bearing the claim does not contain an ingredient that is a gluten-containing grain (e.g., spelt wheat); an ingredient that is derived from a gluten-containing grain and that has not been processed to remove gluten (e.g., wheat flour); or an ingredient that is derived from a gluten-containing grain and that has been processed to remove gluten (e.g., wheat starch), if the use of that ingredient results in the presence of 20 parts per million (ppm) or more gluten in the food (i.e., 20 milligrams (mg) or more gluten per kilogram (kg) of food); or inherently does not contain gluten; and that any unavoidable presence of gluten in the food is below 20 ppm gluten (i.e., below 20 mg gluten per kg of food). A food that bears the claim ``no gluten,'' ``free of gluten,'' or ``without gluten'' in its labeling and fails to meet the requirements for a ``gluten-free'' claim will be deemed to be misbranded. In addition, a food whose labeling includes the term ``wheat'' in the ingredient list or in a separate ``Contains wheat'' statement as required by a section of the Federal Food, Drug, and Cosmetic Act (the FD&C Act) and also bears the claim ``gluten-free'' will be deemed to be misbranded unless its labeling also bears additional language clarifying that the wheat has been processed to allow the food to meet FDA requirements for a ``gluten-free'' claim. Establishing a definition of the term ``gluten-free'' and uniform conditions for its use in food labeling will help ensure that individuals with celiac disease are not misled and are provided with truthful and accurate information with respect to foods so labeled. We are issuing the final rule under the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA).

An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.

PubMed

Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

2015-01-15

A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

Detection of Myoglobin with an Open-Cavity-Based Label-Free Photonic Crystal Biosensor.

PubMed

Zhang, Bailin; Tamez-Vela, Juan Manuel; Solis, Steven; Bustamante, Gilbert; Peterson, Ralph; Rahman, Shafiqur; Morales, Andres; Tang, Liang; Ye, Jing Yong

2013-01-01

The label-free detection of one of the cardiac biomarkers, myoglobin, using a photonic-crystal-based biosensor in a total-internal-reflection configuration (PC-TIR) is presented in this paper. The PC-TIR sensor possesses a unique open optical microcavity that allows for several key advantages in biomolecular assays. In contrast to a conventional closed microcavity, the open configuration allows easy functionalization of the sensing surface for rapid biomolecular binding assays. Moreover, the properties of PC structures make it easy to be designed and engineered for operating at any optical wavelength. Through fine design of the photonic crystal structure, biochemical modification of the sensor surface, and integration with a microfluidic system, we have demonstrated that the detection sensitivity of the sensor for myoglobin has reached the clinically significant concentration range, enabling potential usage of this biosensor for diagnosis of acute myocardial infarction. The real-time response of the sensor to the myoglobin binding may potentially provide point-of-care monitoring of patients and treatment effects.

Label-free histamine detection with nanofluidic diodes through metal ion displacement mechanism.

PubMed

Ali, Mubarak; Ramirez, Patricio; Duznovic, Ivana; Nasir, Saima; Mafe, Salvador; Ensinger, Wolfgang

2017-02-01

We design and characterize a nanofluidic device for the label-free specific detection of histamine neurotransmitter based on a metal ion displacement mechanism. The sensor consists of an asymmetric polymer nanopore fabricated via ion track-etching technique. The nanopore sensor surface having metal-nitrilotriacetic (NTA-Ni 2+ ) chelates is obtained by covalent coupling of native carboxylic acid groups with N α ,N α -bis(carboxymethyl)-l-lysine (BCML), followed by exposure to Ni 2+ ion solution. The BCML immobilization and subsequent Ni 2+ ion complexation with NTA moieties change the surface charge concentration, which has a significant impact on the current-voltage (I-V) curve after chemical modification of the nanopore. The sensing mechanism is based on the displacement of the metal ion from the NTA-Ni 2+ chelates. When the modified pore is exposed to histamine solution, the Ni 2+ ion in NTA-Ni 2+ chelate recognizes histamine through a metal ion coordination displacement process and formation of stable Ni-histamine complexes, leading to the regeneration of metal-free NTA groups on the pore surface, as shown in the current-voltage characteristics. Nanomolar concentrations of the histamine in the working electrolyte can be detected. On the contrary, other neurotransmitters such as glycine, serotonin, gamma-aminobutyric acid, and dopamine do not provoke significant changes in the nanopore electronic signal due to their inability to displace the metal ion and form a stable complex with Ni 2+ ion. The nanofluidic sensor exhibits high sensitivity, specificity and reusability towards histamine detection and can then be used to monitor the concentration of biological important neurotransmitters. Copyright © 2016 Elsevier B.V. All rights reserved.

A label-free, PCR-free and signal-on electrochemical DNA biosensor for Leishmania major based on gold nanoleaves.

PubMed

Moradi, M; Sattarahmady, N; Rahi, A; Hatam, G R; Sorkhabadi, S M Rezayat; Heli, H

2016-12-01

Detection of leishmaniasis is important in clinical diagnoses. In the present study, identification of Leishmania parasites was performed by a label-free, PCR-free and signal-on ultrasensitive electrochemical DNA biosensor. Gold nanoleaves were firstly electrodeposited by an electrodeposition method using spermidine as a shape directing agent. The biosensor was fabricated by immobilization of a Leishmania major specific DNA probe onto gold nanoleaves, and methylene blue was employed as a marker. Hybridization of the complementary single stranded DNA sequence with the biosensor under the selected conditions was then investigated. The biosensor could detect a synthetic DNA target in a range of 1.0×10 -10 to 1.0×10 -19 molL -1 with a limit of detection of 1.8×10 -20 molL -1 , and genomic DNA in a range of 0.5-20ngμL -1 with a limit of detection of 0.07ngμL -1 . The biosensor could distinguish Leishmania major from a non-complementary-sequence oligonucleotide and the tropica species with a high selectivity. The biosensor was applicable to detect Leishmania major in patient samples. Copyright © 2016 Elsevier B.V. All rights reserved.

A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection

NASA Astrophysics Data System (ADS)

Patel, Manoj Kumar; Ali, Md. Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, Abdulaziz A.; Fouad, H.; Ansari, Z. A.; Ansari, S. G.; Malhotra, Bansi D.

2015-11-01

Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R2) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

Novel image processing method study for a label-free optical biosensor

NASA Astrophysics Data System (ADS)

Yang, Chenhao; Wei, Li'an; Yang, Rusong; Feng, Ying

2015-10-01

Optical biosensor is generally divided into labeled type and label-free type, the former mainly contains fluorescence labeled method and radioactive-labeled method, while fluorescence-labeled method is more mature in the application. The mainly image processing methods of fluorescent-labeled biosensor includes smooth filtering, artificial gridding and constant thresholding. Since some fluorescent molecules may influence the biological reaction, label-free methods have been the main developing direction of optical biosensors nowadays. The using of wider field of view and larger angle of incidence light path which could effectively improve the sensitivity of the label-free biosensor also brought more difficulties in image processing, comparing with the fluorescent-labeled biosensor. Otsu's method is widely applied in machine vision, etc, which choose the threshold to minimize the intraclass variance of the thresholded black and white pixels. It's capacity-constrained with the asymmetrical distribution of images as a global threshold segmentation. In order to solve the irregularity of light intensity on the transducer, we improved the algorithm. In this paper, we present a new image processing algorithm based on a reflectance modulation biosensor platform, which mainly comprises the design of sliding normalization algorithm for image rectification and utilizing the improved otsu's method for image segmentation, in order to implement automatic recognition of target areas. Finally we used adaptive gridding method extracting the target parameters for analysis. Those methods could improve the efficiency of image processing, reduce human intervention, enhance the reliability of experiments and laid the foundation for the realization of high throughput of label-free optical biosensors.

A cytokine immunosensor for Multiple Sclerosis detection based upon label-free electrochemical impedance spectroscopy using electroplated printed circuit board electrodes.

PubMed

Bhavsar, Kinjal; Fairchild, Aaron; Alonas, Eric; Bishop, Daniel K; La Belle, Jeffrey T; Sweeney, James; Alford, T L; Joshi, Lokesh

2009-10-15

A biosensor for the serum cytokine, Interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy (EIS) monitoring approach is described. Overexpression of IL-12 has been correlated to the diagnosis of Multiple Sclerosis (MS). An immunosensor has been fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode and immobilizing anti-IL-12 monoclonal antibodies (MAb) onto the surface of the electrode. This approach yields a robust sensor that facilitates reproducible mass fabrication and easy alteration of the electrode shape. Results indicate that this novel PCB sensor can detect IL-12 at physiological levels, <100 fM with f-values of 0.05 (typically <0.0001) in a label-free and rapid manner. A linear (with respect to log concentration) detectable range was achieved. Detection in a complex biological solution is also explored; however, significant loss of dynamic range is noted in the 100% complex solution. The cost effective approach described here can be used potentially for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.

Tapered Optical Fiber Sensor for Label-Free Detection of Biomolecules

PubMed Central

Tian, Ye; Wang, Wenhui; Wu, Nan; Zou, Xiaotian; Wang, Xingwei

2011-01-01

This paper presents a fast, highly sensitive and low-cost tapered optical fiber biosensor that enables the label-free detection of biomolecules. The sensor takes advantage of the interference effect between the fiber’s first two propagation modes along the taper waist region. The biomolecules bonded on the taper surface were determined by demodulating the transmission spectrum phase shift. Because of the sharp spectrum fringe signals, as well as a relatively long biomolecule testing region, the sensor displayed a fast response and was highly sensitive. To better understand the influence of various biomolecules on the sensor, a numerical simulation that varied biolayer parameters such as thickness and refractive index was performed. The results showed that the spectrum fringe shift was obvious to be measured even when the biolayer was only nanometers thick. A microchannel chip was designed and fabricated for the protection of the sensor and biotesting. Microelectromechanical systems (MEMS) fabrication techniques were used to precisely control the profile and depth of the microchannel on the silicon chip with an accuracy of 2 μm. A tapered optical fiber biosensor was fabricated and evaluated with an Immune globulin G (IgG) antibody-antigen pair. PMID:22163821

Tapered optical fiber sensor for label-free detection of biomolecules.

PubMed

Tian, Ye; Wang, Wenhui; Wu, Nan; Zou, Xiaotian; Wang, Xingwei

2011-01-01

This paper presents a fast, highly sensitive and low-cost tapered optical fiber biosensor that enables the label-free detection of biomolecules. The sensor takes advantage of the interference effect between the fiber's first two propagation modes along the taper waist region. The biomolecules bonded on the taper surface were determined by demodulating the transmission spectrum phase shift. Because of the sharp spectrum fringe signals, as well as a relatively long biomolecule testing region, the sensor displayed a fast response and was highly sensitive. To better understand the influence of various biomolecules on the sensor, a numerical simulation that varied biolayer parameters such as thickness and refractive index was performed. The results showed that the spectrum fringe shift was obvious to be measured even when the biolayer was only nanometers thick. A microchannel chip was designed and fabricated for the protection of the sensor and biotesting. Microelectromechanical systems (MEMS) fabrication techniques were used to precisely control the profile and depth of the microchannel on the silicon chip with an accuracy of 2 μm. A tapered optical fiber biosensor was fabricated and evaluated with an Immune globulin G (IgG) antibody-antigen pair.

Epi-detected quadruple-modal nonlinear optical microscopy for label-free imaging of the tooth

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

Wang, Zi; Zheng, Wei; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg

2015-01-19

We present an epi-detected quadruple-modal nonlinear optical microscopic imaging technique (i.e., coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon excited fluorescence (TPEF)) based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of the tooth. We demonstrate that high contrast ps-CARS images covering both the fingerprint (500–1800 cm{sup −1}) and high-wavenumber (2500–3800 cm{sup −1}) regions can be acquired to uncover the distributions of mineral and organic biomaterials in the tooth, while high quality TPEF, SHG, and THG images of the tooth can also be acquired under ps laser excitation without damaging the samples. Themore » quadruple-modal nonlinear microscopic images (CARS/SHG/THG/TPEF) acquired provide better understanding of morphological structures and biochemical/biomolecular distributions in the dentin, enamel, and the dentin-enamel junction of the tooth without labeling, facilitating optical diagnosis and characterization of the tooth in dentistry.« less

Real-Time, Label-Free, All-Electrical Detection of Salmonella typhimurium Using Lead Zirconate Titanate/Gold-Coated Glass Cantilevers at any Relative Humidity

PubMed Central

Zhu, Qing; Shih, Wan Y.; Shih, Wei-Heng

2007-01-01

We have examined non-insulated PZT/gold-coated glass cantilevers for real-time, label-free detection of Salmonella t. by partial dipping at any relative humidity. The PZT/gold-coated glass cantilevers were consisted of a 0.127 mm thick PZT layer about 0.8 mm long, 2 mm wide bonded to a 0.15 mm thick gold-coated glass layer with a 3.0 mm long gold-coated glass tip for detection. We showed that by placing the water level at the nodal point, about 0.8 mm from the free end of the gold-glass tip, there was a 1-hr window in which the resonance frequency was stable despite the water level change by evaporation at 20% relative humidity or higher. By dipping the cantilevers to their nodal point, we were able to do real-time, label-free detection without background resonance frequency corrections at any relative humidity. The partially dipped PZT/gold-coated glass cantilever exhibited mass detection sensitivity, Δm/Δf = −5×10−11g/Hz, and a detection concentration sensitivity, 5×103 cells/ml in 2 ml of liquid, which was about two orders of magnitude lower than that of a 5 MHz QCM. It was also about two orders of magnitude lower than the infection dosage and one order of magnitude lower that the detection limit of a commercial Raptor sensor. PMID:22872784

Ultrasensitive electrochemical aptasensor based on sandwich architecture for selective label-free detection of colorectal cancer (CT26) cells.

PubMed

Hashkavayi, Ayemeh Bagheri; Raoof, Jahan Bakhsh; Ojani, Reza; Kavoosian, Saeid

2017-06-15

Colorectal cancer is one of the most common cancers in the world and has no effective treatment. Therefore, development of new methods for early diagnosis is instantly required. Biological recognition probes such as synthetic receptor and aptamer is one of the candidate recognition layers to detect important biomolecules. In this work, an electrochemical aptasensor was developed by fabricating an aptamer-cell-aptamer sandwich architecture on an SBA-15-3-aminopropyltriethoxysilane (SBA-15-pr-NH 2 ) and Au nanoparticles (AuNPs) modified graphite screen printed electrode (GSPE) surface for the selective, label-free detection of CT26 cancer cells. Based on the incubation of the thiolated aptamer with CT26 cells, the electron-transfer resistance of Fe (CN) 6 3-/4- redox couple increased considerably on the aptasensor surface. The results obtained from cyclic voltammetry and electrochemical impedance spectroscopy studies showed that the fabricated aptasensor can specifically identify CT26 cells in the concentration ranges of 10-1.0×10 5 cells/mL and 1.0×10 5 -6.0×10 6 cells/mL, respectively, with a detection limit of 2cells/mL. Applying the thiol terminated aptamer (5TR1) as a recognition layer led to a sensor with high affinity for CT26 cancer cells, compared to control cancer cells of AGS cells, VERO Cells, PC3 cells and SKOV-3 cells. Therefore a simple, rapid, label free, inexpensive, excellent, sensitive and selective electrochemical aptasensor based on sandwich architecture was developed for detection of CT26 Cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

PubMed

Urmann, Katharina; Reich, Peggy; Walter, Johanna-Gabriela; Beckmann, Dieter; Segal, Ester; Scheper, Thomas

2017-09-10

Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23μM. The apparent dissociation constant was determined as 13.98μM and the LoD is 3.17μM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD. Copyright © 2017 Elsevier B.V. All rights reserved.

First results on label-free detection of DNA and protein molecules using a novel integrated sensor technology based on gravimetric detection principles.

PubMed

Gabl, R; Feucht, H-D; Zeininger, H; Eckstein, G; Schreiter, M; Primig, R; Pitzer, D; Wersing, W

2004-01-15

A novel integrated bio-sensor technology based on thin-film bulk acoustic wave resonators on silicon is presented and the feasibility of detecting DNA and protein molecules proofed. The detection principle of these sensors is label-free and relies on a resonance frequency shift caused by mass loading of an acoustic resonator, a principle very well known from quartz crystal micro balances. Integrated ZnO bulk acoustic wave resonators with resonance frequencies around 2 GHz have been fabricated, employing an acoustic mirror for isolation from the silicon substrate. DNA oligos have been thiol-coupled to the gold electrode by on-wafer dispensing. In a further step, samples have either been hybridised or alternatively a protein has been coupled to the receptor. The measurement results show the new bio-sensor being capable of both, detecting proteins as well as the DNA hybridisation without using a label. Due to the substantially higher oscillation frequency, these sensors already show much higher sensitivity and resolution comparable to quartz crystal micro balances. The potential for these sensors and sensors arrays as well as technological challenges will be discussed in detail.

Study and development of label-free optical biosensors for biomedical applications

NASA Astrophysics Data System (ADS)

Choi, Charles J.

For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of

Patterns of free amino acids in German convenience food products: marked mismatch between label information and composition.

PubMed

Hermanussen, M; Gonder, U; Jakobs, C; Stegemann, D; Hoffmann, G

2010-01-01

Free amino acids affect food palatability. As information on amino acids in frequently purchased pre-packaged food is virtually absent, we analyzed free amino acid patterns of 17 frequently purchased ready-to-serve convenience food products, and compared them with the information obtained from the respective food labels. Quantitative amino acid analysis was performed using ion-exchange chromatography. gamma-Aminobutyric acid (GABA) concentrations were verified using a stable isotope dilution gas chromatography/mass spectrometry (GC-MS) method. The patterns of free amino acids were compared with information obtained from food labels. An obvious mismatch between free amino acid patterns and food label information was detected. Even on considering that tomatoes and cereal proteins are naturally rich in glutamate, the concentrations of free glutamate outranged the natural concentration of this amino acid in several products, and strongly suggested artificial enrichment. Free glutamate was found to be elevated even in dishes that explicitly state 'no glutamate added'. Arginine was markedly elevated in lentils. Free cysteine was generally low, possibly reflecting thermal destruction of this amino acid during food processing. The meat and brain-specific dipeptide carnosine (CARN) was present in most meat-containing products. Some products did not contain detectable amounts of CARN in spite of meat content being claimed on the food labels. We detected GABA at concentrations that contribute significantly to the taste sensation. This investigation highlights a marked mismatch between food label information and food composition.

Label free selective detection of estriol using graphene oxide-based fluorescence sensor

NASA Astrophysics Data System (ADS)

Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

2014-07-01

Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin.

PubMed

Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Wang, Yan; Xie, Shunbi

2012-11-18

For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type electrochemical aptasensor for ultrasensitive detection of thrombin.

Ultrasensitive thrombin detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles as labels and electrocatalysts.

PubMed

Wu, Yongmei; Xu, Wenju; Bai, Lijuan; Yuan, Yali; Yi, Huayu; Chai, Yaqin; Yuan, Ruo

2013-12-15

For the first time, a sandwich-type electrochemical method was proposed for ultrasensitive thrombin (TB) detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles (PtNPs@Hb) as labels and electrocatalysts. The prepared PtNPs@Hb not only exhibited good biocompatibility, excellent electrocatalytic activity, but also presented redox activity of Hb. Thus, it was employed for the fabrication of aptasensor without any extraneous redox mediators, leading to a simple preparation process for the aptasensor. The high loading of Hb spheres as redox mediators could enhance the electrochemical signal. Importantly, the synergetic electrocatalytic behavior of Hb and PtNPs toward H2O2 reduction greatly amplified the electrochemical signal, resulting in the high sensitivity of aptasensor. Consequently, under optimal conditions, the designed aptasensor exhibited a lower detection limit of 0.05 pM and wide dynamic linear range from 0.15 pM to 40 nM for TB detection. Additionally, the proposed mediator-free and signal-amplified electrochemical aptasensor showed great potential in portable and cost-effective TB sensing devices. Copyright © 2013 Elsevier B.V. All rights reserved.

A label-free colorimetric sensor for Pb2+ detection based on the acceleration of gold leaching by graphene oxide.

PubMed

Shi, Xinhao; Gu, Wei; Zhang, Cuiling; Zhao, Longyun; Peng, Weidong; Xian, Yuezhong

2015-03-14

In this work, we developed a novel, label-free, colorimetric sensor for Pb(2+) detection based on the acceleration of gold leaching by graphene oxide (GO) at room temperature. Gold nanoparticles (AuNPs) can be dissolved in a thiosulfate (S2O3(2-)) aqueous environment in the presence of oxygen; however, the leaching rate is very slow due to the high activation energy (27.99 kJ mol(-1)). In order to enhance the reaction rate, some accelerators should be added. In comparison with the traditional accelerators (metal ions or middle ligands), we found that GO could efficiently accelerate the gold leaching reaction. Kinetic data demonstrate that the dissolution rate of gold in the Pb(2+)-S2O3(2-)-GO system is 5 times faster than that without GO at room temperature. In addition, the effects of surface modification and the nanoparticle size on the etching of AuNPs were investigated. Based on the GO-accelerated concentration-dependent colour changes of AuNPs, a colorimetric sensor for Pb(2+) detection was developed with a linear range from 0.1 to 20 μM and the limit of detection (LOD) was evaluated to be 0.05 μM. This colorimetric assay is simple, low-cost, label-free, and has numerous potential applications in the field of environmental chemistry.

Organophosphonate-based PNA-functionalization of silicon nanowires for label-free DNA detection.

PubMed

Cattani-Scholz, Anna; Pedone, Daniel; Dubey, Manish; Neppl, Stefan; Nickel, Bert; Feulner, Peter; Schwartz, Jeffrey; Abstreiter, Gerhard; Tornow, Marc

2008-08-01

We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

PubMed

Zhang, Yan; Wang, Xin-Yan; Zhang, Qianyi; Zhang, Chun-Yang

2017-11-21

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

Developments in label-free microfluidic methods for single-cell analysis and sorting.

PubMed

Carey, Thomas R; Cotner, Kristen L; Li, Brian; Sohn, Lydia L

2018-04-24

Advancements in microfluidic technologies have led to the development of many new tools for both the characterization and sorting of single cells without the need for exogenous labels. Label-free microfluidics reduce the preparation time, reagents needed, and cost of conventional methods based on fluorescent or magnetic labels. Furthermore, these devices enable analysis of cell properties such as mechanical phenotype and dielectric parameters that cannot be characterized with traditional labels. Some of the most promising technologies for current and future development toward label-free, single-cell analysis and sorting include electronic sensors such as Coulter counters and electrical impedance cytometry; deformation analysis using optical traps and deformation cytometry; hydrodynamic sorting such as deterministic lateral displacement, inertial focusing, and microvortex trapping; and acoustic sorting using traveling or standing surface acoustic waves. These label-free microfluidic methods have been used to screen, sort, and analyze cells for a wide range of biomedical and clinical applications, including cell cycle monitoring, rapid complete blood counts, cancer diagnosis, metastatic progression monitoring, HIV and parasite detection, circulating tumor cell isolation, and point-of-care diagnostics. Because of the versatility of label-free methods for characterization and sorting, the low-cost nature of microfluidics, and the rapid prototyping capabilities of modern microfabrication, we expect this class of technology to continue to be an area of high research interest going forward. New developments in this field will contribute to the ongoing paradigm shift in cell analysis and sorting technologies toward label-free microfluidic devices, enabling new capabilities in biomedical research tools as well as clinical diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices. © 2018 Wiley Periodicals, Inc.

Simple and label-free electrochemical impedance Amelogenin gene hybridization biosensing based on reduced graphene oxide.

PubMed

Benvidi, Ali; Rajabzadeh, Nooshin; Mazloum-Ardakani, Mohammad; Heidari, Mohammad Mehdi; Mulchandani, Ashok

2014-08-15

The increasing desire for sensitive, easy, low-cost, and label free methods for the detection of DNA sequences has become a vital matter in biomedical research. For the first time a novel label-free biosensor for sensitive detection of Amelogenin gene (AMEL) using reduced graphene oxide modified glassy carbon electrode (GCE/RGO) has been developed. In this work, detection of DNA hybridization of the target and probe DNA was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The optimum conditions were found for the immobilization of probe on RGO surface and its hybridization with the target DNA. CV and EIS carried out in an aqueous solution containing [Fe(CN)6](3-/4-) redox pair have been used for the biosensor characterization. The biosensor has a wide linear range from 1.0×10(-20) to 1.0×10(-14)M with the lower detection limit of 3.2×10(-21)M. Moreover, the present electrochemical detection offers some unique advantages such as ultrahigh sensitivity, simplicity, and feasibility for apparatus miniaturization in analytical tests. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of RGO, which enhances the probe absorption and promotes direct electron transfer between probe and the electrode surface. This electrochemical DNA sensor could be used for the detection of specific ssDNA sequence in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-free and live cell imaging by interferometric scattering microscopy.

PubMed

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay.

PubMed

Li, Yan; Sun, Shao-kai; Yang, Jia-lin; Jiang, Yan

2011-12-07

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.

Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

2015-03-01

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes.

PubMed

Ding, Jiawang; Chen, Yan; Wang, Xuewei; Qin, Wei

2012-02-21

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

Theoretical analysis of bimetallic nanorod dimer biosensors for label-free molecule detection

NASA Astrophysics Data System (ADS)

Das, Avijit; Talukder, Muhammad Anisuzzaman

2018-02-01

In this work, we theoretically analyze a gold (Au) core within silver (Ag) shell (Au@Ag) nanorod dimer biosensor for label-free molecule detection. The incident light on an Au@Ag nanorod strongly couples to localized surface plasmon modes, especially around the tip region. The field enhancement around the tip of a nanorod or between the tips of two longitudinally aligned nanorods as in a dimer can be exploited for sensitive detection of biomolecules. We derive analytical expressions for the interactions of an Au@Ag nanorod dimer with the incident light. We also study the detail dynamics of an Au@Ag nanorod dimer with the incident light computationally using finite difference time domain (FDTD) technique when core-shell ratio, relative position of the nanorods, and angle of incidence of light change. We find that the results obtained using the developed analytical model match well with that obtained using FDTD simulations. Additionally, we investigate the sensitivity of the Au@Ag nanorod dimer, i.e., shift in the resonance wavelength, when a target biomolecule such as lysozyme (Lys), human serum albumin (HSA), anti-biotin (Abn), human catalase (CAT), and human fibrinogen (Fb) protein molecules are attached to the tips of the nanorods.

Low-picomolar, label-free procalcitonin analytical detection with an electrolyte-gated organic field-effect transistor based electronic immunosensor.

PubMed

Seshadri, Preethi; Manoli, Kyriaki; Schneiderhan-Marra, Nicole; Anthes, Uwe; Wierzchowiec, Piotr; Bonrad, Klaus; Di Franco, Cinzia; Torsi, Luisa

2018-05-01

Herein a label-free immunosensor based on electrolyte-gated organic field-effect transistor (EGOFET) was developed for the detection of procalcitonin (PCT), a sepsis marker. Antibodies specific to PCT were immobilized on the poly-3-hexylthiophene (P3HT) organic semiconductor surface through direct physical adsorption followed by a post-treatment with bovine serum albumin (BSA) which served as the blocking agent to prevent non-specific adsorption. Antibodies together with BSA (forming the whole biorecognition layer) served to selectively capture the procalcitonin target analyte. The entire immunosensor fabrication process was fast, requiring overall 45min to be completed before analyte sensing. The EGOFET immunosensor showed excellent electrical properties, comparable to those of bare P3HT based EGOFET confirming reliable biosensing with bio-functional EGOFET immunosensor. The detection limit of the immunosensor was as low as 2.2pM and within a range of clinical relevance. The relative standard deviation of the individual calibration data points, measured on immunosensors fabricated on different chips (reproducibility error) was below 7%. The developed immunosensor showed high selectivity to the PCT analyte which was evident through control experiments. This report of PCT detection is first of its kind among the electronic sensors based on EGOFETs. The developed sensor is versatile and compatible with low-cost fabrication techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

A sensitive label-free immunosensor for detection α-Fetoprotein in whole blood based on anticoagulating magnetic nanoparticles.

PubMed

Xu, Tingting; Chi, Bo; Wu, Fan; Ma, Shangshang; Zhan, Shuyue; Yi, Meihui; Xu, Hong; Mao, Chun

2017-09-15

Accurate values of tumor markers in blood play an especially important role in the diagnosis of illness. Here, based on the combination of three techniques include anticoagulant technology, nanotechnology and biosensing technology, a sensitive label-free immunosensor with anti-biofouling electrode for detection α-Fetoprotein (AFP) in whole blood was developed by anticoagulating magnetic nanoparticles. The obtained products of Fe 3 O 4 -ɛ-PL-Hep nanoparticles were characterized by fourier transform infrared (FT-IR) spectra, transmission electron microscopy (TEM), ζ-potential and vibrating sample magnetometry (VSM). Moreover, the blood compatibility of anticoagulating magnetic nanoparticles was characterized by in vitro coagulation tests, hemolysis assay and whole blood adhesion tests. Combining the anticoagulant property of heparin (Hep) and the good magnetism of Fe 3 O 4 , the Fe 3 O 4 -ɛ-PL-Hep nanoparticles could improve not only the anti-biofouling property of the electrode surface when they contact with whole blood, but also the stability and reproducibility of the proposed immunosensor. Thus, the prepared anticoagulating magnetic nanoparticles modified immunosensor for the detection of AFP showed excellent electrochemical properties with a wide concentration range from 0.1 to 100ng/mL and a low detection limit of 0.072ng/mL. Furthermore, five blood samples were assayed using the developed immunosensor. The results showed satisfactory accuracy with low relative errors. It indicated that our developed immunoassay was competitive and could be potentially used for the detection of whole blood samples directly. Copyright © 2017 Elsevier B.V. All rights reserved.

Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

He, Shuai; Kah, James C. Y.

2017-04-01

Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

Interferometric nanoimmunosensor for label-free and real-time monitoring of Irgarol 1051 in seawater.

PubMed

Chocarro-Ruiz, Blanca; Herranz, Sonia; Fernández Gavela, Adrián; Sanchís, Josep; Farré, Marinella; Marco, M Pilar; Lechuga, Laura M

2018-05-26

An interferometric nanobiosensor for the specific and label-free detection of the pollutant Irgarol 1051 directly in seawater has been settled. Due to the low molecular weight of Irgarol pollutant and its expected low concentration in seawater, the sensor is based on a competitive inhibition immunoassay. Parameters as surface biofunctionalization, concentration of the selective antibody and regeneration conditions have been carefully evaluated. The optimized immunosensor shows a limit of detection of only 3 ng/L, well below the 16 ng/L set by the EU as the maximum allowable concentration in seawater. It can properly operate during 30 assay-regeneration cycles using the same sensor biosurface and with a time-to-result of only 20 min for each cycle. Moreover, the interferometric nanosensor is able to directly detect low concentrations of Irgarol 1051 in seawater without requiring sample pre-treatments and without showing any background signal due to sea matrix effect. Copyright © 2018 Elsevier B.V. All rights reserved.

A comparative Study of Aptasensor Vs Immunosensor for Label-Free PSA Cancer Detection on GQDs-AuNRs Modified Screen-Printed Electrodes.

PubMed

Srivastava, Monika; Nirala, Narsingh R; Srivastava, S K; Prakash, Rajiv

2018-01-31

Label-free and sensitive detection of PSA (Prostate Specific Antigen) is still a big challenge in the arena of prostate cancer diagnosis in males. We present a comparative study for label-free PSA aptasensor and PSA immunosensor for the PSA-specific monoclonal antibody, based on graphene quantum dots-gold nanorods (GQDs-AuNRs) modified screen-printed electrodes. GQDs-AuNRs composite has been synthesized and used as an electro-active material, which shows fast electron transfer and catalytic property. Aptamer or anti-PSA has immobilized onto the surface of modified screen printed electrodes. Three techniques are used simultaneously, viz. cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedence spectroscopy (EIS) to investigate the analytical performance of both PSA aptasensor and PSA immunosensor with its corresponding PSA antigen. Under optimum conditions, both sensors show comparable results with an almost same limit of detection (LOD) of 0.14 ng mL -1 . The results developed with aptasensor and anti-PSA is also checked through the detection of PSA in real samples with acceptable results. Our study suggests some advantages of aptasensor in terms of better stability, simplicity and cost effectiveness. Further our present work shows enormous potential of our developed sensors for real application using voltammetric and EIS techniques simultaneous to get reliable detection of the disease.

Surface Plasmon Resonance Label-Free Monitoring of Antibody Antigen Interactions in Real Time

ERIC Educational Resources Information Center

Kausaite, Asta; van Dijk, Martijn; Castrop, Jan; Ramanaviciene, Almira; Baltrus, John P.; Acaite, Juzefa; Ramanavicius, Arunas

2007-01-01

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without…

A label-free fluorimetric detection of biothiols based on the oxidase-like activity of Ag+ ions

NASA Astrophysics Data System (ADS)

Li, Ru; Lei, Cuihua; Zhao, Xian-En; Gao, Yue; Gao, Han; Zhu, Shuyun; Wang, Hua

2018-01-01

In this work, a label-free and sensitive fluorimetric method has been developed for the detections of biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), based on the specific biothiol-induced inhibition of the oxidase-like activity of silver ions (Ag+). It is well established that o-phenylenediamine (OPD) can be oxidized by Ag+ ions to generate fluorescent 2,3-diaminophenazine (OPDox). The introduction of biothiols would inhibit the oxidation of OPD by Ag+ due to the strong coordination between biothiols and Ag+. The changes of fluorescence intensities obtained in the Ag+-OPD system exhibited good linear correlations in the ranges of 0.50-30.0 μM for Cys, 1.0-45.0 μM for Hcy and 0.50-40.0 μM for GSH. The detection limits (S/N = 3) of Cys, Hcy and GSH were 110 nM, 200 nM and 150 nM, respectively. Subsequently, the developed fluorimetric method was successfully applied for the detection of biothiols in human serum.

An ATMND/SGI based label-free and fluorescence ratiometric aptasensor for rapid and highly sensitive detection of cocaine in biofluids.

PubMed

Wang, Jiamian; Song, Jie; Wang, Xiuyun; Wu, Shuo; Zhao, Yanqiu; Luo, Pinchen; Meng, Changgong

2016-12-01

A label-free ratiometric fluorescence aptasensor has been developed for the rapid and sensitive detection of cocaine in complex biofluids. The fluorescent aptasensor is composed of a non-labeled GC-38 cocaine aptamer which serves as a basic sensing unit and two fluorophores, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and SYBR Green I (SGI) which serves as a signal reporter and a build-in reference, respectively. The detection principle is based on a specific cocaine mediated ATMND displacement reaction and the corresponding change in the fluorescence ratio of ATMND to SGI. Due to the high affinity of the non-labeled aptamer, the good precision originated from the ratiometric method, and the good fluorescence quantum yield of the fluorophore, the aptasensor shows good analytical performance with respect to cocaine detection. Under optimal conditions, the aptasensor shows a linear range of 0.10-10μM and a low limit of detection of 56nM, with a fast response of 20s. The low limit of detection is comparable to most of the fluorescent aptasensors with signal amplification strategies and much lower than all of the unamplified cocaine aptasensors. Practical sample analysis in a series of complex biofluids, including urine, saliva and serum, also indicates the good precision, stability, and high sensitivity of the aptasensor, which may have great potential for the point-of-care screening of cocaine in complex biofluids. Copyright © 2016 Elsevier B.V. All rights reserved.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

PubMed

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-08-11

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

PubMed Central

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-01-01

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. PMID:27529243

Label-Free Biosensors Based on Bimodal Waveguide (BiMW) Interferometers.

PubMed

Herranz, Sonia; Gavela, Adrián Fernández; Lechuga, Laura M

2017-01-01

The bimodal waveguide (BiMW) sensor is a novel common path interferometric transducer based on the evanescent field detection principle, which in combination with a bio-recognition element allows the direct detection of biomolecular interactions in a label-free scheme. Due to its inherent high sensitivity it has great potential to become a powerful analytical tool for monitoring substances of interest in areas such as environmental control, medical diagnostics and food safety, among others. The BiMW sensor is fabricated using standard silicon-based technology allowing cost-effective production, and meeting the requirements of portability and disposability necessary for implementation in a point-of-care (POC) setting.In this chapter we describe the design and fabrication of the BiMW transducer, as well as its application for bio-sensing purposes. We show as an example the biosensor capabilities two different applications: (1) the immunodetection of Irgarol 1051 biocide useful in the environmental field, and (2) the detection of human growth hormone as used in clinical diagnostics. The detection is performed in real time by monitoring changes in the intensity pattern of light exiting the BiMW transducer resulting from antigen-antibody interactions on the surface of the sensor.

Label-free biosensing of Salmonella enterica serovars at single-cell level

USDA-ARS?s Scientific Manuscript database

Nanotechnology has greatly facilitated the development of label-free biosensors. The atomic force microscopy (AFM) has been used to study the molecular mechanism of the reactions for protein and aptamers. The surface plasmon resonance (SPR) have been used in fast detection of various pathogenic bact...

A fluorescent graphitic carbon nitride nanosheet biosensor for highly sensitive, label-free detection of alkaline phosphatase.

PubMed

Xiang, Mei-Hao; Liu, Jin-Wen; Li, Na; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

2016-02-28

Graphitic C3N4 (g-C3N4) nanosheets provide an attractive option for bioprobes and bioimaging applications. Utilizing highly fluorescent and water-dispersible ultrathin g-C3N4 nanosheets, a highly sensitive, selective and label-free biosensor has been developed for ALP detection for the first time. The developed approach utilizes a natural substrate of ALP in biological systems and thus affords very high catalytic efficiency. This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to 1000 U L(-1) with a low detection limit of 0.08 U L(-1), which is among the most sensitive assays for ALP. It is expected that the developed method may provide a low-cost, convenient, rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications.

"Molecular beacon"-hosted thioflavin T: Applications for label-free fluorescent detection of iodide and logic operations.

PubMed

Li, Yan-Yun; Jiang, Xiao-Qin; Lu, Ling-Fei; Zhang, Min; Shi, Guoyue

2016-04-01

In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I(-)) detection based on "molecular beacon (MB)"-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I(-) ions over other anions interference due to the strong binding force between I(-) ions with Hg(2+). Upon the addition of I(-) ions, it would capture Hg(2+) from a T-Hg(2+)-T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I(-) in real samples such as human urine. Copyright © 2016 Elsevier B.V. All rights reserved.

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

PubMed

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA detection.

PubMed

He, Yi; Huang, Guangming; Cui, Hua

2013-11-13

It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

2014-09-01

A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

A dual marker label free electrochemical assay for Flavivirus dengue diagnosis.

PubMed

Santos, Adriano; Bueno, Paulo R; Davis, Jason J

2018-02-15

Dengue is a RNA viral illness of the genus Flavivirus which can cause, depending on the pervasiveness of the infection, hemorrhagic dengue fever or dengue shock syndrome. Herein we present an electrochemical label free approach enabling the rapid sensitive quantification of NS1 and IgG (supporting an ability to distinguish primary and secondary infections). Using a bifunctional SAM containing PEG moieties and a tethered redox thiol, both markers are detectable across clinically relevant levels by label free impedance derived redox capacitance. A subsequent frequency specific immittance function approach enables assaying (within seconds) with no impairment of analytical quality (linearity, sensitivity and variance). Copyright © 2017 Elsevier B.V. All rights reserved.

Single Zno Nanowire-Based Biofet Sensors for Ultrasensitive, Label-Free and Real-Time Detection of Uric Acid

NASA Astrophysics Data System (ADS)

Lin, Pei; Liu, Xi; Yan, Xiaoqin; Kang, Zhuo; Lei, Yang; Zhao, Yanguang

2012-08-01

Qualitative and quantitative detection of biological and chemical species is crucial in many areas, ranging from clinical diagnosis to homeland security. Due to the advantages of ultrahigh sensitivity, label-free, fast readout and easy fabrication over the traditional detection systems, semiconductor nanowire based electronic devices have emerged as a potential platform. In this paper, we fabricated a single ZnO nanowire-based bioFET sensor for the detection of low and high concentration uric acid solution at the same time. The addition of uric acid with the concentrations from 1 pM to 0.5 mM resulted in the electrical conductance changes of up to 227 nS, and the response time turns out to be in the order of millisecond. The ZnO NW biosensor could easily detect as low as 1 pM of the uric acid with 14.7 nS of conductance increase, which implied that the sensitivity of the biosensor can be below the 1pM concentration.

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.

Robust label-free biosensing using microdisk laser arrays with on-chip references.

PubMed

Wondimu, S F; Hippler, M; Hussal, C; Hofmann, A; Krämmer, S; Lahann, J; Kalt, H; Freude, W; Koos, C

2018-02-05

Whispering-gallery mode (WGM) microdisk lasers show great potential for highly sensitive label-free detection in large-scale sensor arrays. However, when used in practical applications under normal ambient conditions, these devices suffer from temperature fluctuations and photobleaching. Here we demonstrate that these challenges can be overcome by a novel referencing scheme that allows for simultaneous compensation of temperature drift and photobleaching. The technique relies on reference structures protected by locally dispensed passivation materials, and can be scaled to extended arrays of hundreds of devices. We prove the viability of the concept in a series of experiments, demonstrating robust and sensitive label-free detection over a wide range of constant or continuously varying temperatures. To the best of our knowledge, these measurements represent the first demonstration of biosensing in active WGM devices with simultaneous compensation of both photobleaching and temperature drift.

Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

2016-03-01

Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

PubMed

Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

2016-06-15

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Automated selected reaction monitoring software for accurate label-free protein quantification.

PubMed

Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

2012-07-06

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

Direct-detection Free-space Laser Transceiver Test-bed

NASA Technical Reports Server (NTRS)

Krainak, Michael A.; Chen, Jeffrey R.; Dabney, Philip W.; Ferrara, Jeffrey F.; Fong, Wai H.; Martino, Anthony J.; McGarry Jan. F.; Merkowitz, Stephen M.; Principe, Caleb M.; Sun, Siaoli;

Enzyme-free and label-free ultra-sensitive colorimetric detection of Pb(2+) using molecular beacon and DNAzyme based amplification strategy.

PubMed

Yun, Wen; Cai, Dingzhou; Jiang, JiaoLai; Zhao, Pengxiang; Huang, Yu; Sang, Ge

2016-06-15

An enzyme-free and label-free colorimetric Pb(2+) sensor based on DNAzyme and molecular beacon (MB) has been developed and demonstrated by recycle using enzyme strand for signal amplification. The substrate strand DNA (S-DNA) of DNAzyme could be converted into MB structure with base pairs of stem part at the both ends. The MB could hybridize with enzyme strand DNA (E-DNA) to form DNAzyme, and be activated and cleaved in the presence of Pb(2+). The cleaved MB is much less stable, releasing from the DNAzyme as two product pieces. The product pieces of MB are flexible and could bind to unmodified AuNPs to effectively stabilize them against salt-induced aggregation. Then, the E-DNA is liberated to catalyze the next reaction and amplify the response signal. By taking advantage of repeated using of E-DNA, our proposed method exhibited high sensitive for Pb(2+) detection in a linear range from 0.05 to 5 nM with detection limit of 20 pM by UV-vis spectrometer. Moreover, this method was also used for determination of Pb(2+) in river water samples with satisfying results. Importantly, this strategy could reach high sensitivity without any modification and complex enzymatic or hairpins based amplification procedures. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-Free Alignment of Nonmagnetic Particles in a Small Uniform Magnetic Field.

PubMed

Wang, Zhaomeng; Wang, Ying; Wu, Rui Ge; Wang, Z P; Ramanujan, R V

2018-01-01

Label-free manipulation of biological entities can minimize damage, increase viability and improve efficiency of subsequent analysis. Understanding the mechanism of interaction between magnetic and nonmagnetic particles in an inverse ferrofluid can provide a mechanism of label-free manipulation of such entities in a uniform magnetic field. The magnetic force, induced by relative magnetic susceptibility difference between nonmagnetic particles and surrounding magnetic particles as well as particle-particle interaction were studied. Label-free alignment of nonmagnetic particles can be achieved by higher magnetic field strength (Ba), smaller particle spacing (R), larger particle size (rp1), and higher relative magnetic permeability difference between particle and the surrounding fluid (Rμr). Rμr can be used to predict the direction of the magnetic force between both magnetic and nonmagnetic particles. A sandwich structure, containing alternate layers of magnetic and nonmagnetic particle chains, was studied. This work can be used for manipulation of nonmagnetic particles in lab-on-a-chip applications.

A sensitive electrochemical immunosensor based on poly(2-aminobenzylamine) film modified screen-printed carbon electrode for label-free detection of human immunoglobulin G.

PubMed

Putnin, Thitirat; Jumpathong, Watthanachai; Laocharoensuk, Rawiwan; Jakmunee, Jaroon; Ounnunkad, Kontad

2018-08-01

This work focuses on fabricating poly(2-aminobenzylamine)-modified screen-printed carbon electrode as an electrochemical immunosensor for the label-free detection of human immunoglobulin G. To selectively detect immunoglobulin G, the anti-immunoglobulin G antibody with high affinity to immunoglobulin G was covalently linked with the amine group of poly(2-aminobenzylamine) film-deposited screen-printed carbon electrode. The selectivity for immunoglobulin G was subsequently assured by being challenged with redox-active interferences and adventitious adsorption did not significantly interfere the analyte signal. To obviate the use of costly secondary antibody, the [Fe(CN) 6 ] 4-/3- redox probe was instead applied to measure the number of human immunoglobulin G through the immunocomplex formation that is quantitatively related to the level of the differential pulse voltammetric current. The resulting immunosensor exhibited good sensitivity with the detection limit of 0.15 ng mL -1 , limit of quantitation of 0.50 ng mL -1 and the linear range from 1.0 to 50 ng mL -1 . Given those striking analytical performances and the affordability arising from using cheap screen-printed carbon electrode with label-free detection, the immunosensor serves as a promising model for the next-step development of a diagnostic tool.

Label-free detection of protein biomolecules secreted from a heart-on-a-chip model for drug cardiotoxicity evaluation

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Zhang, Yu Shrike; Bustamante, Gilbert; Li, Le; Lauderdale, Matthew; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

2018-02-01

Efficient methods for the accurate analysis of drug toxicities are in urgent demand as failures of newly discovered drug candidates due to toxic side effects have resulted in about 30% of clinical attrition. The high failure rate is partly due to current inadequate models to study drug side effects, i.e., common animal models may fail due to its misrepresentation of human physiology. Therefore, much effort has been allocated in the development of organ-on-a-chip models which offer a variety of human organ models mimicking a multitude of human physiological conditions. However, it is extremely challenging to analyze the transient and long-term response of the organ models to drug treatments during drug toxicity tests, as the proteins secreted from the organ-on-a-chip model are minute due to its volumetric size, and current methods for detecting said biomolecules are not suitable for real-time monitoring. As protein biomolecules are being continuously secreted from the human organ model, fluorescence techniques are practically impossible to achieve real-time fluorescence labeling in the dynamically changing environment, thus making a label-free approach highly desirable for the organ-on-achip applications. In this paper, we report the use of a photonic-crystal biosensor integrated with a microfluidic system for sensitive label-free bioassays of secreted protein biomolecules from a heart-on-the-chip model created with cardiomyocytes derived from human induced pluripotent stem cells.

Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

PubMed

Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

2012-01-13

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

A label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

PubMed Central

Xu, Weichen; Lu, Yi

2009-01-01

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibits high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes. PMID:20017558

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

PubMed

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika; Yki-Järvinen, Hannele; Ikonen, Elina

2016-01-01

Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

PubMed Central

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

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.

Label-free immunosensor based on hyperbranched polyester for specific detection of α-fetoprotein.

PubMed

Niu, Yanlian; Yang, Tian; Ma, Shangshang; Peng, Fang; Yi, Meihui; Wan, Mimi; Mao, Chun; Shen, Jian

2017-06-15

A novel label-free immunosensor based on hyperbranched polyester nanoparticles with nitrite groups (HBPE-NO 2 ), which were synthesized through a simple one-step chemical reaction, was first developed for specific detection of α-fetoprotein (AFP), the tumor marker for liver cancer. The obtained HBPE-NO 2 nanoparticles (NPs) were characterized by the proton nuclear magnetic resonance spectroscopy ( 1 H NMR), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). And the fabricated process of immunosensor was investigated by attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR), static water contact angles, scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical performances of the AFP immunosensor were studied. Results indicated the prepared HBPE-NO 2 -modified immunosensor showed excellent electrochemical properties and satisfactory accuracy for the detection of AFP of the real clinical samples that attributed to the properties of the HBPE-NO 2 NPs, which had nanosized structure to increase the specific surface area and unique chemical reactivity for loading capacity of protein molecules. Construction of biosensors using the structure and properties of hyperbranched molecules will offer ideal electrode substrates, which provided more possibilities for the design of biosensor. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free impedimetric aptasensor for detection of femtomole level acetamiprid using gold nanoparticles decorated multiwalled carbon nanotube-reduced graphene oxide nanoribbon composites.

PubMed

Fei, Airong; Liu, Qian; Huan, Juan; Qian, Jing; Dong, Xiaoya; Qiu, Baijing; Mao, Hanping; Wang, Kun

2015-08-15

Gold nanoparticles (Au NPs) decorated multiwalled carbon nanotube-reduced graphene oxide nanoribbon (Au/MWCNT-rGONR) composites were synthesized by a one-pot reaction. By employing the resulting Au/MWCNT-rGONR composites as the support for aptamer immobilization, we developed an ultrasensitive label-free electrochemical impedimetric aptasensor for acetamiprid detection, which was based on that the variation of electron transfer resistance was relevant to the formation of acetamiprid-aptamer complex at the modified electrode surface. Compared with pure Au NPs and MWCNT-rGONR, the Au/MWCNT-rGONR composites modified electrode was the most sensitive aptasensing platform for the determination of acetamiprid. The proposed aptasensor displayed a linear response for acetamiprid in the range from 5×10(-14) M to 1×10(-5) M with an extremely low detection limit of 1.7×10(-14) M (S/N=3). In addition, this impedimetric aptasensor possessed great advantages including the simple operation process, low-cost, selectivity and sensitivity, which provided a promising model for the aptamer-based detection with a direct impedimetric method. Copyright © 2015 Elsevier B.V. All rights reserved.

A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

NASA Astrophysics Data System (ADS)

Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

2016-03-01

Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

Label-Free Fluorescent DNA Dendrimers for microRNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G-Quadruplex with Low Background Signal.

PubMed

Xue, Qingwang; Liu, Chunxue; Li, Xia; Dai, Li; Wang, Huaisheng

2018-04-18

Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative

High-throughput label-free microcontact printing graphene-based biosensor for valley fever.

PubMed

Tsai, Shih-Ming; Goshia, Tyler; Chen, Yen-Chang; Kagiri, Agnes; Sibal, Angelo; Chiu, Meng-Hsuen; Gadre, Anand; Tung, Vincent; Chin, Wei-Chun

2018-06-18

The highly prevalent and virulent disease in the Western Hemisphere Coccidioidomycosis, also known as Valley Fever, can cause serious illness such as severe pneumonia with respiratory failure. It can also take on a disseminated form where the infection spreads throughout the body. Thus, a serious impetus exists to develop effective detection of the disease that can also operate in a rapid and high-throughput fashion. Here, we report the assembly of a highly sensitive biosensor using reduced graphene oxide (rGO) with Coccidioides(cocci) antibodies as the target analytes. The facile design made possible by the scalable microcontact printing (μCP) surface patterning technique which enables rapid, ultrasensitive detection. It provides a wide linear range and sub picomolar (2.5 pg/ml) detection, while also delivering high selectivity and reproducibility. This work demonstrates an important advancement in the development of a sensitive label-free rGO biosensor for Coccidioidomycosis detection. This result also provides the potential application of direct pathogen diagnosis for the future biosensor development. Copyright © 2018 Elsevier B.V. All rights reserved.

A label-free impedimetric DNA sensing chip integrated with AC electroosmotic stirring.

PubMed

Wu, Ching-Chou; Yang, Dong-Jie

2013-05-15

AC electroosmosis (ACEO) flow and label-free electrochemical impedance spectroscopy are employed to increase the hybridization rate and specifically detect target DNA (tDNA) concentrations. A low-ionic-strength solution, 6.1μS/cm 1mM Tris (pH 9.3), was used to produce ACEO and proved the feasibility of hybridization. Adequate voltage parameters for the simultaneous ACEO driving and DNA hybridization in the 1mM Tris solution were 1.5 Vpp and 200Hz. Moreover, an electrode set with a 1:4 ring width-to-disk diameter ratio exhibited a larger ACEO velocity above the disk electrode surface to improve collecting efficiency. The ACEO-integrated DNA sensing chips could reach 90% saturation hybridization within 117s. The linear range and detection limit of the sensors was 10aM-10pM and 10aM, respectively. The label-free impedimetric DNA sensing chips with integrated ACEO stirring can perform rapid hybridization and highly-sensitive detections to specifically measure tDNA concentrations. Copyright © 2013 Elsevier B.V. All rights reserved.

Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

PubMed

Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

2016-01-15

Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.

PubMed

Wu, Haiyun; Ohnuki, Hitoshi; Ota, Shirei; Murata, Masataka; Yoshiura, Yasutoshi; Endo, Hideaki

2017-07-15

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml -1 (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

NASA Astrophysics Data System (ADS)

Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

2014-11-01

A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml-1 with a limit of detection of 0.16 ng ml-1.

A nuclease-assisted label-free aptasensor for fluorescence turn-on detection of ATP based on the in situ formation of copper nanoparticles.

PubMed

Song, Quanwei; Wang, Ruihua; Sun, Feifei; Chen, Hongkun; Wang, Zoumengke; Na, Na; Ouyang, Jin

2017-01-15

Owing to their promising advantages in biochemical analysis, aptamer-based sensing systems for the fluorescence detection of important biomolecules are being extensively investigated. Herein, we propose a turn-on fluorescent aptasensor for label-free detection of adenosine triphosphate (ATP) by utilizing the in situ formation of copper nanoparticles (CuNPs) and the specific digestion capability of exonuclease I (Exo I). In this assay, the addition of ATP can effectively hinder the digestion of aptamer-derived oligonucleotides due to the G-quadruplex structure. Accordingly, the remaining poly thymine at 5'-terminus of substrate DNA can serve as an efficient template for red-emitting fluorescent CuNPs with a Mega-Stokes shifting in buffered solution, which can be used to evaluate the concentration of ATP. This method is cost-effective and facile, because it avoids the use of traditional dye-labeled DNA strands and complex operation steps. Under optimized conditions, this method achieves a selective response for ATP with a detection limit of 93nM, and exhibits a good detection performance in biological samples. Copyright © 2016 Elsevier B.V. All rights reserved.

A label-free silicon quantum dots-based photoluminescence sensor for ultrasensitive detection of pesticides.

PubMed

Yi, Yinhui; Zhu, Gangbing; Liu, Chang; Huang, Yan; Zhang, Youyu; Li, Haitao; Zhao, Jiangna; Yao, Shouzhuo

2013-12-03

Sensitive, rapid, and simple detection methods for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents are in urgent demand. A novel label-free silicon quantum dots (SiQDs)-based sensor was designed for ultrasensitive detection of pesticides. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce betaine and H2O2 which can quench the photoluminescence (PL) of SiQDs. Upon the addition of pesticides, the activity of AChE is inhibited, leading to the decrease of the generated H2O2, and hence the PL of SiQDs increases. By measuring the increase in SiQDs PL, the inhibition efficiency of pesticide to AChE activity was evaluated. It was found that the inhibition efficiency was linearly dependent on the logarithm of the pesticides concentration. Consequently, pesticides, such as carbaryl, parathion, diazinon, and phorate, were determined with the SiQDs PL sensing method. The lowest detectable concentrations for carbaryl, parathion, diazinon, and phorate reached 7.25 × 10(-9), 3.25 × 10(-8), 6.76 × 10(-8), and 1.9 × 10(-7) g/L, respectively, which were much lower than those previously reported. The detecting results of pesticide residues in food samples via this method agree well with those from high-performance liquid chromatography. The simple strategy reported here should be suitable for on-site pesticides detection, especially in combination with other portable platforms.

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.

PubMed

Husale, Sudhir; Persson, Henrik H J; Sahin, Ozgur

2009-12-24

Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study

PubMed Central

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika

2016-01-01

Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading. PMID:26808140

Evanescent wave absorbance based fiber optic biosensor for label-free detection of E. coli at 280 nm wavelength.

PubMed

Bharadwaj, Reshma; Sai, V V R; Thakare, Kamini; Dhawangale, Arvind; Kundu, Tapanendu; Titus, Susan; Verma, Pradeep Kumar; Mukherji, Soumyo

2011-03-15

A novel label-free technique for the detection of pathogens based on evanescent wave absorbance (EWA) changes at 280 nm from a U-bent optical fiber sensor is demonstrated. Bending a decladded fiber into a U-shaped structure enhances the penetration depth of evanescent waves and hence sensitivity of the probe. We show that the enhanced EWA response from such U-bent probes, caused by the inherent optical absorbance properties of bacterial cells or biomolecules specifically bound to the sensor surface, can be exploited for the detection of pathogens. A portable optical set-up with a UV light emitting diode, a spectrometer and U-bent fiber optic probe of 200 μm core diameter, 0.75 mm bend radius and effective probe length of 1cm demonstrated an ability to detect less than 1000 cfu/ml. Copyright © 2011. Published by Elsevier B.V.

Label-free, real-time interaction and adsorption analysis 1: surface plasmon resonance.

PubMed

Fee, Conan J

2013-01-01

A key requirement for the development of proteins for use in nanotechnology is an understanding of how individual proteins bind to other molecules as they assemble into larger structures. The introduction of labels to enable the detection of biomolecules brings the inherent risk that the labels themselves will influence the nature of biomolecular interactions. Thus, there is a need for label-free interaction and adsorption analysis. In this and the following chapter, two biosensor techniques are reviewed: surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM). Both allow real-time analysis of biomolecular interactions and both are label-free. The first of these, SPR, is an optical technique that is highly sensitive to the changes in refractive index that occur with protein (or other molecule) accumulation near an illuminated gold surface. Unlike QCM ( Chapter 18 ) SPR is not affected by the water that may be associated with the adsorbed layer nor by conformational changes in the adsorbed species. SPR thus provides unique information about the interaction of a protein with its binding partners.

Label-free as-grown double wall carbon nanotubes bundles for Salmonella typhimurium immunoassay.

PubMed

Punbusayakul, Niramol; Talapatra, Saikat; Ajayan, Pulickel M; Surareungchai, Werasak

2013-01-01

A label-free immunosensor from as-grown double wall carbon nanotubes (DW) bundles was developed for detecting Salmonella typhimurium. The immunosensor was fabricated by using the as-grown DW bundles as an electrode material with an anti-Salmonella impregnated on the surface. The immunosensor was electrochemically characterized by cyclic voltammetry. The working potential (100, 200, 300 and 400 mV vs. Ag/AgCl) and the anti-Salmonella concentration (10, 25, 50, 75, and 100 μg/mL) at the electrode were subsequently optimized. Then, chronoamperometry was used with the optimum potential of 100 mV vs. Ag/AgCl) and the optimum impregnated anti-Salmonella of 10 μg/mL to detect S. typhimurium cells (0-10(9) CFU/mL). The DW immunosensor exhibited a detection range of 10(2) to 10(7) CFU/mL for the bacteria with a limit of detection of 8.9 CFU/mL according to the IUPAC recommendation. The electrode also showed specificity to S. typhimurium but no current response to Escherichia coli. These findings suggest that the use of a label-free DW immunosensor is promising for detecting S. typhimurium.

Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

NASA Astrophysics Data System (ADS)

Kunstar, Aliz; Leijten, Jeroen; van Leuveren, Stefan; Hilderink, Janneke; Otto, Cees; van Blitterswijk, Clemens A.; Karperien, Marcel; van Apeldoorn, Aart A.

2012-11-01

Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

A fluorescent aptasensor for amplified label-free detection of adenosine triphosphate based on core-shell Ag@SiO2 nanoparticles.

PubMed

Song, Quanwei; Peng, Manshu; Wang, Le; He, Dacheng; Ouyang, Jin

2016-03-15

The novel, facile and universal aptamer-based methods for the highly sensitive and selective fluorescence detection of important biomolecules have attracted considerable interest. Here, we present a label-free aptasensor for adenosine triphosphate (ATP) detection in aqueous solutions by using an ultra-sensitive nucleic acid stain PicoGreen (PG) as a fluorescent indicator and core-shell Ag@SiO2 nanoparticles (NPs) as a metal-enhanced fluorescence (MEF) platform. In the presence of ATP, the complementary DNA (cDNA)/aptamer duplexes confined onto the Ag@SiO2 NPs surface can release their aptamers into the buffered solution, causing a significant reduction in fluorescence intensity. By virtue of the amplified fluorescence signal, this aptasensor toward ATP can achieve a detection limit of 14.2 nM with a wide linear range and exhibit a good assay performance in complex biological samples. This sensing approach is cost-effective and efficient because it avoids the fluorescence labeling process and the use of any enzymes. Hence, this method may offer an alternative tool for determining the concentrations of ATP in biochemical and biomedical research. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer.

PubMed

Soler, Maria; Estevez, M-Carmen; Villar-Vazquez, Roi; Casal, J Ignacio; Lechuga, Laura M

2016-08-03

Colorectal cancer is treatable and curable when detected at early stages. However there is a lack of less invasive and more specific screening and diagnosis methods which would facilitate its prompt identification. Blood circulating autoantibodies which are immediately produced by the immune system at tumor appearance have become valuable biomarkers for preclinical diagnosis of cancer. In this work, we present the rapid and label-free detection of colorectal cancer autoantibodies directly in blood serum or plasma using a recently developed nanoplasmonic biosensor. Our nanoplasmonic device offers sensitive and real-time quantification of autoantibodies with excellent selectivity and reproducibility, achieving limits of detection around 1 nM (150-160 ng mL(-1)). A preliminary evaluation of clinical samples of colorectal cancer patients has shown good correlation with ELISA. These results demonstrate the reliability of the nanobiosensor strategy and pave the way towards the achievement of a sensitive diagnostic tool for early detection of colorectal cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Gluten-Free Labeling of Foods

MedlinePlus

... Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products Food Home Food Guidance & Regulation Guidance Documents & Regulatory Information by Topic Allergens Gluten-Free Labeling of Foods Share Tweet Linkedin Pin it More sharing options ...

Direct Detection of Biotinylated Proteins by Mass Spectrometry

PubMed Central

2015-01-01

Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h. PMID:25117199

Label-free capacitive biosensor for sensitive detection of multiple biomarkers using gold interdigitated capacitor arrays.

PubMed

Qureshi, Anjum; Niazi, Javed H; Kallempudi, Saravan; Gurbuz, Yasar

2010-06-15

In this study, a highly sensitive and label-free multianalyte capacitive immunosensor was developed based on gold interdigitated electrodes (GID) capacitor arrays to detect a panel of disease biomarkers. C-reactive protein (CRP), TNFalpha, and IL6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of a panel of biomarkers for a disease could enable accurate prediction of a disease risk. The detection of protein biomarkers was based on relative change in capacitive/dielectric properties. Two different lab-on-a-chip formats were employed for multiple biomarker detection on GID-capacitors. In format I, capacitor arrays were immobilized with pure forms of anti-CRP, -TNFalpha, and -IL6 antibodies in which each capacitor array contained a different immobilized antibody. Here, the CRP and IL6 were detected in the range 25 pg/ml to 25 ng/ml and 25 pg/ml to 1 ng/ml for TNFalpha in format I. Sensitive detection was achieved with chips co-immobilized (diluted) with equimolar mixtures of anti-CRP, -IL6, and -TNFalpha antibodies (format II) in which all capacitors in an array were identical and tested for biomarkers with sequential incubation. The resulting response to CRP, IL6, and TNFalpha in format II for all biomarkers was found to be within 25 pg/ml to 25 ng/ml range. The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of biomarker panel in suspected subjects for early diagnosis. Copyright 2010 Elsevier B.V. All rights reserved.

A Nano-Au/C-MWCNT based label free amperometric immunosensor for the detection of capsicum chlorosis virus in bell pepper.

PubMed

Sharma, Anshul; Kaushal, Ankur; Kulshrestha, Saurabh

2017-07-01

Accurate and on time diagnosis of plant viruses is an essential prerequisite for efficient control in field conditions. A number of diagnostic methods have been reported with the required level of sensitivity. Here, we propose a label free immunosensor for efficient and sensitive detection of capsicum chlorosis virus (CaCV) in bell pepper. Antigen was immobilized over the surface of gold nanoparticle/multi-walled carbon nanotube (Nano-Au/C-MWCNT) screen printed electrodes using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross linking chemistry followed by interaction with groundnut bud necrosis virus (GBNV)/CaCV specific polyclonal antibody. The electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) using the redox indicator. Electrode surface characterization was done by performing scanning electron microscopy (SEM). Electrochemical studies showed positive results at different antigenic dilutions ranging from 10 -2 - 8x10 -5 . The sensitivity of the immunosensor developed has been compared with direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) and the results showed that the immunosensor developed was 800-1000 times more sensitive, when compared to DAC-ELISA for CaCV detection. The immunosensor we have developed is economical and sensitive and could be used for immediate determination of the presence of virus in extracts from bell pepper leaves.

Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

PubMed

Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

2016-01-30

Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in

Highly sensitive and label-free electrochemical detection of microRNAs based on triple signal amplification of multifunctional gold nanoparticles, enzymes and redox-cycling reaction.

PubMed

Liu, Lin; Xia, Ning; Liu, Huiping; Kang, Xiaojing; Liu, Xiaoshuan; Xue, Chan; He, Xiaoling

2014-03-15

MicroRNAs (miRNAs) are believed to be important for cancer diagnosis and prognosis, serving as reliable molecular biomarkers. In this work, we presented a label-free and highly sensitive electrochemical genosensor for miRNAs detection with the triple signal amplification of gold nanoparticles (AuNPs), alkaline phosphatase (ALP) and p-aminophenol (p-AP) redox cycling. The label-free strategy is based on the difference in the structures of RNA and DNA. Specifically, miRNAs were first captured by the pre-immobilized DNA probes on a gold electrode. Next, the cis-diol group of ribose sugar at the end of the miRNAs chain allowed 3-aminophenylboronic acid (APBA)/biotin-modified multifunctional AuNPs (denoted as APBA-biotin-AuNPs) to be attached through the formation of a boronate ester covalent bond, which facilitated the capture of streptavidin-conjugated alkaline phosphatase (SA-ALP) via the biotin-streptavidin interaction. After the addition of the 4-aminophenylphosphate (p-APP) substrate, the enzymatic conversion from p-APP to p-AP occurred. The resulting p-AP could be cycled by a chemical reducing reagent after its electro-oxidization on the electrode (known as p-AP redox cycling), thus enabling an increase in the anodic current. As a result, the current increased linearly with the miRNAs concentration over a range of 10 fM-5 pM, and a detection limit of 3 fM was achieved. We believe that this work will be valuable for the design of new types of label-free and sensitive electrochemical biosensors. © 2013 Published by Elsevier B.V.

Label-Free Detection of Cancer Biomarkers Using an In-Line Taper Fiber-Optic Interferometer and a Fiber Bragg Grating

PubMed Central

Sun, Dandan; Wang, Guanjun

2017-01-01

A compact and label-free optical fiber sensor based on a taper interferometer cascaded with a fiber Bragg grating (FBG) is proposed and experimentally demonstrated for detection of a breast cancer biomarker (HER2). The tapered fiber-optic interferometer is extremely sensitive to the ambient refractive index (RI). In addition, being insensitive to the RI variation, the FBG can be applied as a temperature thermometer due to its independent response to the temperature. Surface functionalization to the sensor is carried out to achieve specific targeting of the unlabeled biomarkers. The result shows that the proposed sensor presents a low limit-of-detection (LOD) of 2 ng/mL, enabling its potentials of application in early diagnosis on the breast cancer. PMID:29113127

Label-Free Raman Imaging to Monitor Breast Tumor Signatures

NASA Astrophysics Data System (ADS)

Ciubuc, John

Methods built on Raman spectroscopy have shown major potential in describing and discriminating between malignant and benign specimens. Accurate, real-time medical diagnosis benefits in substantial improvements through this vibrational optical method. Not only is acquisition of data possible in milliseconds and analysis in minutes, Raman allows concurrent detection and monitoring of all biological components. Besides validating a significant Raman signature distinction between non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, this study reveals a label-free method to assess overexpression of epidermal growth factor receptors (EGFR) in tumor cells. EGFR overexpression sires Raman features associated with phosphorylated threonine and serine, and modifications of DNA/RNA characteristics. Investigations by gel electrophoresis reveal EGF induction of phosphorylated Akt, agreeing with the Raman results. The analysis presented is a vital step toward Raman-based evaluation of EGF receptors in breast cancer cells. With the goal of clinically applying Raman-guided methods for diagnosis of breast tumors, the current results lay the basis for proving label-free optical alternatives in making prognosis of the disease.

freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

PubMed

Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

2015-01-01

Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries.

PubMed

Mishra, Rupesh K; Hayat, Akhtar; Catanante, Gaëlle; Ocaña, Cristina; Marty, Jean-Louis

2015-08-19

Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15-2.5 ng/mL, with an acceptable recovery percentage (91-95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free fluorescent aptasensor for potassium ion using structure-switching aptamers and berberine

NASA Astrophysics Data System (ADS)

Guo, Yanqing; Chen, Yanxia; Wei, Yanli; Li, Huanhuan; Dong, Chuan

2015-02-01

A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N = 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust, and stable in rough conditions.

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7.

PubMed

Li, Dujuan; Feng, Yangyang; Zhou, Ling; Ye, Zunzhong; Wang, Jianping; Ying, Yibin; Ruan, Chuanmin; Wang, Ronghui; Li, Yanbin

2011-02-14

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10(2)-10(5) cfu mL(-1) and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor. Copyright © 2010 Elsevier B.V. All rights reserved.

Light-Driven Nano-oscillators for Label-Free Single-Molecule Monitoring of MicroRNA.

PubMed

Chen, Zixuan; Peng, Yujiao; Cao, Yue; Wang, Hui; Zhang, Jian-Rong; Chen, Hong-Yuan; Zhu, Jun-Jie

2018-06-13

Here, we present a mapping tool based on individual light-driven nano-oscillators for label-free single-molecule monitoring of microRNA. This design uses microRNA as a single-molecule damper for nano-oscillators by forming a rigid dual-strand structure in the gap between nano-oscillators and the immobilized surface. The ultrasensitive detection is attributed to comparable dimensions of the gap and microRNA. A developed surface plasmon-coupled scattering imaging technology enables us to directly measure the real-time gap distance vibration of multiple nano-oscillators with high accuracy and fast dynamics. High-level and low-level states of the oscillation amplitude indicate melting and hybridization statuses of microRNA. Lifetimes of two states reveal that the hybridization rate of microRNA is determined by the three-dimensional diffusion. This imaging technique contributes application potentials in a single-molecule detection and nanomechanics study.

Digital microbiology: detection and classification of unknown bacterial pathogens using a label-free laser light scatter-sensing system

NASA Astrophysics Data System (ADS)

Rajwa, Bartek; Dundar, M. Murat; Akova, Ferit; Patsekin, Valery; Bae, Euiwon; Tang, Yanjie; Dietz, J. Eric; Hirleman, E. Daniel; Robinson, J. Paul; Bhunia, Arun K.

2011-06-01

The majority of tools for pathogen sensing and recognition are based on physiological or genetic properties of microorganisms. However, there is enormous interest in devising label-free and reagentless biosensors that would operate utilizing the biophysical signatures of samples without the need for labeling and reporting biochemistry. Optical biosensors are closest to realizing this goal and vibrational spectroscopies are examples of well-established optical label-free biosensing techniques. A recently introduced forward-scatter phenotyping (FSP) also belongs to the broad class of optical sensors. However, in contrast to spectroscopies, the remarkable specificity of FSP derives from the morphological information that bacterial material encodes on a coherent optical wavefront passing through the colony. The system collects elastically scattered light patterns that, given a constant environment, are unique to each bacterial species and/or serovar. Both FSP technology and spectroscopies rely on statistical machine learning to perform recognition and classification. However, the commonly used methods utilize either simplistic unsupervised learning or traditional supervised techniques that assume completeness of training libraries. This restrictive assumption is known to be false for real-life conditions, resulting in unsatisfactory levels of accuracy, and consequently limited overall performance for biodetection and classification tasks. The presented work demonstrates preliminary studies on the use of FSP system to classify selected serotypes of non-O157 Shiga toxin-producing E. coli in a nonexhaustive framework, that is, without full knowledge about all the possible classes that can be encountered. Our study uses a Bayesian approach to learning with a nonexhaustive training dataset to allow for the automated and distributed detection of unknown bacterial classes.

Surface plasmon resonance label-free monitoring of antibody antigen interactions in real time

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

Kausaite, A.; van Dijk, M.; Castrop, J.

2007-01-01

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibodymore » rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.« less

Label-free integrative pharmacology on-target of drugs at the β2-adrenergic receptor

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Fang, Ye

2011-07-01

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the β2-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

Label-free peptide aptamer based impedimetric biosensor for highly sensitive detection of TNT with a ternary assembly layer.

PubMed

Li, Yanyan; Zhao, Manru; Wang, Haiyan

2017-11-01

We report a label-free peptide aptamer based biosensor for highly sensitive detection of TNT which was designed with a ternary assembly layer consisting of anti-TNT peptide aptamer (peptamer), dithiothreitol (DTT), and 6-mercaptohexanol (MCH), forming Au/peptamer-DTT/MCH. A linear relationship between the change in electron transfer resistance and the logarithm of the TNT concentration from 0.44 to 18.92 pM, with a detection limit of 0.15 pM, was obtained. In comparison, the detection limit of the aptasensor with a common binary assembly layer (Au/peptamer/MCH) was 0.15 nM. The remarkable improvement in the detection limit could be ascribed to the crucial role of the ternary assembly layer, providing an OH-richer hydrophilic environment and a highly compact surface layer with minimal surface defects, reducing the non-covalent binding (physisorption) of the peptamer and non-specific adsorption of TNT onto the electrode surface, leading to high sensitivity, and which can serve as a general sensing platform for the fabrication of other biosensors.

An enzyme-free and label-free surface plasmon resonance biosensor for ultrasensitive detection of fusion gene based on DNA self-assembly hydrogel with streptavidin encapsulation.

PubMed

Guo, Bin; Wen, Bo; Cheng, Wei; Zhou, Xiaoyan; Duan, Xiaolei; Zhao, Min; Xia, Qianfeng; Ding, Shijia

2018-07-30

In this research, an enzyme-free and label-free surface plasmon resonance (SPR) biosensing strategy has been developed for ultrasensitive detection of fusion gene based on the heterogeneous target-triggered DNA self-assembly aptamer-based hydrogel with streptavidin (SA) encapsulation. In the presence of target, the capture probes (Cp) immobilized on the chip surface can capture the PML/RARα, forming a Cp-PML/RARα duplex. After that, the aptamer-based network hydrogel nanostructure is formed on the gold surface via target-triggered self-assembly of X shaped polymers. Subsequently, the SA can be encapsulated into hydrogel by the specific binding of SA aptamer, forming the complex with super molecular weight. Thus, the developed strategy achieves dramatic enhancement of the SPR signal. Using PML/RARα "S" subtype as model analyte, the developed biosensing method can detect target down to 45.22 fM with a wide linear range from 100 fM to 10 nM. Moreover, the high efficiency biosensing method shows excellent practical ability to identify the clinical PCR products of PML/RARα. Thus, this proposed strategy presents a powerful platform for ultrasensitive detection of fusion gene and early diagnosis and monitoring of disease. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct detection of free radicals and reactive oxygen species in thylakoids.

PubMed

Hideg, Eva; Kálai, Tamás; Hideg, Kálmán

2011-01-01

In plants, reactive oxygen species (ROS), also known as active oxygen species (AOS), are associated with normal, physiologic processes as well as with responses to adverse conditions. ROS are connected to stress in many ways: as primary elicitors, as products and propagators of oxidative damage, or as signal molecules initiating defense or adaptation. The photosynthetic electron transport is a major site of oxidative stress by visible or ultraviolet light, high or low temperature, pollutants or herbicides. ROS production can be presumed from detecting oxidatively damaged lipids, proteins, or pigments as well as from the alleviating effects of added antioxidants. On the contrary, measuring ROS by special sensor molecules provides more direct information. This chapter focuses on the application of spin trapping electron paramagnetic resonance (EPR) spectroscopy for detecting ROS: singlet oxygen and oxygen free radicals in thylakoid membrane preparations.

Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

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

Singh, Swati; Kumar, Ashok, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com; Academy of Scientific and Innovative Research

A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to itsmore » complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml{sup −1} with a limit of detection of 0.16 ng ml{sup −1}.« less

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

PubMed

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Using label-free screening technology to improve efficiency in drug discovery.

PubMed

Halai, Reena; Cooper, Matthew A

2012-02-01

Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing

NASA Astrophysics Data System (ADS)

Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

2016-05-01

A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R2 = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells.A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori

Label-free specific detection of femtomolar cardiac troponin using an integrated nanoslit array fluidic diode.

PubMed

Liu, Yifan; Yobas, Levent

2014-12-10

We demonstrate here for the first time the utility of an integrated nanofluidic diode for detecting and quantifying physiologically relevant macromolecules. Troponin T, a key human cardiac protein biomarker, was selectively and rapidly detected free of labels for concentrations down to 10 fg/mL (∼ 0.3 fM) in buffer as well as 10 pg/mL (∼ 300 fM) in untreated human serum. This ultrasensitive detection arises from monolithic integration of a unique nanofluidic diode structure that is highly robust and amenable to site-specific surface modification. The structure features a planar nanoslit array where each nanoslit is defined at a nominal width of 70 nm over a micrometer-scale silicon trench without the use of high-resolution patterning techniques. Through vapor deposition, a glass layer is placed at a nonuniform thickness, tapering the trench profile upward and contributing to the triangular nanoslit structure. This asymmetric profile is essential for ionic current rectification noted here at various pH values, ionic strengths, and captured target species, which modulate the surface-charge density within the sensitive region of the nanoslit. The nanoslit, unlike nanopores, offers only 1D confinement, which appears to be adequate for reasonable rectification. The measurements are found in quantitative agreement with the diode simulations for the first time based on a pH- and salt-dependent surface-charge model.

Label-free detection of cardiac troponin-I using gold nanoparticles functionalized single-walled carbon nanotubes based chemiresistive biosensor

NASA Astrophysics Data System (ADS)

Rajesh, Sharma, Vikash; Puri, Nitin K.; Singh, Rajiv K.; Biradar, Ashok M.; Mulchanadani, Ashok

2013-11-01

We report a specific and ultrasensitive, label-free chemiresistive biosensor based on mercaptopropionic acid capped gold nanoparticles (GNP) functionalized single walled carbon nanotube (SWNT) hybrid for the detection of cardiac specific biomarker troponin-I (cTnI). GNPs were attached to SWNTs through a molecular linker 1-pyrenemethylamine. The highly specific cTnI antibody was covalently immobilized on GNPs through capping agent using carbodiimide coupling reaction. The cTnI interaction to its corresponding antibody was studied with respect to changes in conductance in SWNTs channel, and a detailed field-effect transistor characteristic was delineated. The device exhibited a linear response to cTnI from 0.01 to 10 ng ml-1.

Fluorescent trimethyl-substituted naphthyridine as a label-free signal reporter for one-step and highly sensitive fluorescent detection of DNA in serum samples.

PubMed

Wang, Jiamian; Wang, Xiuyun; Wu, Shuo; Che, Ruping; Luo, Pinchen; Meng, Changgong

2017-01-15

A facile label-free sensing method is developed for the one-step and highly sensitive fluorescent detection of DNA, which couples the specific C-C mismatch bonding and fluorescent quenching property of a trimethyl-substituted naphthyridine dye (ATMND) with the exonuclease III (Exo III) assisted cascade target recycling amplification strategy. In the absence of target DNA, the DNA hairpin probe with a C-C mismatch in the stem and more than 4 bases overhung at the 3' terminus could entrap and quench the fluorescence of ATMND and resist the digestion of Exo III, thus showing a low fluorescence background. In the presence of the target, however, the hybridization event between the two protruding segments and the target triggers the digestion reaction of Exo III, recycles the initial target, and simultaneously releases both the secondary target analogue and the ATMND caged in the stem. The released initial and secondary targets take part in another cycle of digestion, thus leading to the release of a huge amount of free ATMND for signal transducing. Based on the fluorescence recovery, the as-proposed label-free fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 10pM to 1μM, a low limit of detection of 6pM, good selectivity, and a facile one-step operation at room temperature. Practical sample analysis in serum samples indicates the method has good precision and accuracy, which may thus have application potentials for point-of-care screening of DNA in complex clinical and environmental samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultrasensitive and label-free detection of pathogenic avian influenza DNA by using CMOS impedimetric sensors.

PubMed

Lai, Wei-An; Lin, Chih-Heng; Yang, Yuh-Shyong; Lu, Michael S-C

2012-05-15

This work presents miniaturized CMOS (complementary metal oxide semiconductor) sensors for non-faradic impedimetric detection of AIV (avian influenza virus) oligonucleotides. The signal-to-noise ratio is significantly improved by monolithic sensor integration to reduce the effect of parasitic capacitances. The use of sub-μm interdigitated microelectrodes is also beneficial for promoting the signal coupling efficiency. Capacitance changes associated with surface modification, functionalization, and DNA hybridization were extracted from the measured frequency responses based on an equivalent-circuit model. Hybridization of the AIV H5 capture and target DNA probes produced a capacitance reduction of -13.2 ± 2.1% for target DNA concentrations from 1 fM to 10 fM, while a capacitance increase was observed when H5 target DNA was replaced with non-complementary H7 target DNA. With the demonstrated superior sensing capabilities, this miniaturized CMOS sensing platform shows great potential for label-free point-of-care biosensing applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

2017-02-01

Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

NASA Astrophysics Data System (ADS)

Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

2017-02-01

Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL-1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL-1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.

Diazonium-based impedimetric aptasensor for the rapid label-free detection of Salmonella typhimurium in food sample.

PubMed

Bagheryan, Zahra; Raoof, Jahan-Bakhsh; Golabi, Mohsen; Turner, Anthony P F; Beni, Valerio

2016-06-15

Fast and accurate detection of microorganisms is of key importance in clinical analysis and in food and water quality monitoring. Salmonella typhimurium is responsible for about a third of all cases of foodborne diseases and consequently, its fast detection is of great importance for ensuring the safety of foodstuffs. We report the development of a label-free impedimetric aptamer-based biosensor for S. typhimurium detection. The aptamer biosensor was fabricated by grafting a diazonium-supporting layer onto screen-printed carbon electrodes (SPEs), via electrochemical or chemical approaches, followed by chemical immobilisation of aminated-aptamer. FTIR-ATR, contact angle and electrochemical measurements were used to monitor the fabrication process. Results showed that electrochemical immobilisation of the diazonium-grafting layer allowed the formation of a denser aptamer layer, which resulted in higher sensitivity. The developed aptamer-biosensor responded linearly, on a logarithm scale, over the concentration range 1 × 10(1) to 1 × 10(8)CFU mL(-1), with a limit of quantification (LOQ) of 1 × 10(1) CFU mL(-1) and a limit of detection (LOD) of 6 CFU mL(-1). Selectivity studies showed that the aptamer biosensor could discriminate S. typhimurium from 6 other model bacteria strains. Finally, recovery studies demonstrated its suitability for the detection of S. typhimurium in spiked (1 × 10(2), 1 × 10(4) and 1 × 10(6) CFU mL(-1)) apple juice samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free detection of protein-ligand interactions by the quartz crystal microbalance.

PubMed

Janshoff, Andreas; Steinem, Claudia

2005-01-01

In recent years the quartz crystal microbalance (QCM) has been accepted as a powerful technique to monitor adsorption processes at interfaces in different chemical and biological research areas. In the last decade, the investigation of adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of the QCM comprising the interaction of nucleic acids, specific molecular recognition of protein-receptor couples, and antigen-antibody reactions realized in immunosensors. The advantage of the QCM technique is that it allows for a label free detection of molecules. This is a result of the fact that the frequency response of the quartz resonator is proportional to the increase in thickness of the adsorbed layer. However, in recent years it became more and more evident that quartz resonators used in fluids are more than mere mass or thickness sensors. The sensor response is also influenced by viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules and surface roughness. These phenomena have been used to get new insights in the adhesion process of living cells and to understand their response to pharmacological substances by determining morphological changes of the cells. In this chapter we describe a protocol to explore the kinetics and thermodynamics of specific interactions of different proteins such as lectins and annexins with their ligands using receptor bearing solid supported lipid bilayers.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

PubMed

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-07-29

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

PubMed Central

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-01-01

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world’s attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg2+ ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

Learning Words from Labeling and Directive Speech

ERIC Educational Resources Information Center

Callanan, Maureen A.; Akhtar, Nameera; Sussman, Lisa

2014-01-01

Despite the common intuition that labeling may be the best way to teach a new word to a child, systematic testing is needed of the prediction that children learn words better from labeling utterances than from directive utterances. Two experiments compared toddlers' label learning in the context of hearing words used in directive versus labeling…

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro

Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

PubMed

Wang, Ying; Zhu, Yingjing; Binyam, Atsebeha; Liu, Misha; Wu, Yinan; Li, Fengting

2016-12-15

A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests. Copyright © 2016 Elsevier B.V. All rights reserved.

Gluten and gluten-free: issues and considerations of labeling regulations, detection methods, and assay validation.

PubMed

Diaz-Amigo, Carmen; Popping, Bert

2012-01-01

Gluten is a commonly used cereal derivative found in bakery products, among other items. In some susceptible individuals, however, it triggers immune responses of different kinds; there is, to a lesser extent, the wheat allergy that is immunoglobulin E (IgE)-mediated and leads to histamine release and typical allergic symptoms. In this case, other water-soluble proteins, like albumins, are also involved. On the other hand, there is, more frequently, celiac disease (CD), where the gluten causes immune reactions in the intestines of certain individuals, leading to degeneration of villi, which typically leads to malabsorption of nutrients and, consequently, malnutrition. The only currently effective health strategy for affected consumers is avoidance of gluten-containing products, based on clear labeling rules. However, despite unanimously accepted Codex definitions by all member jurisdictions, the national implementation of equivalent laws shows significant differences. In the context of CD and in support of the gluten-free statement, regulatory enforcement, as well as manufacturers' quality controls are mostly based on analytical results. However, numerous methods are available, some of which have been validated better than others, and many provide different results on identical samples. Reasons include detection of different gluten components and variability in extraction efficiency due to different buffer compositions, especially from processed foods. Last but not least, the lack of reference materials is hindering the process of generating comparable data across different ELISA kits, as well as other methods. How can such data still be used to support a gluten-free claim? New methodologies, in particular mass spectrometric analysis of gluten derived peptides, are being introduced in numerous laboratories. This methodology is not only capable of detecting gluten derived peptides but can also differentiate between and quantitate wheat, barley, rye, and oat. This

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

NASA Astrophysics Data System (ADS)

Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

2016-09-01

The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice.

PubMed

Miao, Hong; Wang, Lan; Zhuo, Yan; Zhou, Zinan; Yang, Xiaoming

2016-12-15

A facile-green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 13.9% has been built up, while tomato juice served as the carbon source. Interestingly, not only the precursor of CDs and the whole synthesis procedure were environmental-friendly, but this type of CDs also exhibited multiple advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Significantly, a label-free sensitive assay for detecting carcinoembryonic antigen (CEA) in a continuous and recyclable way has been proposed on the basis of adsorption and desorption of aptamers by the surface of CDs through a competitive mechanism. To be specific, the richness of carboxyl groups of the CDs enabled strong adsorption of ssDNA to the surface of CDs through π-π stacking interactions, resulting in the effective fluorescence quenching by forming CDs-aptamer complexes. The stronger binding affinity between CEA and CEA-aptamer than the π-π stacking interactions has been taken advantage to achieve immediate recovery of the fluorescence of CDs once CEA was introduced. Thereby, quantitative evaluation of CEA concentration in a broad range from 1ngmL(-1) to 0.5ngmL(-1) with the detection limit of 0.3ngmL(-1) was realized in this way. This strategy can be applied in a recyclable way, broadening the sensing application of CDs with biocompatibility. Besides, the CDs were used for cell imaging, potentiating them towards diverse purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo and ex vivo EPR detection of spin-labelled ovalbumin in mice.

PubMed

Abramović, Zrinka; Brgles, Marija; Habjanec, Lidija; Tomasić, Jelka; Sentjurc, Marjeta; Frkanec, Ruza

2010-10-01

In this study, spin-labelled ovalbumin (SL-OVA), free or entrapped in liposomes, was administered to mice subcutaneously (s.c.) or intravenously (i.v.) with the aim to determine the conditions for pharmacokinetic studies of spin-labelled proteins by EPR and to measure the time course of SL-OVA distribution in vivo in live mice and ex vivo in isolated organs. Upon s.c. administration, the decay of the EPR signal was followed for 60min at the site of application using an L-band EPR spectrometer. Within this time period, the signal of free SL-OVA was diminished by about 70%. It was estimated with the help of the oxidizing agent K(3)[(FeCN)(6)] that approximately 30% was a consequence of the spin label reduction to EPR non-visible hydroxylamine and about 40% was due to the SL-OVA elimination from the site of measurement. For liposome encapsulated SL-OVA, the intensity diminished only by approx. 40% in the same period, indicating that liposomes successfully protect the protein from reduction. EPR signal could not be detected directly over live mouse organs within 60min after s.c. application of SL-OVA. With the available L-band EPR spectrometer, the measurements at the site of s.c. application are possible if the amount of SL-OVA applied to a mouse is more than 3mg. For the pharmacokinetic studies of the protein distribution in organs after s.c. or i.v. injection the concentration of the spin-labelled protein should be more than 0.5mmol/kg. After i.v. administration, only ex vivo measurements were possible using an X-band EPR spectrometer, since the total amount of SL-OVA was not sufficient for in vivo detection and also because of rapid reduction of nitroxide. After 2min, the protein was preferentially distributed to liver and, to a smaller extent, to spleen.

Whispering Gallery Mode Resonators for Rapid Label-Free Biosensing in Small Volume Droplets

PubMed Central

Wildgen, Sarah M.; Dunn, Robert C.

2015-01-01

Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection. PMID:25806835

Whispering gallery mode resonators for rapid label-free biosensing in small volume droplets.

PubMed

Wildgen, Sarah M; Dunn, Robert C

2015-03-23

Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing

PubMed Central

Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

2015-01-01

Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres. PMID:26287252

Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing.

PubMed

Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

2015-07-22

Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres.

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.

Highly stable porous silicon-carbon composites as label-free optical biosensors.

PubMed

Tsang, Chun Kwan; Kelly, Timothy L; Sailor, Michael J; Li, Yang Yang

2012-12-21

A stable, label-free optical biosensor based on a porous silicon-carbon (pSi-C) composite is demonstrated. The material is prepared by electrochemical anodization of crystalline Si in an HF-containing electrolyte to generate a porous Si template, followed by infiltration of poly(furfuryl) alcohol (PFA) and subsequent carbonization to generate the pSi-C composite as an optically smooth thin film. The pSi-C sensor is significantly more stable toward aqueous buffer solutions (pH 7.4 or 12) compared to thermally oxidized (in air, 800 °C), hydrosilylated (with undecylenic acid), or hydrocarbonized (with acetylene, 700 °C) porous Si samples prepared and tested under similar conditions. Aqueous stability of the pSi-C sensor is comparable to related optical biosensors based on porous TiO(2) or porous Al(2)O(3). Label-free optical interferometric biosensing with the pSi-C composite is demonstrated by detection of rabbit IgG on a protein-A-modified chip and confirmed with control experiments using chicken IgG (which shows no affinity for protein A). The pSi-C sensor binds significantly more of the protein A capture probe than porous TiO(2) or porous Al(2)O(3), and the sensitivity of the protein-A-modified pSi-C sensor to rabbit IgG is found to be ~2× greater than label-free optical biosensors constructed from these other two materials.

Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy

PubMed Central

Lo, Kai-Yin; Sun, Yung-Shin; Landry, James P.; Zhu, Xiangdong; Deng, Wenbin

2012-01-01

Conventional fluorescent microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in ways often uncharacterized. The binding between antibody and antigen might not be in the native situation after such conjugation. Here, we present an oblique-incidence reflectivity difference (OI-RD) microscope as an effective method for label-free, real-time detection of cell surface markers and apply such a technique to analysis of Stage-Specific Embryonic Antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies only bind to the surface of the stem cells while not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. The results concluded that OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells. The information could be another indicator to determine the cell stages. PMID:21781038

Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots.

PubMed

Xu, Xiahong; Liu, Xin; Nie, Zhou; Pan, Yuliang; Guo, Manli; Yao, Shouzhuo

2011-01-01

Herein, we present a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective aggregation of unmodified QDs with anionic surface charge, whereas phosphorylated peptides do not. Phosphorylation by kinase alters the net charge of peptides and subsequently inhibits the aggregation of unmodified QDs, causing an enhanced fluorescence with a 45 nm blue-shift in emission and a yellow-to-green emission color change. Hence the fluorescence response allows this QD-based method to easily probe kinase activity by a spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.47 mU μL(-1)). On the basis of the fluorescence response of QDs on the concentration of PKA inhibitor H-89, the IC(50) value, the half maximal inhibitory concentration, was estimated, which was in agreement with the literature value. Moreover, the system can be applicable to detect the Forskolin/3-isobutyl-1-methylxantine (IBMX)-stimulated activation of PKA in cell lysate. Unlike the existing QD-based enzyme activity assays in which the modification process of QDs is essential, this method relies on unmodified QDs without the requirement of peptide labeling and QDs' modification, presenting a promising candidate for cost-effective kinase activity and inhibitor screening assays.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

PubMed

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands

A Label-Free Porous Silicon Immunosensor for Broad Detection of Opiates in a Blind Clinical Study and Result Comparison to Commercial Analytical Chemistry Techniques

PubMed Central

Bonanno, Lisa M.; Kwong, Tai C.; DeLouise, Lisa A.

2010-01-01

In this work we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n=50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA®) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was < 9% both within-run and between-day at and above the cutoff limit of 300 ng ml−1. This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semi-quantitative demonstration of the technology’s successful clinical use. These results motivate future development of PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting. PMID:21062030

Fiber optic label-free biophotonic diagnostic tool for cardiovascular disease

NASA Astrophysics Data System (ADS)

Rius, Cristina; Ackermann, Tobias N.; Dorado, Beatriz; Muñoz-Berbel, Xavier; Andrés, Vicente; Llobera, Andreu

2015-06-01

A label-free compact method for performing photonic characterization of "healthy" versus "diseased" arteries has been developed. It permits the detection of atherosclerotic lesion in living mouse arteries. Using this prototype, we observed that the spectral response (photonic fingerprint, PIN) obtained from aortas of wild-type mice differs from the response of ApoE-KO mice fed with high-fat diet (an atheroprone mouse model). Benchmark of the results against gold standard was performed by staining the aortas with Oil-Red-O to visualize atherosclerotic plaques.

Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

PubMed Central

Wang, Yaoguang; Zhang, Yong; Wu, Dan; Ma, Hongmin; Pang, Xuehui; Fan, Dawei; Wei, Qin; Du, Bin

2017-01-01

In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe3O4-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe3O4-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe3O4 NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe3O4 NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe3O4-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10−5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers. PMID:28186128

Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

Ultrasensitive Label-free Electronic Chip for DNA Analysis Using Carbon Nanotube Nanoelectrode Arrays

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Ye, Qi; Han, Jie; Meyyappan, M.

2004-01-01

There is a strong need for faster, cheaper, and simpler methods for nucleic acid analysis in today s clinical tests. Nanotechnologies can potentially provide solutions to these requirements by integrating nanomaterials with biofunctionalities. Dramatic improvement in the sensitivity and multiplexing can be achieved through the high-degree miniaturization. Here, we present our study in the development of an ultrasensitive label-free electronic chip for DNA/RNA analysis based on carbon nanotube nanoelectrode arrays. A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in a SiO2 matrix is fabricated using a bottom-up approach. Characteristic nanoelectrode behavior is observed with a low-density MWNT nanoelectrode array in measuring both the bulk and surface immobilized redox species. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes, with a wide potential window, flexible chemical functionalities, and good biocompatibility. A BRCA1 related oligonucleotide probe with 18 bases is covalently functionalized at the open ends of the MWNTs and specifically hybridized with an oligonucleotide target as well as a PCR amplicon. The guanine bases in the target molecules are employed as the signal moieties for the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. This technique has been employed for direct electrochemical detection of label-free PCR amplicon through specific hybridization with the BRCAl probe. The detection limit is estimated to be less than approximately 1000 DNA molecules, approaching the limit of the sensitivity by laser-based fluorescence techniques in DNA microarray. This system provides a general electronic platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, simple sample preparation, and low- cost operation.

Label free detection of lead using impedimetric sensor based on ordered mesoporous carbon-gold nanoparticles and DNAzyme catalytic beacons.

PubMed

Zhou, Yaoyu; Tang, Lin; Zeng, Guangming; Zhang, Chen; Xie, Xia; Liu, Yuanyuan; Wang, Jiajia; Tang, Jing; Zhang, Yi; Deng, Yaocheng

2016-01-01

A novel label-free impedimetric sensing system based on DNAzyme and ordered mesoporous carbon-gold nanoparticle (OMC-GNPs) for the determination of Pb(2+) concentration was developed in the present study. Firstly, gold nanoparticles deposited on the modified electrode surface were employed as a platform for the immobilization of thiolated probe DNA, and then hybridized with DNAzyme catalytic beacons. Subsequently, in the presence of Pb(2+), the DNAzyme could be activated to cleave the substrate strand into two DNA fragments, which causes differences in the electrical properties of the film. Randles equivalent circuit was employed to evaluate the electrochemical impedance spectroscopy (EIS) result. The charge transfer resistance (R(CT)) value for the [Fe(CN)6](3-/4-) redox indicator was remarkably decline after hybridization with Pb(2+). The difference in RCT values before and after hybridization with Pb(2+) showed a linear relation with the concentration of the Pb(2+) in a range of 5×10(-10)-5×10(-5) M, with a detection limit of 2×10(-10) M (S/N=3). Furthermore, with the application of Pb(2+) dependent 8-17DNAzyme, the proposed sensing system exhibited high selectivity without using any labeled probes. This biosensor demonstrated a promising potential for Pb(2+) detection in real sample. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Biological and Chemical Sensing Using Whispering Gallery Mode Optical Resonators: Past, Present, and Future

PubMed Central

Su, Judith

2017-01-01

Sensitive and rapid label-free biological and chemical sensors are needed for a wide variety of applications including early disease diagnosis and prognosis, the monitoring of food and water quality, as well as the detection of bacteria and viruses for public health concerns and chemical threat sensing. Whispering gallery mode optical resonator based sensing is a rapidly developing field due to the high sensitivity and speed of these devices as well as their label-free nature. Here, we describe the history of whispering gallery mode optical resonator sensors, the principles behind detection, the latest developments in the fields of biological and chemical sensing, current challenges toward widespread adoption of these devices, and an outlook for the future. In addition, we evaluate the performance capabilities of these sensors across three key parameters: sensitivity, selectivity, and speed. PMID:28282881

Label-free SERS study of galvanic replacement reaction on silver nanorod surface and its application to detect trace mercury ion

PubMed Central

Wang, Yaohui; Wen, Guiqing; Ye, Lingling; Liang, Aihui; Jiang, Zhiliang

2016-01-01

It is significant to explore a rapid and highly sensitive galvanic replacement reaction (GRR) surface enhanced Raman scattering (SERS) method for detection of trace mercury ions. This article was reported a new GRR SERS analytical platform for detecting Hg(II) with label-free molecular probe Victoria blue B (VBB). In HAc-NaCl-silver nanorod (AgNR) substrate, the molecular probe VBB exhibited a strong SERS peak at 1609 cm−1. Upon addition of Hg(II), the GRR occurred between the AgNR and Hg(II), and formed a weak SERS activity of Hg2Cl2 that deposited on the AgNR surfaces to decrease the SERS intensity at 1609 cm−1. The decreased SERS intensity was linear to Hg(II) concentration in the range of 1.25–125 nmol/L, with a detection limit of 0.2 nmol/L. The GRR was studied by SERS, transmission electron microscopy and other techniques, and the GRR mechanism was discussed. PMID:26792071

Label-free in situ SERS imaging of biofilms.

PubMed

Ivleva, Natalia P; Wagner, Michael; Szkola, Agathe; Horn, Harald; Niessner, Reinhard; Haisch, Christoph

2010-08-12

Surface-enhanced Raman scattering (SERS) is a promising technique for the chemical characterization of biological systems. It yields highly informative spectra, can be applied directly in aqueous environment, and has high sensitivity in comparison with normal Raman spectroscopy. Moreover, SERS imaging can provide chemical information with spatial resolution in the micrometer range (chemical imaging). In this paper, we report for the first time on the application of SERS for in situ, label-free imaging of biofilms and demonstrate the suitability of this technique for the characterization of the complex biomatrix. Biofilms, being communities of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), represent the predominant mode of microbial life. Knowledge of the chemical composition and the structure of the biofilm matrix is important in different fields, e.g., medicine, biology, and industrial processes. We used colloidal silver nanoparticles for the in situ SERS analysis. Good SERS measurement reproducibility, along with a significant enhancement of Raman signals by SERS (>10(4)) and highly informative SERS signature, enables rapid SERS imaging (1 s for a single spectrum) of the biofilm matrix. Altogether, this work illustrates the potential of SERS for biofilm analysis, including the detection of different constituents and the determination of their distribution in a biofilm even at low biomass concentration.

Deep Learning in Label-free Cell Classification

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

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

Deep Learning in Label-free Cell Classification

PubMed Central

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-01-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

Deep Learning in Label-free Cell Classification

DOE PAGES

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

2016-03-15

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

Deep Learning in Label-free Cell Classification

NASA Astrophysics Data System (ADS)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-03-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

Novel electrochemiluminescence of silver nanoclusters fabricated on triplex DNA scaffolds for label-free detection of biothiols.

PubMed

Feng, Lingyan; Wu, Li; Xing, Feifei; Hu, Lianzhe; Ren, Jinsong; Qu, Xiaogang

2017-12-15

Electrochemiluminescence (ECL) of metal nanoclusters and their application have been widely reported due to the good biocompatibility, fascinating electrocatalytic activity and so on. Using DNA as synthesis template opens new opportunities to modulate the physical properties of AgNCs. Triplex DNA has been reported for the site-specific, homogeneous and highly stable silver nanoclusters (AgNCs) fabrication from our recent research. Here we further explore their extraordinary ECL properties and applications in biosensor utilization. By reasonable design of DNA sequence, AgNCs were obtained in the predefined position of CG.C + sites of triplex DNA, and the ECL emission at a low potential was observed with this novel DNA template. Finally, a simple and label-free method was developed for biothiols detection based on the enhanced catalytic reaction and a robust interaction between the triplex-AgNCs and cysteine, by influencing the microenvironment provided by DNA template. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free detection and identification of waterborne parasites using a microfluidic multi-angle laser scattering system

NASA Astrophysics Data System (ADS)

Huang, Wei; Yang, Limei; Lei, Lei; Li, Feng

2017-10-01

A microfluidic-based multi-angle laser scattering (MALS) system capable of acquiring scattering patterns of a single particle is designed and demonstrated. The system includes a sheathless nozzle microfluidic glass chip, and an on-chip MALS unit being in alignment with the nozzle exit in the chip. The size and relative refractive indices (RI) of polystyrene (PS) microspheres were deduced with accuracies of 60 nm and 0.002 by comparing the experimental scattering patterns with theoretical ones. We measured scattering patterns of waterborne parasites i.e., Cryptosporidium parvum (C.parvum) and Giardia lamblia (G. lamblia), and some other representative species suspended in deionized water at a maximum flow rate of 12 μL/min, and a maximum of 3000 waterborne parasites can be identified within one minute with a mean accuracy higher than 96% by classification of distinctive scattering patterns using a support-vector-machine (SVM) algorithm. The system provides a promising tool for label-free detection of waterborne parasites and other biological contaminants.

Exonuclease III-assisted cascade signal amplification strategy for label-free and ultrasensitive electrochemical detection of nucleic acids.

PubMed

Xiong, Erhu; Yan, Xiaoxia; Zhang, Xiaohua; Liu, Yunqing; Zhou, Jiawan; Chen, Jinhua

2017-01-15

In this work, a simple, signal-on and label-free electrochemical biosensor for ultrasensitive DNA detection is reported on the basis of an autocatalytic and exonuclease III (Exo III)-assisted cascade signal amplification strategy. In the presence of target DNA (T-DNA), the hybridization between the 3'-protruding DNA fragment of hairpin DNA probe (HP1) and T-DNA triggered the Exo III cleavage process, accompanied by the releasing of T-DNA and autonomous generation of new DNA fragment which was used for the successive hybridization with the another hairpin DNA (HP2) on the electrode. After the Exo III cleavage process, numerous quadruplex-forming oligomers which caged in HP2 were liberated on the electrode surface and folded into G-quadruplex-hemin complexes with the help of K + and hemin to give a remarkable electrochemical response. As a result, a low detection limit of 4.83fM with an excellent selectivity toward T-DNA was achieved. The developed electrochemical biosensor should be further extended for the detection of a wide spectrum of analytes and has great potential for the development of ultrasensitive biosensing platform for early diagnosis in gene-related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Cost-effective and label-free holographic biosensor for detection of herpes simplex virus (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ray, Aniruddha; Ho, Ha; Daloglu, Mustafa; Torres, Avee; McLeod, Euan; Ozcan, Aydogan

2017-03-01

Herpes is one of the most widespread sexually transmitted viral diseases. Timely detection of Herpes Simplex Virus (HSV) can help prevent the rampant spreading of the virus. Current detection techniques such as viral culture, immuno-assays or Polymerase-Chain-Reaction, are time extensive and require expert handling. Here we present a field-portable, easy-to-use, and cost-effective biosensor for the detection of HSV based on holographic imaging. The virus is first captured from a target solution onto specifically developed substrates, prepared by coating glass coverslips with HSV-specific antibodies, and imaged using a lensfree holographic microscope. Several light-emitting-diodes (LEDs), coupled to multi-mode optical-fibers, are used to illuminate the sample containing the viruses. A micro-controller is used to activate the LEDs one at a time and in-line holograms are recorded using a CMOS imager placed immediately above the substrate. These sub-pixel shifted holograms are used to generate a super-resolved hologram, which is reconstructed to obtain the phase and amplitude images of the viruses. The signal of the viruses is enhanced using self-assembled PEG-based nanolenses, formed around the viral particles. Based on the phase information of the reconstructed images we can estimate the size of the viral particles, with an accuracy of +/- 11 nm, as well as quantify the viral load. The limit-of-detection of this system is estimated to be <500 viral copies per 100 μL sample volume that is imaged over 30 mm^2 field-of-view. This holographic microscopy based biosensor is label-free, cost-effective and field-portable, providing results in 2 hours, including sample preparation and imaging time.

Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

PubMed

Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

2018-05-09

The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

Label-free serum ribonucleic acid analysis for colorectal cancer detection by surface-enhanced Raman spectroscopy and multivariate analysis

NASA Astrophysics Data System (ADS)

Chen, Yanping; Chen, Gang; Feng, Shangyuan; Pan, Jianji; Zheng, Xiongwei; Su, Ying; Chen, Yan; Huang, Zufang; Lin, Xiaoqian; Lan, Fenghua; Chen, Rong; Zeng, Haishan

2012-06-01

Studies with circulating ribonucleic acid (RNA) not only provide new targets for cancer detection, but also open up the possibility of noninvasive gene expression profiling for cancer. In this paper, we developed a surface-enhanced Raman scattering (SERS), platform for detection and differentiation of serum RNAs of colorectal cancer. A novel three-dimensional (3-D), Ag nanofilm formed by dry MgSO4 aggregated silver nanoparticles, Ag NP, as the SERS-active substrate was presented to effectively enhance the RNA Raman signals. SERS measurements were performed on two groups of serum RNA samples. One group from patients, n=55 with pathologically diagnosed colorectal cancer and the other group from healthy controls, n=45. Tentative assignments of the Raman bands in the normalized SERS spectra demonstrated that there are differential expressions of cancer-related RNAs between the two groups. Linear discriminate analysis, based on principal component analysis, generated features can differentiate the colorectal cancer SERS spectra from normal SERS spectra with sensitivity of 89.1 percent and specificity of 95.6 percent. This exploratory study demonstrated great potential for developing serum RNA SERS analysis into a useful clinical tool for label-free, noninvasive screening and detection of colorectal cancers.

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

PubMed

Wang, Guanghui; Wu, Wells W; Zeng, Weihua; Chou, Chung-Lin; Shen, Rong-Fong

2006-05-01

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative

Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

PubMed

Donmez, Soner; Arslan, Fatma; Arslan, Halit

2016-05-01

In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Label-free protein assay based on a nanomechanical cantilever array

NASA Astrophysics Data System (ADS)

Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

2003-01-01

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Label-free surface plasmon sensing towards cancer diagnostics

NASA Astrophysics Data System (ADS)

Sankaranarayanan, Goutham

The main objective of this thesis is to develop a conventional, home-built SPR bio-sensor to demonstrate bio-sensing applications. This emphasizes the understanding of basic concepts of Surface Plasmon Resonance and various interrogation techniques. Intensity Modulation was opted to perform the label-free SPR bio-sensing experiments due to its cost-efficient and compact setup. Later, label-free surface plasmon sensing was carried out to study and understand the bio-molecular interactions between (1). BSA and Anti BSA molecules and (2). Exosome/Liposome on thin metal (Au) films. Exosomes are cell-derived vesicles present in bodily fluids like blood, saliva, urine, epididymal fluid containing miRNAs, RNA, proteins, etc., at stable quantities during normal health conditions. The exosomes comprise varied constituents based on their cell origin from where they are secreted and is specific to that particular origin. However an exacerbated release is observed during tumor or cancer conditions. This increased level of exosomes present in the sample, can be detected using the SPR bio-sensor demonstrated in this thesis and effective thickness of adsorption on Au surface can be estimated. Also, chemically synthesized liposome particles were studied to determine if they can generate an equivalent sensor response to that of exosomes to consider them as an alternate. Finally a 10ppb Mercury (Hg) sensing was performed as part of Environment Monitoring application and results have been tabulated and compared.

Label-free biodetection using a smartphone.

PubMed

Gallegos, Dustin; Long, Kenneth D; Yu, Hojeong; Clark, Peter P; Lin, Yixiao; George, Sherine; Nath, Pabitra; Cunningham, Brian T

2013-06-07

Utilizing its integrated camera as a spectrometer, we demonstrate the use of a smartphone as the detection instrument for a label-free photonic crystal biosensor. A custom-designed cradle holds the smartphone in fixed alignment with optical components, allowing for accurate and repeatable measurements of shifts in the resonant wavelength of the sensor. Externally provided broadband light incident upon an entrance pinhole is subsequently collimated and linearly polarized before passing through the biosensor, which resonantly reflects only a narrow band of wavelengths. A diffraction grating spreads the remaining wavelengths over the camera's pixels to display a high resolution transmission spectrum. The photonic crystal biosensor is fabricated on a plastic substrate and attached to a standard glass microscope slide that can easily be removed and replaced within the optical path. A custom software app was developed to convert the camera images into the photonic crystal transmission spectrum in the visible wavelength range, including curve-fitting analysis that computes the photonic crystal resonant wavelength with 0.009 nm accuracy. We demonstrate the functionality of the system through detection of an immobilized protein monolayer, and selective detection of concentration-dependent antibody binding to a functionalized photonic crystal. We envision the capability for an inexpensive, handheld biosensor instrument with web connectivity to enable point-of-care sensing in environments that have not been practical previously.

Gold nanoclusters-based chemiluminescence resonance energy transfer method for sensitive and label-free detection of trypsin.

PubMed

You, Xiaoying; Li, Yinhuan; Li, Baoping; Ma, Jie

2016-01-15

A chemiluminescence resonance energy transfer (CRET) platform was developed for sensitive and label-free detection of protease by using trypsin as a model analyte. In this CRET platform, bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide chemiluminescence (CL) reaction was utilized as an energy donor and bovine serum albumin (BSA)-stabilized gold nanoclusters (Au NCs) as an energy acceptor. The BSA-stabilized Au NCs triggered the CRET phenomenon by accepting the energy from TCPO-H2O2 CL reaction, thus producing intense CL. In the presence of trypsin, the protein template of BSA-stabilized Au NCs was digested, which frustrated the energy transfer efficiency between the CL donor and the BSA-stabilized Au NCs, leading to a significant decrease in the CL signal. The decreased CL signal was proportional to the logarithm of trypsin concentration in the range of 0.01-50.0µg mL(-1). The detection limit for trypsin was 9ng mL(-)(1) and the relative standard deviations were lesser than 3% (n=11). This Au NCs-based CRET platform was successfully applied to the determination of trypsin in human urine samples, demonstrating its potential application in clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free immunosensor based on gold nanoparticle silver enhancement.

PubMed

Yang, Minghui; Wang, Cunchang

2009-02-01

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

PubMed

Uckermann, Ortrud; Galli, Roberta; Leupold, Susann; Coras, Roland; Meinhardt, Matthias; Hallmeyer-Elgner, Susanne; Mayer, Thomas; Storch, Alexander; Schackert, Gabriele; Koch, Edmund; Blümcke, Ingmar; Steiner, Gerald; Kirsch, Matthias

2017-01-01

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

A label-free electrochemical impedance immunosensor based on AuNPs/PAMAM-MWCNT-Chi nanocomposite modified glassy carbon electrode for detection of Salmonella typhimurium in milk.

PubMed

Dong, Jing; Zhao, Han; Xu, Minrong; Ma, Qiang; Ai, Shiyun

2013-12-01

A sensitive and stable label-free electrochemical impedance immunosensor for the detection of Salmonella typhimurium was developed by immobilising anti-Salmonella antibodies onto the gold nanoparticles and poly(amidoamine)-multiwalled carbon nanotubes-chitosan nanocomposite film modified glassy carbon electrode (AuNPs/PAMAM-MWCNT-Chi/GCE). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to verify the stepwise assembly of the immunosensor. Co-addition of MWCNT, PAMAM and AuNPs greatly enhanced the sensitivity of the immunosensor. The immobilisation of antibodies and the binding of Salmonella cells to the modified electrode increased the electron-transfer resistance (Ret), which was directly measured with EIS using [Fe(CN)6](3-/4-) as a redox probe. A linear relationship of Ret and Salmonella concentration was obtained in the Salmonella concentration range of 1.0×10(3) to 1.0×10(7) CFU mL(-1) with a detection limit of 5.0×10(2) CFU mL(-1). Additionally, the proposed method was successfully applied to determine S. typhimurium content in milk samples with satisfactory results. Copyright © 2013 Elsevier Ltd. All rights reserved.

Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.

PubMed

Martin, Julio

2010-09-01

Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electrical impedance, are versatile platforms for detecting phenotypic changes in both engineered and native cells. Their non-invasive nature allows for the kinetic monitoring of multiple real-time cellular responses to external stimuli, as well as for the use of successive pharmacological challenges. The temporal signature recorded for a particular stimulus is characteristic of the cell type and the signaling pathway activated upon binding of a ligand to its receptor. Cellular label-free technology is an important technical advance in the study of functional pharmacological selectivity. Described in this overview are some of the hurdles encountered in modern drug discovery and the ways in which label-free technologies can be used to overcome these obstacles.

Label-free resistive-pulse cytometry.

PubMed

Chapman, M R; Sohn, L L

2011-01-01

Numerous methods have recently been developed to characterize cells for size, shape, and specific cell-surface markers. Most of these methods rely upon exogenous labeling of the cells and are better suited for large cell populations (>10,000). Here, we review a label-free method of characterizing and screening cells based on the Coulter-counter technique of particle sizing: an individual cell transiting a microchannel (or "pore") causes a downward pulse in the measured DC current across that "pore". Pulse magnitude corresponds to the cell size, pulse width to the transit time needed for the cell to pass through the pore, and pulse shape to how the cell traverses across the pore (i.e., rolling or tumbling). When the pore is functionalized with an antibody that is specific to a surface-epitope of interest, label-free screening of a specific marker is possible, as transient binding between the two results in longer time duration than when the pore is unfunctionalized or functionalized with a nonspecific antibody. While this method cannot currently compete with traditional technology in terms of throughput, there are a number of applications for which this technology is better suited than current commercial cytometry systems. Applications include the rapid and nondestructive analysis of small cell populations (<100), which is not possible with current technology, and a platform for providing true point-of-care clinical diagnostics, due to the simplicity of the device, low manufacturing costs, and ease of use. Copyright © 2011 Elsevier Inc. All rights reserved.

Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy.

PubMed

You, Sixian; Tu, Haohua; Chaney, Eric J; Sun, Yi; Zhao, Youbo; Bower, Andrew J; Liu, Yuan-Zhi; Marjanovic, Marina; Sinha, Saurabh; Pu, Yang; Boppart, Stephen A

2018-05-29

Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.

PubMed

Kusić, Dragana; Rösch, Petra; Popp, Jürgen

2016-03-01

Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species. Copyright © 2016 Elsevier GmbH. All rights reserved.

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

PubMed Central

Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

2009-01-01

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A.

PubMed

Park, Jin-Ho; Byun, Ju-Young; Mun, Hyoyoung; Shim, Won-Bo; Shin, Yong-Beom; Li, Taihua; Kim, Min-Gon

2014-09-15

Binding of an analyte on the surface of a nanoparticle typically promotes a change in the local refractive index, which gives rise to a shift in the wavelength of the localized surface plasmon resonance (LSPR) absorption band. The magnitude of the LSPR wavelength change is dependent on both the location of the analyte relative to the surface of the nanoparticle and the degree of alteration of the refractive index. We have employed this phenomenon as the basis for designing a new, label-free approach for the detection of the toxic mold mycotoxin, ochratoxin A (OTA) that employs a gold nanorod (GNR) and an aptamer target binding mechanism. In this system, binding of OTA causes an accumulation of OTA and G-quadruplex structure of the aptamer. This process results in a longitudinal wavelength shift of the LSPR peak associated with a change in the local refractive index near the GNR surface. By using this method, OTA can be quantitatively detected at concentrations lower than 1 nM. In addition, the results of this effort show that aptamer functionalized GNR substrate is robust in that it can be regenerated for reuse over seven times by heating in methanol at 70 °C to remove OTA. Moreover, the proposed biosensor system exhibits high selectivity for OTA over other mycotoxins. Finally, the sensor can be employed to detect OTA in ground corn samples with excellent recovery levels. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-free photoacoustic nanoscopy

PubMed Central

Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

2014-01-01

Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

A sensitive label-free amperometric CEA immunosensor based on graphene-nafion nanocomposite film as an enhanced sensing platform.

PubMed

Li, Yan; Yang, Wei-Kang; Fan, Man-Qi; Liu, Ao

2011-01-01

A novel approach to fabricate a label-free amperometric immunosensor for the detection of carcinoembryonic antigen (CEA) was described. Herein, methylene blue (MB), gold nanoparticles (AuNPs) and carcinoembryonic antibody (anti-CEA) were layer-by-layer assembled on the graphene-Nafion nanocomposite film-modified electrode by means of a self-assembling technique and the opposite-charged adsorption. Subsequently, the stepwise self-assembling procedure of the immunosensor was further characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The factors influencing the performance of the resulting immunosensor were studied in detail. The developed procedure showed improved features, including larger amount and higher immunoactivity of the immobilized antibody and repeatable regeneration of the sensor, as well as direct, rapid and simple determination for the antigen without multiple separation and labeling steps. The immunosensor could detect the target protein in a range of 0.5 to 120 ng/mL with a limit of 0.17 ng/mL (at 3σ). Finally, the immunosensing system was evaluated on several clinical samples. Analytical results were found to be in satisfactory agreement with those detected by the enzyme-linked immunosorbent assay (ELISA) method, indicating that this new method was a promising alternative tool for clinical diagnosis.

Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline

NASA Astrophysics Data System (ADS)

He, Yi; Peng, Rufang

2014-11-01

In this work, luminol functionalized gold nanoparticles (LuAuNPs) were used as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of minocycline (MC). The LuAuNPs were prepared by simple one-pot reduction of HAuCl4 with luminol, which exhibited a good chemiluminescence (CL) activity owing to the presence of luminol molecules on their surface and surface plasmon resonance absorption. In the absence of MC, the color of LuAuNPs was wine red and their size was relatively small (˜25 nm), which could react with silver nitrate, producing a strong CL emission. Upon the addition of MC at acidic buffer solutions, the electrostatic interaction between positively charged MC and negatively charged LuAuNPs caused the aggregation of LuAuNPs, generating a purple or blue color. Simultaneously, the aggregated LuAuNPs did not effectively react with silver nitrate, producing a weak CL emission. The signal change was linearly dependent on the logarithm of MC concentration in the range from 30 ng to 1.0 μg for colorimetric detection and from 10 ng to 1.0 μg for CL detection. With colorimetry, a detection limit of 22 ng was achieved, while the detection limit for CL detection modality was 9.7 ng.

Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

NASA Astrophysics Data System (ADS)

Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

2016-07-01

A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

A cascade signal amplification strategy for sensitive and label-free DNA detection based on Exo III-catalyzed recycling coupled with rolling circle amplification.

PubMed

Liu, Xingti; Xue, Qingwang; Ding, Yongshun; Zhu, Jing; Wang, Lei; Jiang, Wei

2014-06-07

A sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining exonuclease III (Exo III)-catalyzed recycling with rolling circle amplification. In this assay, probe DNA hybridized with template DNA was coupled onto magnetic nanoparticles to prepare a magnetic bead-probe (MNB-probe)-template complex. The complex could hybridize with the target DNA, which transformed the protruding 3' terminus of template DNA into a blunt end. Exo III could then digest template DNA, liberating the MNB-probe and target DNA. The intact target DNA then hybridized with other templates and released more MNB-probes. The liberated MNB-probe captured the primer, circular DNA and then initiated the rolling circle amplification (RCA) reaction, realizing a cascade signal amplification. Using this cascade amplification strategy, a sensitive DNA detection method was developed which was superior to many existing Exo III-based signal amplification methods. Moreover, N-methyl mesoporphyrin IX, which had a pronounced structural selectivity for the G-quadruplex, was used to combine with the G-quadruplex RCA products and generate a fluorescence signal, avoiding the need for any fluorophore-label probes. The spike and recovery experiments in a human serum sample indicated that our assay also had great potential for DNA detection in real biological samples.

Paper-based microfluidic sensing device for label-free immunoassay demonstrated by biotin-avidin binding interaction.

PubMed

Lei, Kin Fong; Yang, Shih-I; Tsai, Shiao-Wen; Hsu, Hsiao-Ting

2015-03-01

Efficient diagnosis is very important for the prevention and treatment of diseases. Rapid disease screening in ambulatory environment is one of the most pressing needs for disease control. Despite there are many methods to detect the results of immunoassays, quantitative measurement for rapid disease screening is still a great challenge for point-of-care applications. In this study, a fabrication method for depositing carbon nanotube bundles has been successfully developed for realization of functional paper-based microfluidic sensing device. Quantitative detection of label-free immunoassay, i.e., biotin-avidin binding interaction, was demonstrated by direct measurement of the current change of the biosensor after single application of the target analyte. Sensitivity of 0.33 μA/ng mL(-1) and minimal detectable analyte concentration of 25 ng/mL were achieved. The time necessary for the detection was 500 s which is a large reduction compared with the conventional immunoassay. Such paper-based biosensor has the benefits of portability, fast response, simple operation, and low cost and has the potential for the development of rapid disease screening devices. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid Quantitative Detection of Brucella melitensis by a Label-Free Impedance Immunosensor Based on a Gold Nanoparticle-Modified Screen-Printed Carbon Electrode

PubMed Central

Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

2013-01-01

A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 104 to 4 × 106 CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 104 and 4 × 105 CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis. PMID:23881126

Rapid quantitative detection of Brucella melitensis by a label-free impedance immunosensor based on a gold nanoparticle-modified screen-printed carbon electrode.

PubMed

Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

2013-07-04

A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 10(4) to 4 × 10(6) CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 10(4) and 4 × 10(5) CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis.

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

PubMed

Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

2016-11-15

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free optical imaging of nonfluorescent molecules by stimulated radiation.

PubMed

Min, Wei

2011-12-01

Imaging contrasts other than fluorescence are highly desirable for label-free detection and interrogation of nonfluorescent molecular species inside live cells, tissues, and organisms. The recently developed stimulated Raman scattering (SRS) and stimulated emission microscopy techniques provide sensitive and specific contrast mechanisms for nonfluorescent species, by employing the light amplification aspect of stimulated radiation. Compared to their spontaneous counterparts, stimulated radiation can enhance the imaging performance significantly, making the previously 'dark' molecules observable. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Copyright © 2011 Elsevier Ltd. All rights reserved.

Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

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

Lee, Ai Cheng; Du, Dan; Chen, Baowei

2014-09-07

Here we describe an ultrasensitive electrochemical nucleic acids assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL) related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation, and used to label and amplify target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The effect of DP and HRP loading of the CNT-based labels on its signal-to-noise ratio of electrochemical detection wasmore » studied systematically for the first time. Under optimized conditions, the signal-amplified assay achieved a detection limit of 83 fM targets oligonuecleotides and a 4-order wide dynamic range of target concentration. The resulting assay allowed a robust discrimination between the perfect match and a three-base mismatch sequence. When subjected to full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of approximately 33 pg of the target gene. The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60. The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, point-of-care and early disease diagnostic applications.« less

Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detection.

PubMed

Xu, Yuanhong; Li, Jing; Wang, Erkang

2008-05-01

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00x10(-6), 2x10(-6), 7x10(-7), and 5x10(-7) mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips.

Dextran hydrogel coated surface plasmon resonance imaging (SPRi) sensor for sensitive and label-free detection of small molecule drugs

NASA Astrophysics Data System (ADS)

Li, Shaopeng; Yang, Mo; Zhou, Wenfei; Johnston, Trevor G.; Wang, Rui; Zhu, Jinsong

2015-11-01

The label-free and sensitive detection of small molecule drugs on SPRi is still a challenging task, mainly due to the limited surface immobilization capacity of the sensor. In this research, a dextran hydrogel-coated gold sensor chip for SPRi was successfully fabricated via photo-cross-linking for enhanced surface immobilization capacity. The density of the dextran hydrogel was optimized for protein immobilization and sensitive small molecule detection. The protein immobilization capacity of the hydrogel was 10 times greater than a bare gold surface, and 20 times greater than an 11-mercaptoundecanoic acid (MUA) surface. Such a drastic improvement in immobilization capacity allowed the SPRi sensor to detect adequate response signals when probing small molecule binding events. The binding signal of 4 nM liquid-phase biotin to streptavidin immobilized on the dextran surface reached 435 RU, while no response was observed on bare gold or MUA surfaces. The dextran hydrogel-coated SPRi sensor was also applied in a kinetic study of the binding between an immunosuppressive drug (FK506) and its target protein (FKBP12) in a high-throughput microarray format. The measured binding affinity was shown to be consistent with reported literature values, and a detection limit of 0.5 nM was achieved.

Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Shao, Jin-Yu; Wang, Lihong V.

2017-04-01

Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification in vivo. The feasibility was first demonstrated by imaging CTC cluster ex vivo. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats in vivo. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters in vivo and showed its potential for tumor metastasis study and cancer therapy.

Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

NASA Astrophysics Data System (ADS)

Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

2016-02-01

We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

Label-free optical biosensors based on aptamer-functionalized porous silicon scaffolds.

PubMed

Urmann, Katharina; Walter, Johanna-Gabriela; Scheper, Thomas; Segal, Ester

2015-02-03

A proof-of-concept for a label-free and reagentless optical biosensing platform based on nanostructured porous silicon (PSi) and aptamers is presented in this work. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensor design. Here we describe the fabrication and characterization of aptamer-conjugated PSi biosensors, where a previously characterized his-tag binding aptamer (6H7) is used as model system. Exposure of the aptamer-functionalized PSi to the target proteins as well as to complex fluids (i.e., bacteria lysates containing target proteins) results in robust and well-defined changes in the PSi optical interference spectrum, ascribed to specific aptamer-protein binding events occurring within the nanoscale pores, monitored in real time. The biosensors show exceptional stability and can be easily regenerated by a short rinsing step for multiple biosensing analyses. This proof-of-concept study demonstrates the possibility of designing highly stable and specific label-free optical PSi biosensors, employing aptamers as capture probes, holding immense potential for application in detection of a broad range of targets, in a simple yet reliable manner.

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

Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

PubMed

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

Labeling of indocyanine green with carrier-free iodine-123

DOEpatents

Ansari, Azizullah N.; Lambrecht, Richard M.; Redvanly, Carol S.; Wolf, Alfred P.

1976-01-01

The method of labeling indocyanine green (ICG) with carrier-free iodine-123 comprising the steps of condensing xenon-123 on crystals of ICG followed by permitting decay of the .sup.123 Xe a sufficient length of time to produce .sup.123 I-electronically excited ions and atoms which subsequently label ICG.

Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay.

PubMed

Wang, Xiu-Li; Zhu, Ying; Fang, Qun

2014-01-07

In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensin I and angiotensin II as model samples, and the repeatabilities of peak area for angiotensin I and angiotensin II were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.

Label-free and non-contact optical biosensing of glucose with quantum dots.

PubMed

Khan, Saara A; Smith, Gennifer T; Seo, Felix; Ellerbee, Audrey K

2015-02-15

We present a label-free, optical sensor for biomedical applications based on changes in the visible photoluminescence (PL) of quantum dots in a thin polymer film. Using glucose as the target molecule, the screening of UV excitation due to pre-absorption by the product of an enzymatic assay leads to quenching of the PL of quantum dots (QDs) in a non-contact scheme. The irradiance changes in QD PL indicate quantitatively the level of glucose present. The non-contact nature of the assay prevents surface degradation of the QDs, which yields an efficient, waste-free, cost-effective, portable, and sustainable biosensor with attractive market features. The limit of detection of the demonstrated biosensor is ~3.5 µm, which is competitive with existing contact-based bioassays. In addition, the biosensor operates over the entire clinically relevant range of glucose concentrations of biological fluids including urine and whole blood. The comparable results achieved across a range of cost-affordable detectors, including a spectrophotometer, portable spectrometer, and iPhone camera, suggest that label-free and visible quantification of glucose with QD films can be applied to low-cost, point-of-care biomedical sensing as well as scientific applications in the laboratory for characterizing glucose or other analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

Slow light Mach-Zehnder interferometer as label-free biosensor with scalable sensitivity

DOE PAGES

Qin, Kun; Hu, Shuren; Retterer, Scott T.; ...

2016-02-05

Our design, fabrication, and characterization of a label-free Mach–Zehnder interferometer (MZI) optical biosensor that incorporates a highly dispersive one-dimensional (1D) photonic crystal in one arm are presented. The sensitivity of this slow light MZI-based sensor scales with the length of the slow light photonic crystal region. The numerically simulated sensitivity of a MZI sensor with a 16 μm long slow light region is 115,000 rad/RIU-cm, which is sevenfold higher than traditional MZI biosensors with millimeter-length sensing regions. Moreover, the experimental bulk refractive index detection sensitivity of 84,000 rad/RIU-cm is realized and nucleic acid detection is also demonstrated.

A universal label-free fluorescent aptasensor based on Ru complex and quantum dots for adenosine, dopamine and 17β-estradiol detection.

PubMed

Huang, Hailiang; Shi, Shuo; Gao, Xing; Gao, Ruru; Zhu, Ying; Wu, Xuewen; Zang, Ruimin; Yao, Tianming

2016-05-15

Based on specific aptamer binding properties, a strategy for adenosine, dopamine and 17β-estradiol detection was realised by employing Ru complex and quantum dots (QDs) as fluorescence probes. Ru complex, which could quench the fluorescence of QDs, preferred to bind with aptamer DNA and resulted in the fluorescence rise of QDs. When the aptamer DNA was incubated with the target first, it could not bind with Ru complex and the fluorescence of QDs was quenched. Under the optimal condition, the fluorescence intensity was linearly proportional to the concentration of adenosine, dopamine and 17β-estradiol with a limit of detection (LOD) of 101 nM, 19 nM and 37 nM, respectively. The experiments in fetal bovine serum were also carried out with good results. This universal method was rapid, label-free, low-cost, easy-operating and highly repeatable for the detection of adenosine, dopamine and 17β-estradiol. Qualitative detection by naked eyes was also available without complex instruments. It could also be extended to detect various analytes, such as metal ions, proteins and small molecules by using appropriate aptamers. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Sensors Based on Graphene Field-Effect Transistors for the Detection of Human Chorionic Gonadotropin Cancer Risk Biomarker

PubMed Central

Haslam, Carrie; Damiati, Samar; Whitley, Toby; Ifeachor, Emmanuel

2018-01-01

We report on the development of label-free chemical vapour deposition (CVD) graphene field effect transistor (GFET) immunosensors for the sensitive detection of Human Chorionic Gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFET sensors were fabricated on Si/SiO2 substrate using photolithography with evaporated chromium and sputtered gold contacts. GFET channels were functionalised with a linker molecule to an immobile anti-hCG antibody on the surface of graphene. The binding reaction of the antibody with varying concentration levels of hCG antigen demonstrated the limit of detection of the GFET sensors to be below 1 pg/mL using four-probe electrical measurements. We also show that annealing can significantly improve the carrier transport properties of GFETs and shift the Dirac point (Fermi level) with reduced p-doping in back-gated measurements. The developed GFET biosensors are generic and could find applications in a broad range of medical diagnostics in addition to cancer, such as neurodegenerative (Alzheimer’s and Parkinson’s) and cardiovascular disorders. PMID:29316718

Glucose-oxidase label-based redox cycling for an incubation period-free electrochemical immunosensor.

PubMed

Singh, Amardeep; Park, Seonhwa; Yang, Haesik

2013-05-21

Catalytic reactions of enzyme labels in enzyme-linked immunosorbent assays require a long incubation period to obtain high signal amplification. We present herein a simple immunosensing scheme in which the incubation period is minimized without a large increase in the detection limit. This scheme is based on electrochemical-enzymatic (EN) redox cycling using glucose oxidase (GOx) as an enzyme label, Ru(NH3)6(3+) as a redox mediator, and glucose as an enzyme substrate. Fast electron mediation of Ru(NH3)6(3+) between the electrode and the GOx label attached to the electrode allows high signal amplification. The acquisition of chronocoulometric charges at a potential in the mass transfer-controlled region excludes the influence of the kinetics of Ru(NH3)6(2+) electrooxidation and also facilitates high signal-to-background ratios. The reaction between reduced GOx and Ru(NH3)6(3+) is rapid even in air-saturated Tris buffer, where the faster competitive reaction between reduced GOx and dissolved oxygen also occurs. The direct electrooxidation of glucose at the electrode and the direct electron transfer between glucose and Ru(NH3)6(3+) that undesirably increase background levels occur relatively slowly. The detection limit for the EN redox cycling-based detection of cancer antigen 125 (CA-125) in human serum is slightly higher than 0.1 U/mL for the incubation period of 0 min, and the detection limits for the incubation periods of 5 and 10 min are slightly lower than 0.1 U/mL, indicating that the detection limits are almost similar irrespective of the incubation period and that the immunosensor is highly sensitive.

Linear-array-based photoacoustic tomography for label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Wang, Lihong V.

2017-03-01

Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

PubMed

Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

2014-08-15

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

NASA Astrophysics Data System (ADS)

Sohn, Lydia L.

2013-03-01

Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine

Label free sensing of creatinine using a 6 GHz CMOS near-field dielectric immunosensor.

PubMed

Guha, S; Warsinke, A; Tientcheu, Ch M; Schmalz, K; Meliani, C; Wenger, Ch

2015-05-07

In this work we present a CMOS high frequency direct immunosensor operating at 6 GHz (C-band) for label free determination of creatinine. The sensor is fabricated in standard 0.13 μm SiGe:C BiCMOS process. The report also demonstrates the ability to immobilize creatinine molecules on a Si3N4 passivation layer of the standard BiCMOS/CMOS process, therefore, evading any further need of cumbersome post processing of the fabricated sensor chip. The sensor is based on capacitive detection of the amount of non-creatinine bound antibodies binding to an immobilized creatinine layer on the passivated sensor. The chip bound antibody amount in turn corresponds indirectly to the creatinine concentration used in the incubation phase. The determination of creatinine in the concentration range of 0.88-880 μM is successfully demonstrated in this work. A sensitivity of 35 MHz/10 fold increase in creatinine concentration (during incubation) at the centre frequency of 6 GHz is gained by the immunosensor. The results are compared with a standard optical measurement technique and the dynamic range and sensitivity is of the order of the established optical indication technique. The C-band immunosensor chip comprising an area of 0.3 mm(2) reduces the sensing area considerably, therefore, requiring a sample volume as low as 2 μl. The small analyte sample volume and label free approach also reduce the experimental costs in addition to the low fabrication costs offered by the batch fabrication technique of CMOS/BiCMOS process.

A Method for Label-Free, Differential Top-Down Proteomics.

PubMed

Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

2016-01-01

Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

Label-free cell separation and sorting in microfluidic systems

PubMed Central

Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

2010-01-01

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Biconically tapered fiber optic probes for rapid label-free immunoassays.

PubMed

Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Ortiz, Adrian; Almaz, Ekrem; Almaz, Zuleyha Turkoglu; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

2015-04-01

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-based visual detection of nucleic acids

NASA Astrophysics Data System (ADS)

Lee, Ai Cheng; Ye, Jian-Shan; Ngin Tan, Swee; Poenar, Daniel P.; Sheu, Fwu-Shan; Kiat Heng, Chew; Meng Lim, Tit

2007-11-01

A novel carbon nanotube (CNT) derived label capable of dramatic signal amplification of nucleic acid detection and direct visual detection of target hybridization has been developed. Highly sensitive colorimetric detection of human acute lymphocytic leukemia (ALL) related oncogene sequences amplified by the novel CNT-based label was demonstrated. Atomic force microscope (AFM) images confirmed that a monolayer of horseradish peroxidase and detection probe molecules was immobilized along the carboxylated CNT carrier. The resulting CNT labels significantly enhanced the nucleic acid assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA). An excellent detection limit of 1 × 10-12 M (60 × 10-18 mol in 60 µl) and a four-order wide dynamic range of target concentration were achieved. Hybridizations using these labels were coupled to a concentration-dependent formation of visible dark aggregates. Targets can thus be detected simply with visual inspection, eliminating the need for expensive and sophisticated detection systems. The approach holds promise for ultrasensitive and low cost visual inspection and colorimetric nucleic acid detection in point-of-care and early disease diagnostic application.

Dual-primer self-generation SERS signal amplification assay for PDGF-BB using label-free aptamer.

PubMed

Ye, SuJuan; Zhai, XiaoMo; Wu, YanYing; Kuang, ShaoPing

2016-05-15

Highly sensitive detection of proteins, especially those associated with cancers, is essential to biomedical research as well as clinical diagnosis. In this work, a simple and novel one-two-three signal amplification surface-enhanced Raman scattering (SERS) method for the detection of protein is fabricated by using label-free aptamer and dual-primer self-generation. Platelet-derived growth factor B-chain (PDGF-BB) is selected as the model protein. The one-two-three cascade DNA amplification means one target-aptamer binding event, two hairpin DNA switches and three DNA amplification reactions. This strategy possesses some remarkable features compared to conventional signal amplification methods: (i) A smart probe including a label-free aptamer is fabricated, for suitable hybridization without hindering the affinity of the aptamer toward its target. (ii) Using the unique structure switch of the aptamer and cooperator, a one-two-three working mode is developed to amplify the SERS signal. The amplification efficiency is enhanced. Given the unique and attractive characteristics, a simple and universal strategy is designed to accomplish ultrasensitive detection of proteins. The detection limit of PDGF-BB via SERS detection is 0.42 pM, with the linear range from 1.0×10(-12)M to 10(-8)M. It is potentially universal because the aptamer can be easily designed for biomolecules whose aptamers undergo similar conformational changes. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

PubMed

Brito, Mayara F; Auler, Patrícia A; Tavares, Guilherme C; Rezende, Cristiana P; Almeida, Gabriel M F; Pereira, Felipe L; Leal, Carlos A G; Moura, Arlindo de Alencar; Figueiredo, Henrique C P; Henry, Marc

2018-06-11

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728. © 2018 Blackwell Verlag GmbH.

Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification.

PubMed

Zhang, Li-rong; Zhu, Guichi; Zhang, Chun-yang

2014-07-01

MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10(-13) M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

Silicon photonic resonator for label-free bio-sensing application

NASA Astrophysics Data System (ADS)

Udomsom, Suruk; Mankong, Ukrit; Theera-Umpon, Nipon; Ittipratheep, Nattapol; Umezawa, Toshimasa; Matsumoto, Atsushi; Yamamoto, Naokatsu

2018-03-01

In medical diagnostics there is an increasing demand for biosensors that can specifically detect biological analytes in a fluid. Especially label-free sensing, consistings of a transducer with biorecognition molecules immobilized on its surface without relying on fluorescent dye. In this paper we study the design and fabrication of a silicon nanowire photonic ring resonator and its feasibility as a biosensor. We have simulated and fabricated racetrack ring resonators which have a few tenths of micrometer gap, up to 0.5 μm between the input / output waveguides and the resonators. It is found that the devices can be designed with large Q factors. Sensitivity to biomaterial detection has been simulated for antibody (goat anti-mouse IgG) - antigen (mouse IgG) using 3-dimensional Finite Difference Time Domain technique. The simulated results show that the ring resonator has a response 15 nm resonance shift per refractive index unit. Antibody coating method is also discussed in this paper which can be applied to other antibody-antigen types.

A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles.

PubMed

Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang

2016-08-17

In this work, we report a novel label-free fluorescence "turn off-on" biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS2 quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS2 QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS2 QDs surface were interacted with the amino groups (NH2), carboxyl groups (COOH) and hydroxyl groups (OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively "turned on". Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I0 (I and I0 were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2-192.5 nmol L(-1), And the detection limit could be down to 0.08 nmol L(-1). Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free voltammetric detection of MicroRNAs at multi-channel screen printed array of electrodes comparison to graphite sensors.

PubMed

Erdem, Arzum; Congur, Gulsah

2014-01-01

The multi-channel screen-printed array of electrodes (MUX-SPE16) was used in our study for the first time for electrochemical monitoring of nucleic acid hybridization related to different miRNA sequences (miRNA-16, miRNA-15a and miRNA-660, i.e, the biomarkers for Alzheimer disease). The MUX-SPE16 was also used for the first time herein for the label-free electrochemical detection of nucleic acid hybridization combined magnetic beads (MB) assay in comparison to the disposable pencil graphite electrode (PGE). Under the principle of the magnetic beads assay, the biotinylated inosine substituted DNA probe was firstly immobilized onto streptavidin coated MB, and then, the hybridization process between probe and its complementary miRNA sequence was performed at MB surface. The voltammetric transduction was performed using differential pulse voltammetry (DPV) technique in combination with the single-use graphite sensor technologies; PGE and MUX-SPE16 for miRNA detection by measuring the guanine oxidation signal without using any external indicator. The features of single-use sensor technologies, PGE and MUX-SPE16, were discussed concerning to their reproducibility, detection limit, and selectivity compared to the results in the earlier studies presenting the electrochemical miRNA detection related to different miRNA sequences. © 2013 Elsevier B.V. All rights reserved.

Label propagation algorithm for community detection based on node importance and label influence

NASA Astrophysics Data System (ADS)

Zhang, Xian-Kun; Ren, Jing; Song, Chen; Jia, Jia; Zhang, Qian

2017-09-01

Recently, the detection of high-quality community has become a hot spot in the research of social network. Label propagation algorithm (LPA) has been widely concerned since it has the advantages of linear time complexity and is unnecessary to define objective function and the number of community in advance. However, LPA has the shortcomings of uncertainty and randomness in the label propagation process, which affects the accuracy and stability of the community. For large-scale social network, this paper proposes a novel label propagation algorithm for community detection based on node importance and label influence (LPA_NI). The experiments with comparative algorithms on real-world networks and synthetic networks have shown that LPA_NI can significantly improve the quality of community detection and shorten the iteration period. Also, it has better accuracy and stability in the case of similar complexity.

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

PubMed

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Optofluidic analysis system for amplification-free, direct detection of Ebola infection

NASA Astrophysics Data System (ADS)

Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

2015-09-01

The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

Determination of reduced homocysteine in human serum by elemental labelling and liquid chromatography with ICP-MS and ESI-MS detection.

PubMed

Espina, Juan Gómez; Montes-Bayón, Maria; Blanco-González, Elisa; Sanz-Medel, Alfredo

2015-10-01

Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 μM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.

Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

PubMed

Wu, Enqi; Peng, Yuan; Zhang, Xihao; Bai, Jialei; Song, Yanqiu; He, Houluo; Fan, Longxing; Qu, Xiaochen; Gao, Zhixian; Liu, Ying; Ning, Baoan

2017-02-22

A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10 -3 ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.

Nanophotonic label-free biosensors for environmental monitoring.

PubMed

Chocarro-Ruiz, Blanca; Fernández-Gavela, Adrián; Herranz, Sonia; Lechuga, Laura M

2017-06-01

The field of environmental monitoring has experienced a substantial progress in the last years but still the on-site control of contaminants is an elusive problem. In addition, the growing number of pollutant sources is accompanied by an increasing need of having efficient early warning systems. Several years ago biosensor devices emerged as promising environmental monitoring tools, but their level of miniaturization and their fully operation outside the laboratory prevented their use on-site. In the last period, nanophotonic biosensors based on evanescent sensing have emerged as an outstanding choice for portable point-of-care diagnosis thanks to their capability, among others, of miniaturization, multiplexing, label-free detection and integration in lab-on-chip platforms. This review covers the most relevant nanophotonic biosensors which have been proposed (including interferometric waveguides, grating-couplers, microcavity resonators, photonic crystals and localized surface plasmon resonance sensors) and their recent application for environmental surveillance. Copyright © 2017 Elsevier Ltd. All rights reserved.

Carbon nanostructure-based field-effect transistors for label-free chemical/biological sensors.

PubMed

Hu, PingAn; Zhang, Jia; Li, Le; Wang, Zhenlong; O'Neill, William; Estrela, Pedro

2010-01-01

Over the past decade, electrical detection of chemical and biological species using novel nanostructure-based devices has attracted significant attention for chemical, genomics, biomedical diagnostics, and drug discovery applications. The use of nanostructured devices in chemical/biological sensors in place of conventional sensing technologies has advantages of high sensitivity, low decreased energy consumption and potentially highly miniaturized integration. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene based electrical devices have been widely developed for high performance label-free chemical/biological sensors. Here, we review the latest developments of carbon nanostructure-based transistor sensors in ultrasensitive detection of chemical/biological entities, such as poisonous gases, nucleic acids, proteins and cells.

Flexible-rate optical packet generation/detection and label swapping for optical label switching networks

NASA Astrophysics Data System (ADS)

Wu, Zhongying; Li, Juhao; Tian, Yu; Ge, Dawei; Zhu, Paikun; Chen, Yuanxiang; Chen, Zhangyuan; He, Yongqi

2017-03-01

In recent years, optical label switching (OLS) gains lots of attentions due to its intrinsic advantages to implement protocol, bit-rate, granularity and data format transparency packet switching. In this paper, we propose a novel scheme to realize flexible-rate optical packet switching for OLS networks. At the transmitter node, flexible-rate packet is generated by parallel modulating different combinations of optical carriers generated from the optical multi-carrier generator (OMCG), among which the low-speed optical label occupies one carrier. At the switching node, label is extracted and re-generated in label processing unit (LPU). The payloads are switched based on routing information and new label is added after switching. At the receiver node, another OMCG serves as local oscillators (LOs) for optical payloads coherent detection. The proposed scheme offers good flexibility for dynamic optical packet switching by adjusting the payload bandwidth and could also effectively reduce the number of lasers, modulators and receivers for packet generation/detection. We present proof-of-concept demonstrations of flexible-rate packet generation/detection and label swapping in 12.5 GHz grid. The influence of crosstalk for cascaded label swapping is also investigated.

Label-Free SERS Selective Detection of Dopamine and Serotonin Using Graphene-Au Nanopyramid Heterostructure.

PubMed

Wang, Pu; Xia, Ming; Liang, Owen; Sun, Ke; Cipriano, Aaron F; Schroeder, Thomas; Liu, Huinan; Xie, Ya-Hong

2015-10-20

Ultrasensitive detection and spatially resolved mapping of neurotransmitters, dopamine and serotonin, are critical to facilitate understanding brain functions and investigate the information processing in neural networks. In this work, we demonstrated single molecule detection of dopamine and serotonin using a graphene-Au nanopyramid heterostructure platform. The quasi-periodic Au structure boosts high-density and high-homogeneity hotspots resulting in ultrahigh sensitivity with a surface enhanced Raman spectroscopic (SERS) enhancement factor ∼10(10). A single layer graphene superimposed on a Au structure not only can locate SERS hot spots but also modify the surface chemistry to realize selective enhancement Raman yield. Dopamine and serotonin could be detected and distinguished from each other at 10(-10) M level in 1 s data acquisition time without any pretreatment and labeling process. Moreover, the heterostructure realized nanomolar detection of neurotransmitters in the presence of simulated body fluids. These findings represent a step forward in enabling in-depth studies of neurological processes including those closely related to brain activity mapping (BAM).

Simple and label-free liquid crystal-based sensor for detecting trypsin coupled to the interaction between cationic surfactant and BSA.

PubMed

Wang, Yi; Zhou, Lele; Kang, Qi; Yu, Li

2018-06-01

Trypsin plays a central role in catalyzing the hydrolysis of peptide bonds, so a technique with simple operation is needed to monitor the activity of trypsin. Here a simple and label-free senor based on liquid crystals (LCs) was developed by employing bovine serum albumin (BSA) as the enzyme substrate and dodecyl trimethyl ammonium bromide (DTAB) as the controller for the alignment of LC. It was found that DTAB could form a self-assembled monolayer at the aqueous/LC interface to produce the dark optical images of LCs. And the addition of BSA could disturb the monolayer, so that the optical signal of LCs turned bright from dark. But the hydrolysis of BSA by trypsin resulted in the dark appearance. The sensing platform allows detection as low as 1 U/mL under the polarized light microscope based on at least three measurements. Moreover, this method was successfully applied in the detection of trypsin in human urines, suggesting its potential applications in clinic diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

NASA Astrophysics Data System (ADS)

Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

2015-01-01

This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

PubMed

Winn-Deen

1998-12-01

Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.

A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

PubMed

Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

2017-03-08

K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS 2- by H 2 O 2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

NASA Astrophysics Data System (ADS)

Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

2015-07-01

Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

Cost-effective flow-through nanohole array-based biosensing platform for the label-free detection of uropathogenic E. coli in real time.

PubMed

Gomez-Cruz, Juan; Nair, Srijit; Manjarrez-Hernandez, Angel; Gavilanes-Parra, Sandra; Ascanio, Gabriel; Escobedo, Carlos

2018-05-30

Rapid, inexpensive and sensitive detection of uropathogenic Escherichia coli (UPEC), a common cause of ascending urinary tract infections (UTIs) including cystitis and pyelonephritis, is critical given the increasing number of cases and its recurrence worldwide. In this paper, we present a label-free nanoplasmonic sensing platform, built with off-the-shelf optical and electronic components, which can detect intact UPEC at concentrations lower than the physiological limit for UTI diagnosis, in real time. The sensing platform consists of a red LED light source, lens assembly, CMOS detector, Raspberry Pi interface in conjugation with a metallic flow-through nanohole array-based sensor. Detection is achieved exploiting nanoplasmonic phenomena from the nanohole arrays through surface plasmon resonance imaging (SPRi) technique. The platform has a bulk sensitivity of 212 pixel intensity unit (PIU)/refractive index unit (RIU), and a resolution in the order of 10 -6 RIU. We demonstrate capture and detection of UPEC with a detection limit of ~100 CFU/ml - a concentration well below the threshold limit for UTI diagnosis in clinical samples. We also demonstrate detection of UPEC in spiked human urine samples for two different concentrations of bacteria. This work is particularly relevant for point-of-care applications, especially for regions around the world where accessibility to medical facilities is heavily dependent upon economy, and availability. Copyright © 2018 Elsevier B.V. All rights reserved.

A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

PubMed

Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

2015-07-07

Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.

PubMed

Sabaté Del Río, Jonathan; Steylaerts, Tim; Henry, Olivier Y F; Bienstman, Peter; Stakenborg, Tim; Van Roy, Wim; O'Sullivan, Ciara K

2015-11-15

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts. Published by Elsevier B.V.

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

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

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection ofmore » target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.« less

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

DOE PAGES

Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; ...

2015-08-31

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based onmore » changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Moreover, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. In conclusion, our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.« less

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

PubMed Central

Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; Hoang, Mai P.; Ji, Minbiao; Fu, Dan; Holtom, Gary R.; Neel, Victor A.; Freudiger, Christian W.; Fisher, David E.; Xie, X. Sunney

2015-01-01

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time. PMID:26324899

Hybrid integrated label-free chemical and biological sensors.

PubMed

Mehrabani, Simin; Maker, Ashley J; Armani, Andrea M

2014-03-26

Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits). This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach.

Stem Cell Monitoring with a Direct or Indirect Labeling Method.

PubMed

Kim, Min Hwan; Lee, Yong Jin; Kang, Joo Hyun

2016-12-01

The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

NASA Astrophysics Data System (ADS)

Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

2016-02-01

One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 - 2.0 × 108 cells mL-1), a low limit of detection (79 cells mL-1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

Label-Free Biosensor Imaging on Photonic Crystal Surfaces.

PubMed

Zhuo, Yue; Cunningham, Brian T

2015-08-28

We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in "digital" diagnostics with single molecule sensing resolution. We will review PCEM's development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity.

Label-Free Biosensor Imaging on Photonic Crystal Surfaces

PubMed Central

Zhuo, Yue; Cunningham, Brian T.

2015-01-01

We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in “digital” diagnostics with single molecule sensing resolution. We will review PCEM’s development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity. PMID:26343684

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

PubMed

Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

1988-03-15

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

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.

Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles.

PubMed

Li, Guiyin; Li, Shanshan; Wang, Zhihong; Xue, Yewei; Dong, Chenyang; Zeng, Junxiang; Huang, Yong; Liang, Jintao; Zhou, Zhide

2018-04-15

Sensitive and accurate detection of tumor markers is critical to early diagnosis, point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an important clinical tumor marker for hepatocellular carcinoma (HCC), and the concentration of AFP in human serum is related to the stage of HCC. In this paper, a label-free electrochemical aptasensor for AFP detection was fabricated using AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic property and large specific surface area, RGO and Au NPs were employed on the screen-printed carbon electrode to load TH molecules. The TH not only acted as a bridging molecule to effectively capture and immobilize AFP-aptamer, but as the electron transfer mediator to provide the electrochemical signal. The AFP detection was based on the monitoring of the electrochemical current response change of TH by the differential pulse voltammetry. Under optimal conditions, the electrochemical responses were proportional to the AFP concentration in the range of 0.1-100.0 μg/mL. The limit of detection was 0.050 μg/mL at a signal-to-noise ratio of 3. The proposed method may provide a promising application of aptamer with the properties of facile procedure, low cost, high selectivity in clinic. Copyright © 2018. Published by Elsevier Inc.

Microfluidic and Label-Free Multi-Immunosensors Based on Carbon Nanotube Microelectrodes

NASA Astrophysics Data System (ADS)

Tsujita, Yuichi; Maehashi, Kenzo; Matsumoto, Kazuhiko; Chikae, Miyuki; Takamura, Yuzuru; Tamiya, Eiichi

2009-06-01

We fabricated microfluidic and label-free multi-immunosensors by the integration of carbon nanotube (CNT)-arrayed electrodes and microchannels with pneumatic micropumps made of poly(dimethylsiloxane). In the microfluidic systems, four kinds of sample solutions were transported from each liquid inlet to microchannels using six pneumatic micropumps. As a result, two kinds of antibodies were immobilized onto different CNT electrodes using the microfluidic systems. Next, two kinds of cancer markers, prostate specific antigen and human chorionic gonadotropin in phosphate buffer solution, were simultaneously detected by differential pulse voltammetry. Therefore, microfludic multi-immunosensors based on CNT electrodes and pneumatic micropumps are useful for the development of multiplex hand-held biosensors.

A multicenter study benchmarks software tools for label-free proteome quantification.

PubMed

Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

2016-11-01

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

Non-aggregation based label free colorimetric sensor for the detection of Cu2+ based on catalyzing etching of gold nanorods by dissolve oxygen.

PubMed

Liu, Jia-Ming; Jiao, Li; Lin, Li-Ping; Cui, Ma-Lin; Wang, Xin-Xing; Zhang, Li-Hong; Zheng, Zhi-Yong; Jiang, Shu-Lian

2013-12-15

A label-free non-aggregation colorimetric sensor has been designed for the detection of Cu(2+), based on Cu(2+) catalyzing etching of gold nanorods (AuNRs) along longitudinal axis induced by dissolve oxygen in the presence of S2O3(2-), which caused the aspect ratio (length/width) of AuNRs to decrease and the color of the solution to distinctly change. The linear range and the detection limit (LD, calculated by 10 Sb/k, n=11) of this sensor were 0.080-4.8 µM Cu(2+) and 0.22 µM Cu(2+), respectively. This sensor has been utilized to detect Cu(2+) in tap water and human serum samples with the results agreeing well with those of inductively coupled plasma-mass spectroscopy (ICP-MS), showing its remarkable practicality. In order to prove the possibility of catalyzing AuNRs non-aggregation colorimetric sensor for the detection of Cu(2+), the morphological structures of AuNRs were characterized by high resolution transmission electron microscopy (HRTEM) and the sensing mechanism of colorimetric sensor for the detection of Cu(2+) was also discussed. © 2013 Elsevier B.V. All rights reserved.

Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

PubMed

Gao, Ran; Lu, Dan-Feng; Cheng, Jin; Jiang, Yi; Jiang, Lan; Xu, Jian-Dong; Qi, Zhi-Mei

2016-12-15

An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

OAM-labeled free-space optical flow routing.

PubMed

Gao, Shecheng; Lei, Ting; Li, Yangjin; Yuan, Yangsheng; Xie, Zhenwei; Li, Zhaohui; Yuan, Xiaocong

2016-09-19

Space-division multiplexing allows unprecedented scaling of bandwidth density for optical communication. Routing spatial channels among transmission ports is critical for future scalable optical network, however, there is still no characteristic parameter to label the overlapped optical carriers. Here we propose a free-space optical flow routing (OFR) scheme by using optical orbital angular moment (OAM) states to label optical flows and simultaneously steer each flow according to their OAM states. With an OAM multiplexer and a reconfigurable OAM demultiplexer, massive individual optical flows can be routed to the demanded optical ports. In the routing process, the OAM beams act as data carriers at the same time their topological charges act as each carrier's labels. Using this scheme, we experimentally demonstrate switching, multicasting and filtering network functions by simultaneously steer 10 input optical flows on demand to 10 output ports. The demonstration of data-carrying OFR with nonreturn-to-zero signals shows that this process enables synchronous processing of massive spatial channels and flexible optical network.

Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives.

PubMed

Cima, Igor; Wen Yee, Chay; Iliescu, Florina S; Phyo, Wai Min; Lim, Kiat Hon; Iliescu, Ciprian; Tan, Min Han

2013-01-01

This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to "marker-based" techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics.

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

PubMed

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Kobayashi, Hirofumi; Aisaka, Yuri; Ito, Takuro; Guo, Baoshan; Nitta, Nao; Kutsuna, Natsumaro; Ozeki, Yasuyuki; Nakagawa, Atsuhiro; Yatomi, Yutaka; Goda, Keisuke

2017-07-11

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

Quantitative label-free multimodality nonlinear optical imaging for in situ differentiation of cancerous lesions

NASA Astrophysics Data System (ADS)

Xu, Xiaoyun; Li, Xiaoyan; Cheng, Jie; Liu, Zhengfan; Thrall, Michael J.; Wang, Xi; Wang, Zhiyong; Wong, Stephen T. C.

2013-03-01

The development of real-time, label-free imaging techniques has recently attracted research interest for in situ differentiation of cancerous lesions from normal tissues. Molecule-specific intrinsic contrast can arise from label-free imaging techniques such as Coherent Anti-Stokes Raman Scattering (CARS), Two-Photon Excited AutoFluorescence (TPEAF), and Second Harmonic Generation (SHG), which, in combination, would hold the promise of a powerful label-free tool for cancer diagnosis. Among cancer-related deaths, lung carcinoma is the leading cause for both sexes. Although early treatment can increase the survival rate dramatically, lesion detection and precise diagnosis at an early stage is unusual due to its asymptomatic nature and limitations of current diagnostic techniques that make screening difficult. We investigated the potential of using multimodality nonlinear optical microscopy that incorporates CARS, TPEAF, and SHG techniques for differentiation of lung cancer from normal tissue. Cancerous and non-cancerous lung tissue samples from patients were imaged using CARS, TPEAF, and SHG techniques for comparison. These images showed good pathology correlation with hematoxylin and eosin (H and E) stained sections from the same tissue samples. Ongoing work includes imaging at various penetration depths to show three-dimensional morphologies of tumor cell nuclei using CARS, elastin using TPEAF, and collagen using SHG and developing classification algorithms for quantitative feature extraction to enable lung cancer diagnosis. Our results indicate that via real-time morphology analyses, a multimodality nonlinear optical imaging platform potentially offers a powerful minimally-invasive way to differentiate cancer lesions from surrounding non-tumor tissues in vivo for clinical applications.

Grazing incidence angle based sensing approach integrated with fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy for remote and label-free detection of medical device contaminations.

PubMed

Hassan, Moinuddin; Ilev, Ilko

2014-10-01

Contamination of medical devices has become a critical and prevalent public health safety concern since medical devices are being increasingly used in clinical practices for diagnostics, therapeutics and medical implants. The development of effective sensing methods for real-time detection of pathogenic contamination is needed to prevent and reduce the spread of infections to patients and the healthcare community. In this study, a hollow-core fiber-optic Fourier transform infrared spectroscopy methodology employing a grazing incidence angle based sensing approach (FO-FTIR-GIA) was developed for detection of various biochemical contaminants on medical device surfaces. We demonstrated the sensitivity of FO-FTIR-GIA sensing approach for non-contact and label-free detection of contaminants such as lipopolysaccharide from various surface materials relevant to medical device. The proposed sensing system can detect at a minimum loading concentration of approximately 0.7 μg/cm(2). The FO-FTIR-GIA has the potential for the detection of unwanted pathogen in real time.

Grazing incidence angle based sensing approach integrated with fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy for remote and label-free detection of medical device contaminations

NASA Astrophysics Data System (ADS)

Hassan, Moinuddin; Ilev, Ilko

2014-10-01

Contamination of medical devices has become a critical and prevalent public health safety concern since medical devices are being increasingly used in clinical practices for diagnostics, therapeutics and medical implants. The development of effective sensing methods for real-time detection of pathogenic contamination is needed to prevent and reduce the spread of infections to patients and the healthcare community. In this study, a hollow-core fiber-optic Fourier transform infrared spectroscopy methodology employing a grazing incidence angle based sensing approach (FO-FTIR-GIA) was developed for detection of various biochemical contaminants on medical device surfaces. We demonstrated the sensitivity of FO-FTIR-GIA sensing approach for non-contact and label-free detection of contaminants such as lipopolysaccharide from various surface materials relevant to medical device. The proposed sensing system can detect at a minimum loading concentration of approximately 0.7 μg/cm2. The FO-FTIR-GIA has the potential for the detection of unwanted pathogen in real time.

Grazing incidence angle based sensing approach integrated with fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy for remote and label-free detection of medical device contaminations

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

Hassan, Moinuddin, E-mail: moinuddin.hassan@fda.hhs.gov; Ilev, Ilko

2014-10-15

Contamination of medical devices has become a critical and prevalent public health safety concern since medical devices are being increasingly used in clinical practices for diagnostics, therapeutics and medical implants. The development of effective sensing methods for real-time detection of pathogenic contamination is needed to prevent and reduce the spread of infections to patients and the healthcare community. In this study, a hollow-core fiber-optic Fourier transform infrared spectroscopy methodology employing a grazing incidence angle based sensing approach (FO-FTIR-GIA) was developed for detection of various biochemical contaminants on medical device surfaces. We demonstrated the sensitivity of FO-FTIR-GIA sensing approach for non-contactmore » and label-free detection of contaminants such as lipopolysaccharide from various surface materials relevant to medical device. The proposed sensing system can detect at a minimum loading concentration of approximately 0.7 μg/cm{sup 2}. The FO-FTIR-GIA has the potential for the detection of unwanted pathogen in real time.« less

A label-free nanostructured plasmonic biosensor based on Blu-ray discs with integrated microfluidics for sensitive biodetection.

PubMed

López-Muñoz, Gerardo A; Estevez, M-Carmen; Peláez-Gutierrez, E Cristina; Homs-Corbera, Antoni; García-Hernandez, M Carmen; Imbaud, J Ignacio; Lechuga, Laura M

2017-10-15

Nanostructure-based plasmonic biosensors have quickly positioned themselves as interesting candidates for the design of portable optical biosensor platforms considering the potential benefits they can offer in integration, miniaturization, multiplexing, and real-time label-free detection. We have developed a simple integrated nanoplasmonic sensor taking advantage of the periodic nanostructured array of commercial Blu-ray discs. Sensors with two gold film thicknesses (50 and 100nm) were fabricated and optically characterized by varying the oblique-angle of the incident light in optical reflectance measurements. Contrary to the use normal light incidence previously reported with other optical discs, we observed an enhancement in sensitivity and a narrowing of the resonant linewidths as the light incidence angle was increased, which could be related to the generation of Fano resonant modes. The new sensors achieve a figure of merit (FOM) up to 35 RIU -1 and a competitive bulk limit of detection (LOD) of 6.3×10 -6 RIU. These values significantly improve previously reported results obtained with normal light incidence reflectance measurements using similar structures. The sensor has been combined with versatile, simple, ease to-fabricate microfluidics. The integrated chip is only 1cm 2 (including a PDMS flow cell with a 50µm height microfluidic channel fabricated with double-sided adhesive tape) and all the optical components are mounted on a 10cm×10cm portable prototype, illustrating its facile miniaturization, integration and potential portability. Finally, to assess the label-free biosensing capability of the new sensor, we have evaluated the presence of specific antibodies against the GTF2b protein, a tumor-associate antigen (TAA) related to colorectal cancer. We have achieved a LOD in the pM order and have assessed the feasibility of directly measuring biological samples such as human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

Myelin basic protein immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy.

PubMed

Derkus, Burak; Emregul, Emel; Yucesan, Canan; Cebesoy Emregul, Kaan

2013-08-15

A novel highly sensitive impedimetric Myelin Basic Protein (MBP) immunosensor for the determination of a Multiple Sclerosis (MS) autoantibody, Anti-Myelin Basic Protein (Anti-MBP) was developed by immobilization of MBP on Gelatin and Gelatin-Titanium Dioxide (TiO₂) modified platinium electrode. Cyclic voltammetric (CV) and Electrochemical Impedance Spectroscopic (EIS) methods were employed in determination of the electrode responses and applicability. Gelatin-MBP and gelatin-TiO₂-MBP electrodes were prepared by chemical immobilization of the substrates onto the platinium electrodes. The formal potentials of MBP confined on gelatin-MBP and gelatin-TiO₂-MBP surfaces are estimated to be 195 and 205 mV, respectively. Thus, a little more reversible electron transfer reaction occurs on the gelatin-TiO₂-MBP immunosensor surface. The peak separations of MBP (150 mV and 110 mV s(-1) at 100 mV s(-1)) and the asymmetric anodic and cathodic peak currents indicate that the electron transfer between Anti-MBP and gelatin-MBP/gelatin-TiO₂-MBP immunosensor is quasireversible. Control samples containing a nonspecific human immunoglobulin G (hIgG) antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors. Gelatin-MBP and gelatin-TiO₂-MBP immunosensors have detection limit of 0.1528 ng ml(-1) and 0.1495 ng ml(-1) respectively. This immunosensor exhibits high sensitivity and low response times (58 s for gelatin-MBP and 46 s for gelatin-TiO₂-MBP immunosensor). The developed label-free impedimetric immunosensors also provide a simple and sensitive detection method for the specific determination of Anti-MBP in human cerebrospinal fluid (CSF) and serum samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Microchannel DNA Sequencing by End-Labelled Free Solution Electrophoresis

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

Barron, A.

2005-09-29

The further development of End-Labeled Free-Solution Electrophoresis will greatly simplify DNA separation and sequencing on microfluidic devices. The development and optimization of drag-tags is critical to the success of this research.

Nanostructured plasmonic interferometers for ultrasensitive label-free biosensing

NASA Astrophysics Data System (ADS)

Gao, Yongkang

Optical biosensors that utilize surface plasmon resonance (SPR) technique to analyze the biomolecular interactions have been extensively explored in the last two decades and have become the gold standard for label-free biosensing. These powerful sensing tools allow fast, highly-sensitive monitoring of the interaction between biomolecules in real time, without the need for laborious fluorescent labeling, and have found widely ranging applications from biomedical diagnostics and drug discovery, to environmental sensing and food safety monitoring. However, the prism-coupling SPR geometry is complex and bulky, and has severely limited the integration of this technique into low-cost portable biomedical devices for point-of-care diagnostics and personal healthcare applications. Also, the complex prism-coupling scheme prevents the use of high numerical aperture (NA) optics to increase the spatial resolution for multi-channel, high-throughput detection in SPR imaging mode. This dissertation is focused on the design and fabrication of a promising new class of nanopatterned interferometric SPR sensors that integrate the strengths of miniaturized nanoplasmonic architectures with sensitive optical interferometry techniques to achieve bold advances in SPR biosensing. The nanosensor chips developed provide superior sensing performance comparable to conventional SPR systems, but employing a far simpler collinear optical transmission geometry, which largely facilitates system integration, miniaturization, and low-cost production. Moreover, the fabricated nanostructure-based SPR sensors feature a very small sensor footprint, allowing massive multiplexing on a chip for high-throughput detection. The successful transformation of SPR technique from bulky prism-coupling setup into this low-cost compact plasmonic platform would have a far-reaching impact on point-of-care diagnostic tools and also lead to advances in high-throughput sensing applications in proteomics, immunology, drug

Hybrid Integrated Label-Free Chemical and Biological Sensors

PubMed Central

Mehrabani, Simin; Maker, Ashley J.; Armani, Andrea M.

2014-01-01

Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits). This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach. PMID:24675757

CACA-TOCSY with alternate 13C–12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

PubMed Central

Takeuchi, Koh; Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian

2010-01-01

We present a 13C direct detection CACA-TOCSY experiment for samples with alternate 13C–12C labeling. It provides inter-residue correlations between 13Cα resonances of residue i and adjacent Cαs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to Cα nuclei separated by more than one residue. The experiment also provides Cα-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate 13C–12C labeling with [1,3-13C] glycerol or [2-13C] glycerol, which allows utilizing the small scalar 3JCC couplings that are masked by strong 1JCC couplings in uniformly 13C labeled samples. PMID:20383561

Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

PubMed

Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

2014-07-15

A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

2011-01-01

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID

Label-free visualization of ultrastructural features of artificial synapses via cryo-EM.

PubMed

Gopalakrishnan, Gopakumar; Yam, Patricia T; Madwar, Carolin; Bostina, Mihnea; Rouiller, Isabelle; Colman, David R; Lennox, R Bruce

2011-12-21

The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

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

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

DOE PAGES

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

2017-02-16

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

Chemical Methods for the Direct Detection and Labeling of S-Nitrosothiols

PubMed Central

Bechtold, Erika

2012-01-01

Abstract Significance: Posttranslational modification of proteins through phosphorylation, glycosylation, and oxidation adds complexity to the proteome by reversibly altering the structure and function of target proteins in a highly controlled fashion. Recent Advances: The study of reversible cysteine oxidation highlights a role for this oxidative modification in complex signal transduction pathways. Nitric oxide (NO), and its respective metabolites (including reactive nitrogen species), participates in a variety of these cellular redox processes, including the reversible oxidation of cysteine to S-nitrosothiols (RSNOs). RSNOs act as endogenous transporters of NO, but also possess beneficial effects independent of NO-related signaling, which suggests a complex and versatile biological role. In this review, we highlight the importance of RSNOs as a required posttranslational modification and summarize the current methods available for detecting S-nitrosation. Critical Issues: Given the limitations of these indirect detection methods, the review covers recent developments toward the direct detection of RSNOs by phosphine-based chemical probes. The intrinsic properties that dictate this phosphine/RSNO reactivity are summarized. In general, RSNOs (both small molecule and protein) react with phosphines to yield reactive S-substituted aza-ylides that undergo further reactions leading to stable RSNO-based adducts. Future Directions: This newly explored chemical reactivity forms the basis of a number of exciting potential chemical methods for protein RSNO detection in biological systems. Antioxid. Redox Signal. 17, 981–991. PMID:22356122

Assessment of Free Dye in Solutions of Dual-Labeled Antibody Conjugates for In Vivo Molecular Imaging

PubMed Central

Aldrich, Melissa B.; Wang, XueJuan; Hart, Amy; Sampath, Lakshmi; Marshall, Milton V.; Sevick-Muraca, Eva M.

2017-01-01

PURPOSE Recent preclinical and clinical studies show dyes that excite and fluoresce in the near infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic. PROCEDURES Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800CW in (DTPA)n-trastuzumab—(IRDye 800)m conjugate sample preparations in which HPLC assessment of free dye was not possible. RESULTS The SDS-PAGE assay was accurate and valid for free IRDye 800CW amounts between 38 and 4 molar percent of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined. CONCLUSION This method may be applicable to other near infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging. PMID:20458634

Label-free all-electronic biosensing in microfluidic systems

NASA Astrophysics Data System (ADS)

Stanton, Michael A.

Label-free, all-electronic detection techniques offer great promise for advancements in medical and biological analysis. Electrical sensing can be used to measure both interfacial and bulk impedance changes in conducting solutions. Electronic sensors produced using standard microfabrication processes are easily integrated into microfluidic systems. Combined with the sensitivity of radiofrequency electrical measurements, this approach offers significant advantages over competing biological sensing methods. Scalable fabrication methods also provide a means of bypassing the prohibitive costs and infrastructure associated with current technologies. We describe the design, development and use of a radiofrequency reflectometer integrated into a microfluidic system towards the specific detection of biologically relevant materials. We developed a detection protocol based on impedimetric changes caused by the binding of antibody/antigen pairs to the sensing region. Here we report the surface chemistry that forms the necessary capture mechanism. Gold-thiol binding was utilized to create an ordered alkane monolayer on the sensor surface. Exposed functional groups target the N-terminus, affixing a protein to the monolayer. The general applicability of this method lends itself to a wide variety of proteins. To demonstrate specificity, commercially available mouse anti- Streptococcus Pneumoniae monoclonal antibody was used to target the full-length recombinant pneumococcal surface protein A, type 2 strain D39 expressed by Streptococcus Pneumoniae. We demonstrate the RF response of the sensor to both the presence of the surface decoration and bound SPn cells in a 1x phosphate buffered saline solution. The combined microfluidic sensor represents a powerful platform for the analysis and detection of cells and biomolecules.

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

PubMed Central

Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

2016-01-01

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor.

PubMed

Adams, André A; Okagbare, Paul I; Feng, Juan; Hupert, Matuesz L; Patterson, Don; Göttert, Jost; McCarley, Robin L; Nikitopoulos, Dimitris; Murphy, Michael C; Soper, Steven A

2008-07-09

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor

PubMed Central

Adams, André A.; Okagbare, Paul I.; Feng, Juan; Hupert, Matuesz L.; Patterson, Don; Göttert, Jost; McCarley, Robin L.; Nikitopoulos, Dimitris; Murphy, Michael C.; Soper, Steven A.

2008-01-01

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 μm width × 150 μm depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation. PMID:18557614

Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

PubMed

Manciu, Felicia S; Ciubuc, John D; Parra, Karla; Manciu, Marian; Bennet, Kevin E; Valenzuela, Paloma; Sundin, Emma M; Durrer, William G; Reza, Luis; Francia, Giulio

2017-08-01

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for

Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

PubMed

Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

2017-09-15

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients.

PubMed

Maldonado, Jesús; González-Guerrero, Ana Belén; Domínguez, Carlos; Lechuga, Laura M

2016-11-15

Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free immunosensor based on Pd nanoplates for amperometric immunoassay of alpha-fetoprotein.

PubMed

Wang, Huan; Li, He; Zhang, Yihe; Wei, Qin; Ma, Hongmin; Wu, Dan; Li, Yan; Zhang, Yong; Du, Bin

2014-03-15

In this paper, Pd nanoplates were used as a kind of electrode materials for fabrication of an electrochemical immunosensor, which was applied for detection of cancer biomarker alpha-fetoprotein (AFP). Thanks to the unique structure and properties of Pd nanoplates, the antibody of AFP (Ab) was effectively immobilized onto the surface of the Pd nanoplates modified glassy carbon electrode (GCE). Moreover, the good electrochemical properties of Pd nanoplates greatly improved the electronic transmission rate and enhanced the electrochemical signal, which led to an increase of the detection sensitivity. Based on the specific antibody-antigen interaction, a label-free immunosensor based on Pd nanoplates was developed for sensing of AFP. The current method allows us to detect AFP over a wide concentration range from 0.01 to 75.0 ng/mL with a detection limit of 4 pg/mL. The proposed immunosensor has been used to determine AFP in human serum with satisfactory results. © 2013 Elsevier B.V. All rights reserved.

CACA-TOCSY with alternate 13C-12C labeling: a 13Calpha direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification.

PubMed

Takeuchi, Koh; Frueh, Dominique P; Sun, Zhen-Yu J; Hiller, Sebastian; Wagner, Gerhard

2010-05-01

We present a (13)C direct detection CACA-TOCSY experiment for samples with alternate (13)C-(12)C labeling. It provides inter-residue correlations between (13)C(alpha) resonances of residue i and adjacent C(alpha)s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C(alpha) nuclei separated by more than one residue. The experiment also provides C(alpha)-to-sidechain correlations, some amino acid type identifications and estimates for psi dihedral angles. The power of the experiment derives from the alternate (13)C-(12)C labeling with [1,3-(13)C] glycerol or [2-(13)C] glycerol, which allows utilizing the small scalar (3)J(CC) couplings that are masked by strong (1)J(CC) couplings in uniformly (13)C labeled samples.

Track-before-detect labeled multi-bernoulli particle filter with label switching

NASA Astrophysics Data System (ADS)

Garcia-Fernandez, Angel F.

2016-10-01

This paper presents a multitarget tracking particle filter (PF) for general track-before-detect measurement models. The PF is presented in the random finite set framework and uses a labelled multi-Bernoulli approximation. We also present a label switching improvement algorithm based on Markov chain Monte Carlo that is expected to increase filter performance if targets get in close proximity for a sufficiently long time. The PF is tested in two challenging numerical examples.

Extended Solution Gate OFET-based Biosensor for Label-free Glial Fibrillary Acidic Protein Detection with Polyethylene Glycol-Containing Bioreceptor Layer.

PubMed

Song, Jian; Dailey, Jennifer; Li, Hui; Jang, Hyun-June; Zhang, Pengfei; Wang, Jeff Tza-Huei; Everett, Allen D; Katz, Howard E

2017-05-25

A novel organic field effect transistor (OFET) -based biosensor is described for label-free glial fibrillary acidic protein (GFAP) detection. We report the first use of an extended solution gate structure where the sensing area and the organic semiconductor are separated, and a reference electrode is not needed. Different molecular weight polyethylene glycols (PEGs) are mixed into the bio-receptor layer to help extend the Debye screening length. The drain current change was significantly increased with the help of higher molecular weight PEGs, as they are known to reduce the dielectric constant. We also investigated the sensing performance under different gate voltage (V g ). The sensitivity increased after we decreased V g from -5 V to -2 V, because the lower V g is much closer to the OFET threshold voltage and the influence of attached negatively charged proteins become more apparent. Finally, the selectivity experiments toward different interferents were performed. The stability and selectivity are promising for clinical applications.

A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing.

PubMed

Ewald, Melanie; Fechner, Peter; Gauglitz, Günter

2015-05-01

For the first time, a multi-analyte biosensor platform has been developed using the label-free 1-lambda-reflectometry technique. This platform is the first, which does not use imaging techniques, but is able to perform multi-analyte measurements. It is designed to be portable and cost-effective and therefore allows for point-of-need testing or on-site field-testing with possible applications in diagnostics. This work highlights the application possibilities of this platform in the field of animal testing, but is also relevant and transferable to human diagnostics. The performance of the platform has been evaluated using relevant reference systems like biomarker (C-reactive protein) and serology (anti-Salmonella antibodies) as well as a panel of real samples (animal sera). The comparison of the working range and limit of detection shows no loss of performance transferring the separate assays to the multi-analyte setup. Moreover, the new multi-analyte platform allows for discrimination between sera of animals infected with different Salmonella subtypes.

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Presley, Andrew D; Fuller, Kathryn M; Arriaga, Edgar A

2003-08-05

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.

Label free biosensor incorporating a replica-molded, vertically emitting distributed feedback laser

NASA Astrophysics Data System (ADS)

Lu, M.; Choi, S. S.; Wagner, C. J.; Eden, J. G.; Cunningham, B. T.

2008-06-01

A label free biosensor based upon a vertically emitting distributed feedback (DFB) laser has been demonstrated. The DFB laser comprises a replica-molded, one-dimensional dielectric grating coated with laser dye-doped polymer as the gain medium. Adsorption of biomolecules onto the laser surface alters the DFB laser emission wavelength, thereby permitting the kinetic adsorption of a protein polymer monolayer or the specific binding of small molecules to be quantified. A bulk sensitivity of 16.6nm per refractive index unit and the detection of a monolayer of the protein polymer poly(Lys, Phe) have been observed with this biosensor. The sensor represents a departure from conventional passive resonant optical sensors from the standpoint that the device actively generates its own narrowband high intensity output without stringent requirements on the coupling alignments, resulting in a simple, robust illumination and detection configuration.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis

PubMed Central

Noothalapati, Hemanth; Sasaki, Takahiro; Kaino, Tomohiro; Kawamukai, Makoto; Ando, Masahiro; Hamaguchi, Hiro-o; Yamamoto, Tatsuyuki

2016-01-01

Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, β-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and β-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future. PMID:27278218

A novel label-free cell-based assay technology using biolayer interferometry.

PubMed

Verzijl, D; Riedl, T; Parren, P W H I; Gerritsen, A F

2017-01-15

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β 2 -adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Label-free vascular imaging in a spontaneous hamster cheek pouch carcinogen model for pre-cancer detection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hu, Fangyao; Morhard, Robert; Liu, Heather; Murphy, Helen; Farsiu, Sina; Ramanujam, Nimmi

2016-03-01

Inducing angiogenesis is one hallmark of cancer. Tumor induced neovasculature is often characterized as leaky, tortuous and chaotic, unlike a highly organized normal vasculature. Additionally, in the course of carcinogenesis, angiogenesis precedes a visible lesion. Tumor cannot grow beyond 1-2 mm in diameter without inducing angiogenesis. Therefore, capturing the event of angiogenesis may aid early detection of pre-cancer -important for better treatment prognoses in regions that lack the resources to manage invasive cancer. In this study, we imaged the neovascularization in vivo in a spontaneous hamster cheek pouch carcinogen model using a, non-invasive, label-free, high resolution, reflected-light spectral darkfield microscope. Hamsters' cheek pouches were painted with 7,12-Dimethylbenz[a]anthracene (DMBA) to induce pre-cancerous to cancerous changes, or mineral oil as control. High resolution spectral darkfield images were obtained over the course of pre-cancer development and in control cheek pouches. The vasculature was segmented with a multi-scale Gabor filter with an 85% accuracy compared with manually traced masks. Highly tortuous vasculature was observed only in the DMBA treated cheek pouches as early as 6 weeks of treatment. In addition, the highly tortuous vessels could be identified before a visible lesion occurred later during the treatment. The vessel patterns as determined by the tortuosity index were significantly different from that of the control cheek pouch. This preliminary study suggests that high-resolution darkfield microscopy is promising tool for pre-cancer and early cancer detection in low resource settings.

Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples

NASA Astrophysics Data System (ADS)

Velmanickam, Logeeshan; Laudenbach, Darrin; Nawarathna, Dharmakeerthi

2016-10-01

The current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The detection limit of ELISA is about 250 pg/ml. However, to quantify analytes that are related to various stages of tumors including early detection requires detecting well below the current limit of the ELISA test. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are <100 pg/ml and the prostate specific antigen level of the early stage of prostate cancer is about 1 ng/ml. Further, it has been reported that there are significantly less than 1 pg /mL of analytes in the early stage of tumors. Therefore, depending on the tumor type and the stage of the tumors, it is required to quantify various levels of analytes ranging from ng/ml to pg/ml. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes (label-free, high-throughput technique based on dielectrophoresis. This technique is capable of quantifying target analytes down to a few thousands of molecules (˜zmoles ).

Label-free surface plasmon resonance biosensing with titanium nitride thin film.

PubMed

Qiu, Guangyu; Ng, Siu Pang; Wu, Chi-Man Lawrence

2018-05-30

In this report, titanium nitride thin film synthesized with reactive magneto-sputtering technique is proposed as an alternative surface plasmon resonance sensing material. The physical and chemical natures were initially studied by atomic force microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. In virtue of white-light common-path sensing system, the wavelength modulated TiN films achieved tunable evanescent plasmonic field from 573 nm to 627 nm. The optimized TiN film with 29.8 nm thickness exhibited good differential phase sensitivity (i.e. 1.932 × 10 -7 RIU) to refractive index alteration, which is comparable to the performance of gold film. We have also attained direct measurement of biotin adsorption on the TiN and monitored sub-sequential biotin-streptavidin conjugation. It was found that TiN films have significantly higher binding affinity toward biotin than that of gold in experiments, so we are able to detect biotin directly to 0.22 µg/ml (0.90 µM) in label-free manner. The adsorption mechanism of biotin on TiN(200) are also explored with periodic density functional theory (DFT) via computer simulation and it was found that the exceptional biotin-TiN affinity may be due to the stacking formation of both N-Ti and O-Ti bonds. Also, the adsorption energy of biotin-TiN was found to be - 1.85 eV, which was two times higher than that of biotin-gold. Both experimental and computational results indicate, for the first time, that the TiN film can be directly functionalized with biotin molecules, thus it serves as an alternative plasmonic material to existing gold-based SPR biosensors. Copyright © 2018 Elsevier B.V. All rights reserved.

Label-Free Pyrophosphate Recognition with Functionalized Asymmetric Nanopores.

PubMed

Ali, Mubarak; Ahmed, Ishtiaq; Ramirez, Patricio; Nasir, Saima; Niemeyer, Christof M; Mafe, Salvador; Ensinger, Wolfgang

2016-04-01

The label-free detection of pyrophosphate (PPi) anions with a nanofluidic sensing device based on asymmetric nanopores is demonstrated. The pore surface is functionalized with zinc complexes based on two di(2-picolyl)amine [bis(DPA)] moieties using carbodiimide coupling chemistry. The complexation of zinc (Zn(2+) ) ion is achieved by exposing the modified pore to a solution of zinc chloride to form bis(Zn(2+) -DPA) complexes. The chemical functionalization is demonstrated by recording the changes in the observed current-voltage (I-V) curves before and after pore modification. The bis(Zn(2+) -DPA) complexes on the pore walls serve as recognition sites for pyrophosphate anion. The experimental results show that the proposed nanofluidic sensor has the ability to sense picomolar concentrations of PPi anion in the surrounding environment. On the contrary, it does not respond to other phosphate anions, including monohydrogen phosphate, dihydrogen phosphate, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate. The experimental results are described theoretically by using a model based on the Poisson-Nernst-Planck equations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

2009-01-01

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

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

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

A multi-center study benchmarks software tools for label-free proteome quantification

PubMed Central

Gillet, Ludovic C; Bernhardt, Oliver M.; MacLean, Brendan; Röst, Hannes L.; Tate, Stephen A.; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I.; Aebersold, Ruedi; Tenzer, Stefan

2016-01-01

The consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows setups. For consistent evaluation we developed LFQbench, an R-package to calculate metrics of precision and accuracy in label-free quantitative MS, and report the identification performance, robustness and specificity of each software tool. Our reference datasets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics. PMID:27701404

A label-free fluorescent biosensor for the detection of protein kinase activity based on gold nanoclusters/graphene oxide hybrid materials.

PubMed

Liu, Qing; Li, Ning; Wang, Mengke; Wang, Lei; Su, Xingguang

2018-07-12

Protein kinase (PKA) can regulate many cellular biological processes by phosphorylation substrate peptide or protein. A new fluorescent biosensing method for the detection of PKA activity was developed by using 11-mercaptoundecanoic acid-capped gold nanoclusters (MUA-Au NCs) and graphene oxide (GO) with low background noise. In this strategy, the special designed peptide could be anchored on the surface of MUA-Au NCs by the Au-S bond and also adsorbed on the surface of GO owing to the electrostatic interaction. As a result, the fluorescence of MUA-Au NCs was quenched leading to low background fluorescence due to the forster resonance energy transfer (FRET) between MUA-Au NCs and GO via peptide as a bridge. However, when the substrate peptide was phosphorylated by PKA, the FRET between GO and MUA-Au NCs was disrupted because of the weakened interaction between the phosphorylated peptide and the GO, resulting in recovery of the fluorescence intensity. The developed label-free fluorescence "turn-off-on" method can detect protein kinase activity in the range of 0.6-2.0 U mL -1 with a detection limit of 0.17 U mL -1 (3σ). The feasibility of this present method for kinase inhibitor screening was also studied by assessment of H-89 kinase inhibition with an IC 50 value of 0.049 μmol L -1 . Copyright © 2018. Published by Elsevier B.V.

Label-free, non-derivatization CRET detection platform for 6-mercaptopurine based on the distance-dependent optical properties of gold nanoparticles.

PubMed

Du, Jianxiu; Wang, Yadi; Zhang, Weimin

2012-07-02

A label-free, non-derivatization chemiluminescence resonance energy transfer (CRET) detection platform has been developed for the detection of the non-fluorescent small molecule 6-mercaptopurine. This CRET process arose from a chemiluminescent (CL) donor-acceptor system in which the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2)-fluorescein (maximum emission at 521.6 nm) served as the donor and gold nanoparticles (AuNPs, maximum absorption at 520.0 nm) served as the acceptor. This process caused a significant decrease in the CL signal of the TCPO-H(2)O(2)-fluorescein reaction. The presence of 6-mercaptopurine induced an aggregation of AuNPs with the assistance of Cu(2+) ions through cooperative metal-ligand interactions that was accompanied by a distinct change in color and optical properties. The maximum absorption band of the AuNPs was red-shifted to 721.0 nm and no longer overlapped with the CL spectrum of the reaction; as a result, the CL signal was restored. This CRET system exhibited a wide linear range, from 9.0 nmol L(-1) to 18.0 μmol L(-1), and a low detection limit (0.62 nmol L(-1)) for 6-mercaptopurine. The applicability of the proposed CRET system was evaluated by analysis of 6-mercaptopurine in spiked human plasma samples. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improving label-free detection of circulating melanoma cells by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Huan; Wang, Qiyan; Pang, Kai; Zhou, Quanyu; Yang, Ping; He, Hao; Wei, Xunbin

2018-02-01

Melanoma is a kind of a malignant tumor of melanocytes with the properties of high mortality and high metastasis rate. The circulating melanoma cells with the high content of melanin can be detected by light absorption to diagnose and treat cancer at an early stage. Compared with conventional detection methods such as in vivo flow cytometry (IVFC) based on fluorescence, the in vivo photoacoustic flow cytometry (PAFC) utilizes melanin cells as biomarkers to collect the photoacoustic (PA) signals without toxic fluorescent dyes labeling in a non-invasive way. The information of target tumor cells is helpful for data analysis and cell counting. However, the raw signals in PAFC system contain numerous noises such as environmental noise, device noise and in vivo motion noise. Conventional denoising algorithms such as wavelet denoising (WD) method and means filter (MF) method are based on the local information to extract the data of clinical interest, which remove the subtle feature and leave many noises. To address the above questions, the nonlocal means (NLM) method based on nonlocal data has been proposed to suppress the noise in PA signals. Extensive experiments on in vivo PA signals from the mice with the injection of B16F10 cells in caudal vein have been conducted. All the results indicate that the NLM method has superior noise reduction performance and subtle information reservation.

Direct optical detection of protein-ligand interactions.

PubMed

Gesellchen, Frank; Zimmermann, Bastian; Herberg, Friedrich W

2005-01-01

Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.

Label-free sensitive luminescence biosensor for immunoglobulin G based on Ag6Au6 ethisterone cluster-estrogen receptor α aggregation and graphene.

PubMed

Chen, Nannan; Guo, Wenjing; Lin, Zhixiang; Wei, Qiaohua; Chen, Guonan

2018-08-01

A specific and label-free "on-off-on" luminescence biosensor based on a novel heterometallic cluster [Ag 6 Au 6 (ethisterone) 12 ]-estrogen receptor α (Ag 6 Au 6 Eth-ERα) aggregation utilizing graphene oxide (GO) as a quencher to lead a small background signal was firstly constructed to detect immunoglobulin G (IgG) with a simple process and high selectivity. The efficient photoluminescent (PL) Ag 6 Au 6 Eth-ERα aggregation is strongly quenched by GO. In the presence of IgG, the PL of this system will be restored, and perceivable by human eyes under UV lamp excitation (365 nm). The quenching mechanism of GO on Ag 6 Au 6 Eth-ERα and enhancement mechanism of IgG on Ag 6 Au 6 Eth-ERα-GO were investigated in detail. Under the optimum conditions, the biosensor for high sensitive IgG detection expressed a wider linear range of 0.0078-10 ng/mL and a lower detection limit of 0.65 pg/mL with good stability and repeatability, which provided a new approach for label-free IgG detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid quantification of free cholesterol in tears using direct insertion/electron ionization-mass spectrometry.

PubMed

Wei, Xiaojia Eric; Korth, John; Brown, Simon H J; Mitchell, Todd W; Truscott, Roger J W; Blanksby, Stephen J; Willcox, Mark D P; Zhao, Zhenjun

2013-12-09

To establish a simple and rapid analytical method, based on direct insertion/electron ionization-mass spectrometry (DI/EI-MS), for measuring free cholesterol in tears from humans and rabbits. A stable-isotope dilution protocol employing DI/EI-MS in selected ion monitoring mode was developed and validated. It was used to quantify the free cholesterol content in human and rabbit tear extracts. Tears were collected from adult humans (n = 15) and rabbits (n = 10) and lipids extracted. Screening, full-scan (m/z 40-600) DI/EI-MS analysis of crude tear extracts showed that diagnostic ions located in the mass range m/z 350 to 400 were those derived from free cholesterol, with no contribution from cholesterol esters. DI/EI-MS data acquired using selected ion monitoring (SIM) were analyzed for the abundance ratios of diagnostic ions with their stable isotope-labeled analogues arising from the D6-cholesterol internal standard. Standard curves of good linearity were produced and an on-probe limit of detection of 3 ng (at 3:1 signal to noise) and limit of quantification of 8 ng (at 10:1 signal to noise). The concentration of free cholesterol in human tears was 15 ± 6 μg/g, which was higher than in rabbit tears (10 ± 5 μg/g). A stable-isotope dilution DI/EI-SIM method for free cholesterol quantification without prior chromatographic separation was established. Using this method demonstrated that humans have higher free cholesterol levels in their tears than rabbits. This is in agreement with previous reports. This paper provides a rapid and reliable method to measure free cholesterol in small-volume clinical samples.

Direct single-molecule dynamic detection of chemical reactions.

PubMed

Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N; Zhang, Deqing; Guo, Xuefeng

2018-02-01

Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry.

Direct single-molecule dynamic detection of chemical reactions

PubMed Central

Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N.; Zhang, Deqing; Guo, Xuefeng

2018-01-01

Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry. PMID:29487914

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

PubMed

Wang, Ye; Gan, Ning; Zhou, You; Li, Tianhua; Hu, Futao; Cao, Yuting; Chen, Yinji

2017-11-15

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL -1 to 10ngmL -1 , with the detection limits of 0.7pgmL -1 and 0.9pgmL -1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Specific labeling of zinc finger proteins using noncanonical amino acids and copper-free click chemistry.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A; Schroeder, Charles M

2012-09-19

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays, and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry.

A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe.

PubMed

Gao, Zhong Feng; Chen, Dong Mei; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

2015-10-15

Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparing model-based and model-free analysis methods for QUASAR arterial spin labeling perfusion quantification.

PubMed

Chappell, Michael A; Woolrich, Mark W; Petersen, Esben T; Golay, Xavier; Payne, Stephen J

2013-05-01

Amongst the various implementations of arterial spin labeling MRI methods for quantifying cerebral perfusion, the QUASAR method is unique. By using a combination of labeling with and without flow suppression gradients, the QUASAR method offers the separation of macrovascular and tissue signals. This permits local arterial input functions to be defined and "model-free" analysis, using numerical deconvolution, to be used. However, it remains unclear whether arterial spin labeling data are best treated using model-free or model-based analysis. This work provides a critical comparison of these two approaches for QUASAR arterial spin labeling in the healthy brain. An existing two-component (arterial and tissue) model was extended to the mixed flow suppression scheme of QUASAR to provide an optimal model-based analysis. The model-based analysis was extended to incorporate dispersion of the labeled bolus, generally regarded as the major source of discrepancy between the two analysis approaches. Model-free and model-based analyses were compared for perfusion quantification including absolute measurements, uncertainty estimation, and spatial variation in cerebral blood flow estimates. Major sources of discrepancies between model-free and model-based analysis were attributed to the effects of dispersion and the degree to which the two methods can separate macrovascular and tissue signal. Copyright © 2012 Wiley Periodicals, Inc.

Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia

DTIC Science & Technology

2015-10-01

eyes and image choroidal vessels/capillaries using CARS intravital microscopy Subtask 3: Measure oxy-hemoglobin levels in PBI test and control eyes...AWARD NUMBER: W81XWH-14-1-0537 TITLE: Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia...4. TITLE AND SUBTITLE Mobile, Multimodal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia 5a. CONTRACT NUMBER W81XWH

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

PubMed

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

PubMed Central

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-01-01

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring. PMID:28335318

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

PubMed

Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

2017-05-15

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10 5 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy

PubMed Central

Small, David M.; Jones, Jason S.; Tendler, Irwin I.; Miller, Paul E.; Ghetti, Andre; Nishimura, Nozomi

2017-01-01

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool. PMID:29359098

Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels

DOEpatents

Mathies, Richard A.; Singhal, Pankaj; Xie, Jin; Glazer, Alexander N.

2002-01-01

This invention relates to a microfabricated capillary electrophoresis chip for detecting multiple redox-active labels simultaneously using a matrix coding scheme and to a method of selectively labeling analytes for simultaneous electrochemical detection of multiple label-analyte conjugates after electrophoretic or chromatographic separation.

Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

PubMed

Ma, Jin-Liang; Yin, Bin-Cheng; Le, Huynh-Nhu; Ye, Bang-Ce

2015-06-17

We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

Nanoribbon field-effect transistors as direct and label-free sensors of enzyme-substrate interactions

NASA Astrophysics Data System (ADS)

Mu, Luye; Droujinine, Ilia; Rajan, Nitin; Sawtelle, Sonya; Reed, Mark

2015-03-01

The ability to measure enzyme-substrate interactions is essential in areas such as diagnostics, treatment, and biochemical screens. Many enzymatic reactions alter the pH of its environment, suggesting of a simple and direct method for detection. We show the ability of Al2O3-coated Si nanoribbon field-effect transistor biosensors to sensitively measure various aspects of enzyme-substrate interactions through measuring the pH. Urea in phosphate buffered saline (PBS) and penicillinase in PBS and urine were measured to limits of <200 μM and 0.02 units/mL, respectively. We also show the ability to extract accurate kinetics from the interaction of acetylcholine and its esterase. Prior work on FET sensors has been limited by the use of surface functionalization, which not only alters enzyme-substrate affinity, but also makes enzyme activity quantification difficult. Our method involves direct detection of reactions in solution without requiring alteration to the reactants, allowing us to obtain repeatable results and sensitive limits of detection. This method is a simple, inexpensive, and effective platform for detection of enzymatic reactions, and can be readily generalized to many unrelated classes of reactants. This work was supported in part by U.S. Army Research Office and Air Force Research Laboratory.

PSEA-Quant: a protein set enrichment analysis on label-free and label-based protein quantification data.

PubMed

Lavallée-Adam, Mathieu; Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

2014-12-05

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights.

PSEA-Quant: A Protein Set Enrichment Analysis on Label-Free and Label-Based Protein Quantification Data

PubMed Central

2015-01-01

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766