Poly(o-phenylenediamine) colloid-quenched fluorescent oligonucleotide as a probe for fluorescence-enhanced nucleic acid detection.

PubMed

Tian, Jingqi; Li, Hailong; Luo, Yonglan; Wang, Lei; Zhang, Yingwei; Sun, Xuping

2011-02-01

In this Letter, we demonstrate that chemical oxidation polymerization of o-phenylenediamine (OPD) by potassium bichromate at room temperature results in the formation of submicrometer-scale poly(o-phenylenediamine) (POPD) colloids. Such colloids can absorb and quench dye-labeled single-stranded DNA (ssDNA) very effectively. In the presence of a target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. With the use of an oligonucleotide (OND) sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch.

Nine New Fluorescent Probes

NASA Astrophysics Data System (ADS)

Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

1989-06-01

Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

PubMed Central

Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; Sørensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

2001-01-01

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

Poly-adenine-mediated fluorescent spherical nucleic acid probes for live-cell imaging of endogenous tumor-related mRNA.

PubMed

Zhu, Dan; Zhao, Dongxia; Huang, Jiaxuan; Zhu, Yu; Chao, Jie; Su, Shao; Li, Jiang; Wang, Lihua; Shi, Jiye; Zuo, Xiaolei; Weng, Lixing; Li, Qian; Wang, Lianhui

2018-05-16

Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent "reporter" strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the "reporter" strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging. Copyright © 2018. Published by Elsevier Inc.

A colorimetric and fluorescent probe for detecting intracellular biothiols.

PubMed

Chen, Chunyang; Liu, Wei; Xu, Cong; Liu, Weisheng

2016-11-15

A new rapid and highly sensitive coumarin-based probe (probe 1) has been designed and synthesized for detecting intracellular thiols. Probe 1 was prepared by a 4-step procedure as a latent fluorescence probe to achieve high sensitivity and fluorescence turn-on response toward cysteine and homocysteine over GSH and other various natural amino acids under physiological conditions. Owing to specific cyclization between thiols and aldehyde group, probe 1 displayed a highly selectivity toward cysteine and homocysteine. Above all, probe 1 was successfully used for fluorescence imaging of biothiols in Hela cells, and quantitative determination had been achieved within a certain range. Then specific fluorescence imaging of mice organ tissues was obtained for proving the permeability of probe 1. Simultaneously, the viability was measured to be more than 80%, which shows probe 1 can be a rapid and biocompatible probe for biothiols in cells. Furthermore, the measurement of thiols detection in 5 kinds of animal serum showed that probe 1 can be used in determination of biothiols in blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

PubMed

Shahmuradyan, Anna; Krull, Ulrich J

2016-03-15

Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

PubMed

Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

2016-04-13

Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

PubMed

Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

2017-05-15

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and evaluation of a photoresponsive quencher for fluorescent hybridization probes.

PubMed

Kovaliov, Marina; Wachtel, Chaim; Yavin, Eylon; Fischer, Bilha

2014-10-21

Nowadays, most nucleic acid detections using fluorescent probes rely on quenching of fluorescence by energy transfer from one fluorophore to another or to a non-fluorescent molecule (quencher). The most widely used quencher in fluorescent probes is 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL). We targeted a nucleoside-DABCYL analogue which could be incorporated anywhere in an oligonucleotide sequence and in any number, and used as a quencher in different hybridization sensitive probes. Specifically, we introduced a 5-(4-((dimethylamino)phenyl)azo)benzene)-2'-deoxy-uridine (dU(DAB)) quencher. The photoisomerization and dU(DAB)'s ability to quench fluorescein emission have been investigated. We incorporated dU(DAB) into a series of oligonucleotide (ON) probes including strand displacement probes, labeled with both fluorescein (FAM) and dU(DAB), and TaqMan probes bearing one or two dU(DAB) and a FAM fluorophore. We used these probes for the detection of a DNA target in real-time PCR (RT-PCR). All probes showed amplification of targeted DNA. A dU(DAB) modified TaqMan RT-PCR probe was more efficient as compared to a DABCYL bearing probe (93% vs. 87%, respectively). Furthermore, dU(DAB) had a stabilizing effect on the duplex, causing an increase in Tm up to 11 °C. In addition we showed the photoisomerisation of the azobenzene moiety of dU(DAB) and the dU(DAB) triply-labeled oligonucleotide upon irradiation. These findings suggest that dU(DAB) modified probes are promising probes for gene quantification in real-time PCR detection and as photoswitchable devices.

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

Design and synthesis of a novel lanthanide fluorescent probe (TbIII-dtpa-bis(2,6-diaminopurine)) and its application to the detection of uric acid in urine sample.

PubMed

Yang, Fan; Yu, Zhiyue; Li, Xinyi; Ren, Peipei; Liu, Guanhong; Song, Youtao; Wang, Jun

2018-06-03

In this study, a novel fluorescent probe, Tb III -dtpa-bis(2,6-diaminopurine) (Tb-dtpa-bdap), is designed based on the principle of complementary base pairing and synthesized for uric acid detection. The synthesized fluorescent probe is characterized by 1 H NMR, 13 C NMR, infra-red (IR) spectrum and ultraviolet-visible (UV-vis) spectra. It is found that the fluorescence of Tb-dtpa-bdap solution can be quenched obviously in the presence of uric acid. The affecting factors, including solution acidity, uric acid concentration and interfering substances, on the detection of uric acid using this probe are examined. Under optimized conditions, the fluorescence intensities of Tb-dtpa-bdap solution towards different uric acid concentrations show a linear response in the range from 1.00 × 10 -5  mol·L -1 to 5.00 × 10 -5  mol·L -1 with a linear correlation coefficient (R 2 ) of 0.9877. And the obtained limit of detection (LOD) is about 5.80 × 10 -6  mol·L -1 , which is lower than the level of uric acid in actual urine. The mechanism on the detection of uric acid by using Tb-dtpa-bdap is inferred from the experimental results. The facts demonstrate that the proposed fluorescent probe can be successfully applied for the determination of uric acid in human urine samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

PubMed

Pierzyńska-Mach, Agnieszka; Janowski, Paweł A; Dobrucki, Jurek W

2014-08-01

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. © 2014 International Society for Advancement of Cytometry.

An off-on fluorescence probe targeting mitochondria based on oxidation-reduction response for tumor cell and tissue imaging

NASA Astrophysics Data System (ADS)

Yao, Hanchun; Cao, Li; Zhao, Weiwei; Zhang, Suge; Zeng, Man; Du, Bin

2017-10-01

In this study, a tumor-targeting poly( d, l-lactic-co-glycolic acid) (PLGA) loaded "off-on" fluorescent probe nanoparticle (PFN) delivery system was developed to evaluate the region of tumor by off-on fluorescence. The biodegradability of the nanosize PFN delivery system readily released the probe under tumor acidic conditions. The probe with good biocompatibility was used to monitor the intracellular glutathione (GSH) of cancer cells and selectively localize to mitochondria for tumor imaging. The incorporated tumor-targeting probe was based on the molecular photoinduced electron transfer (PET) mechanism preventing fluorescence ("off" state) and could be easily released under tumor acidic conditions. However, the released tumor-targeting fluorescence probe molecule was selective towards GSH with high selectivity and an ultra-sensitivity for the mitochondria of cancer cells and tissues significantly increasing the probe molecule fluorescence signal ("on" state). The tumor-targeting fluorescence probe showed sensitivity to GSH avoiding interference from cysteine and homocysteine. The PFNs could enable fluorescence-guided cancer imaging during cancer therapy. This work may expand the biological applications of PFNs as a diagnostic reagent, which will be beneficial for fundamental research in tumor imaging. [Figure not available: see fulltext.

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

PubMed Central

Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

2000-01-01

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

A new fluorescent pH probe for imaging lysosomes in living cells.

PubMed

Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

2014-01-15

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations.

PubMed

Mochida, Ai; Ogata, Fusa; Nagaya, Tadanobu; Choyke, Peter L; Kobayashi, Hisataka

2018-02-15

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy. Published by Elsevier Ltd.

Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

NASA Technical Reports Server (NTRS)

Weinstein, S.; Pane, D.; Warren-Rhodes, K.; Cockell, C.; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.

2005-01-01

We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins.

PubMed

Murata, Atsushi; Arai, Satoshi; Yoon, Su-In; Takabayashi, Masao; Ozaki, Miwako; Takeoka, Shinji

2010-12-01

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

Fluorescent probes and bioimaging: alkali metals, alkaline earth metals and pH.

PubMed

Yin, Jun; Hu, Ying; Yoon, Juyoung

2015-07-21

All living species and life forms have an absolute requirement for bio-functional metals and acid-base equilibrium chemistry owing to the critical roles they play in biological processes. Hence, a great need exists for efficient methods to detect and monitor biometals and acids. In the last few years, great attention has been paid to the development of organic molecule based fluorescent chemosensors. The availability of new synthetic fluorescent probes has made fluorescence microscopy an indispensable tool for tracing biologically important molecules and in the area of clinical diagnostics. This review highlights the recent advances that have been made in the design and bioimaging applications of fluorescent probes for alkali metals and alkaline earth metal cations, including lithium, sodium and potassium, magnesium and calcium, and for pH determination within biological systems.

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

PubMed

Shibata, Aya

2017-01-01

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

Recent Progress in Fluorescent Imaging Probes

PubMed Central

Pak, Yen Leng; Swamy, K. M. K.; Yoon, Juyoung

2015-01-01

Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn2+, Hg2+, Cu2+ and Au3+, and anions including cyanide and adenosine triphosphate (ATP). PMID:26402684

Recent Progress in Fluorescent Imaging Probes.

PubMed

Pak, Yen Leng; Swamy, K M K; Yoon, Juyoung

2015-09-22

Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn(2+), Hg(2+), Cu(2+) and Au(3+), and anions including cyanide and adenosine triphosphate (ATP).

An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells.

PubMed

Sun, Mingtai; Du, Libo; Yu, Huan; Zhang, Kui; Liu, Yang; Wang, Suhua

2017-01-01

It is crucial to monitor intracellular pH values and their fluctuation since the organelles of cells have different pH distribution. Herein we construct a new small molecule fluorescent probe HBT-O for monitoring the subtle pH values within the scope of neutral to acid in living cells. The probe exhibited good water solubility, a marked turquoise to olivine emission color change in response to pH, and tremendous fluorescence hypochromatic shift of ∼50nm (1718cm -1 ) as well as the increased fluorescence intensity when the pH value changed from neutral to acid. Thus, the probe HBT-O can distinguish the subtle changes in the range of normal pH values from neutral to acid with significant fluorescence changes. These properties can be attributed to the intramolecular charge transfer (ICT) process of the probe upon protonation in buffer solutions at varied pH values. Moreover, the probe was reversible and nearly non-toxic for living cells. Then the probe was successfully used to detect pH fluctuation in living cells by exhibiting different fluorescence colors and intensity. These findings demonstrate that the probe will find useful applications in biology and biomedical research. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and characterization of the fluorescent probes for the labeling of Microthrix parvicella.

PubMed

Li, Songya; Fei, Xuening; Jiao, Xiumei; Lin, Dayong; Zhang, Baolian; Cao, Lingyun

2016-03-01

Although the fluorescent in situ hybridization (FISH) has been widely used to identify the Microthrix parvicella (M. parvicella), there are a few disadvantages and difficulties, such as complicated process, time consuming, etc. In this work, a series of fluorescent probes, which were modified by long-chain alkane with hydrophobic property and based on the property of M. parvicella utilizing long-chain fatty acids (LCFA), for the labeling of M. parvicella in bulking sludge were designed, synthesized, and characterized. The probes were characterized by ultraviolet-visible (UV-Vis) absorption spectra, fluorescence spectra, (1)H NMR spectra, and mass spectra, and the photostability and hydrophobic property of probes were investigated. All the results showed that the probes were quite stable and suitable for the fluorescent labeling. The probes had a large stoke shift of 98-137 nm, which was benefit for the fluorescent labeling. In the fluorescent labeling of M. parvicella by the synthesized probes, the probes had excellent labeling effects. By comparison of the images and the Image Pro Plus 6.0 analysis, the optimal concentration of the probes in the activated sludge sample for labeling was 0.010 mmol/L and the probe 3d had the best labeling. In addition, the effect of the duration time of probes was also investigated, and the results showed that the fluorescent intensity of probes hardly changed in a long period of time and it was suitable for labeling.

A rapid microwave synthesis of nitrogen-sulfur co-doped carbon nanodots as highly sensitive and selective fluorescence probes for ascorbic acid.

PubMed

Duan, Junxia; Yu, Jie; Feng, Suling; Su, Li

2016-06-01

A ultrafast one-step microwave-assisted method was developed for the synthesis of nitrogen-sulfur co-doped carbon nanodots (N,S-CDs) by using ethylenediamine as the carbon source and sulfamic acid as the surface passivation reagent. The morphology and the properties of N,S-CDs were explored by a series of techniques, such as high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV-vis absorption and fluorescence spectroscopy. The prepared N,S-CDs exhibit bright blue photoluminescence with a high fluorescence quantum yield (FLQY) up to 28%, and high stability and excellent water solubility. A N,S-CDs-based fluorescent probe was developed for sensitive detection ascorbic acid (AA) in the presence of Cu(2+), based on the mechanism that AA reduces Cu(2+) to Cu(+), then Cu(+) quenches the fluorescence of N,S-CDs through electron or energy transfer due to the interaction between Cu(+) and thiol ligand on the N,S-CDs surface. The observed linear response concentration range was from 0.057 to 4.0μM to AA with a detection limit as low as 18nM. The probe exhibited a highly selective response toward AA even in the presence of possible interfering substances, such as uric acid and citric acid. Moreover, these promising features made the sensing system used for the analysis of human serum and urine samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Design strategy for photoinduced electron transfer-based small-molecule fluorescent probes of biomacromolecules.

PubMed

Zhang, Wei; Ma, Zhao; Du, Lupei; Li, Minyong

2014-06-07

As the cardinal support of innumerable biological processes, biomacromolecules such as proteins, nucleic acids and polysaccharides are of importance to living systems. The key to understanding biological processes is to realize the role of these biomacromolecules in thte localization, distribution, conformation and interaction with other molecules. With the current development and adaptation of fluorescent technologies in biomedical and pharmaceutical fields, the fluorescence imaging (FLI) approach of using small-molecule fluorescent probes is becoming an up-to-the-minute method for the detection and monitoring of these imperative biomolecules in life sciences. However, conventional small-molecule fluorescent probes may provide undesirable results because of their intrinsic deficiencies such as low signal-to-noise ratio (SNR) and false-positive errors. Recently, small-molecule fluorescent probes with a photoinduced electron transfer (PET) "on/off" switch for biomacromolecules have been thoroughly considered. When recognized by the biomacromolecules, these probes turn on/off the PET switch and change the fluorescence intensity to present a high SNR result. It should be emphasized that these PET-based fluorescent probes could be advantageous for understanding the pathogenesis of various diseases caused by abnormal expression of biomacromolecules. The discussion of this successful strategy involved in this review will be a valuable guide for the further development of new PET-based small-molecule fluorescent probes for biomacromolecules.

Design and preparation of quantum dots fluorescent probes for in situ identification of Microthrix parvicella in bulking sludge.

PubMed

Fei, Xuening; Sun, Wenke; Cao, Lingyun; Jiao, Xiumei; Lin, Dayong; Jia, Guozhi

2016-01-01

A series of quantum dots (QDs) fluorescent probes for the in situ identification of Microthrix parvicella (M. parvicella) in bulking sludge were designed and prepared. In the preparation of CdTe/CdS QDs, the 11-mercaptoundecanoic acid (11-acid) and 16-mercaptohexadecanoic acid (16-acid) were used as the stabilizer. The prepared QDs probes were characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and transmission electron microscopy (TEM), and the results showed that the CdTe/CdS QDs formed a core-shell structure and the long carbon chain was successfully grafted onto its surface. And the three QDs probes had different crystallinity and particle size, which was due to the inhibition effect of long carbon chain. The optical properties test results showed that although the formed core-shell structure and long carbon chain affected the fluorescent intensity, adsorption, and emission spectra of the QDs probes, the probes B and C had a large stokes-shift of 82 and 101 nm, which was a benefit for their fluorescent labeling property. In the fluorescent identification of M. parvicella, the probes B and C effectively adsorbed onto the surface of M. parvicella through a hydrophobic bond, and then identified M. parvicella by their unique fluorescence. In addition, it was found that a better hydrophobic property resulted in better identification efficiency.

Unusual Fluorescent Responses of Morpholine-functionalized Fluorescent Probes to pH via Manipulation of BODIPY’s HOMO and LUMO Energy Orbitals for Intracellular pH Detection

PubMed Central

Zhang, Jingtuo; Yang, Mu; Mazi, Wafa; Adhikari, Kapil; Fang, Mingxi; Xie, Fei; Valenzano, Loredana; Tiwari, Ashutosh; Luo, Fen-Tair; Liu, Haiying

2016-01-01

Three uncommon morpholine-based fluorescent probes (A, B and C) for pH were prepared by introducing morpholine residues to BODIPY dyes at 4,4’- and 2,6-positions, respectively. In contrast to morpholine-based fluorescent probes for pH reported in literature, these fluorescent probes display high fluorescence in a basic condition while they exhibit very weak fluorescence in an acidic condition. The theoretical calculation confirmed that morpholine is unable to function as either an electron donor or an electron acceptor to quench the BODIPY fluorescence in the neutral and basic condition via photo-induced electron transfer (PET) mechanism because the LUMO energy of morpholine is higher than those of the BODIPY dyes while its HOMO energy is lower than those of the BODIPY dyes. However, the protonation of tertiary amines of the morpholine residues in an acidic environment leads to fluorescence quenching of the BODIPY dyes via d-PET mechanism. The fluorescence quenching is because the protonation effectively decreases the LUMO energy which locates between the HOMO and LUMO energies of the BODIPY dyes. Fluorescent probe C with deep-red emission has been successfully used to detect pH changes in mammalian cells. PMID:27547822

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

PubMed

Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

2012-01-01

We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side-chains and Rational Design of Fluorescence Probes of α-Helix Formation

PubMed Central

Taskent-Sezgin, Humeyra; Marek, Peter; Thomas, Rosanne; Goldberg, Daniel; Chung, Juah; Carrico, Isaac; Raleigh, Daniel P.

2011-01-01

p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure which can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions and amyloid formation, however the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study on the effects of different side-chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 M−1 and 22.1 M−1, respectively. The quenching of p-cyanophenylalanine fluorescence by specific side-chains is exploited to develop specific, high sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i+4. The p-cyanophenylalanine-His pair is most useful when the His side-chain is deprotonated and is, thus, complimentary to Trp-His pair which is most sensitive when the His side-chain is protonated. PMID:20565125

[1,10]Phenanthroline based cyanine dyes as fluorescent probes for ribonucleic acids in live cells

NASA Astrophysics Data System (ADS)

Kovalska, Vladyslava; Kuperman, Marina; Varzatskii, Oleg; Kryvorotenko, Dmytro; Kinski, Elisa; Schikora, Margot; Janko, Christina; Alexiou, Christoph; Yarmoluk, Sergiy; Mokhir, Andriy

2017-12-01

A series of monomethine, trimethine- and styrylcyanine dyes based on a [1,10]phenanthroline moiety was synthesized, characterized and investigated as potential fluorescent probes for nucleic acids in cell free settings and in cells. The dyes were found to be weakly fluorescent in the unbound state, whereas upon the binding to dsDNA or RNA their emission intensity raised up to 50 times (for monomethine benzothiazole derivative FT1 complexed with RNA). The strongest fluorescence intensity in assemblies with dsDNA and RNA was observed for the trimethine benzothiazole derivative FT4. The quantum yield of FT4 fluorescence in its complex with dsDNA was found to be 1.5% and the binding constant (K b) was estimated to be 7.9 × 104 M-1 that is a typical value for intercalating molecules. The FT4 dye was found to be cell membrane permeable. It stains RNA rich components—the nucleoli and most probably the cytoplasmic RNA. FT4 bound to RNAs delivers a very strong fluorescence signal, which makes this easily accessible dye a potentially useful alternative to known RNA stains, e.g. expensive SYTO® 83. The advantage of FT4 is its easy synthetic access including no chromatographic purification steps, which will be reflected in its substantially lower price.

Discovery of a novel calcium-sensitive fluorescent probe for α-ketoglutarate.

PubMed

Gan, Lin-Lin; Chen, Lin-Hai; Nan, Fa-Jun

2017-12-01

α-Ketoglutarate (α-KG), a pivotal metabolite in energy metabolism, has been implicated in nonalcoholic fatty liver disease (NAFLD) and several cancers. It is recently proposed that plasma α-KG is a surrogate biomarker of NAFLD. Here, we report the development of a novel "turn-on" chemosensor for α-KG that contains a coumarin moiety as a fluorophore. Using benzothiazole-coumarin (BTC) as inspiration, we designed a probe for calcium ion recognition that possesses a unique fluorophore compared with previously reported probes for α-KG measurement. This chemosensor is based on the specific Schiff base reaction and the calcium ion recognition property of the widely used calcium indicator BTC. The probe was synthesized, and a series of parallel experiments were conducted to optimize the chemical recognition process. Compared to the initial weak fluorescence, a remarkable 7.6-fold enhancement in fluorescence intensity (I/I 0 at 495 nm) was observed for the conditions in which the probe (1 μmol/L), α-KG (50 μmol/L), and Ca 2+ (100 μmol/L) were incubated at 30 °C in EtOH. The probe displayed good selectivity for α-KG even in an environment with an abundance of amino acids and other interfering species such as glutaric acid. We determined that the quantitative detection range of α-KG in EtOH was between 5 and 50 μmol/L. Finally, probe in serum loaded with α-KG (10 mmol/L) showed a 7.4-fold fluorescence enhancement. In summary, a novel probe for detecting the biomarker α-KG through a typical Schiff base reaction has been discovered. With further optimization, this probe may be a good alternative for detecting the physiological metabolite α-KG.

Fluorescent-protein-based probes: general principles and practices.

PubMed

Ai, Hui-Wang

2015-01-01

An important application of fluorescent proteins is to derive genetically encoded fluorescent probes that can actively respond to cellular dynamics such as pH change, redox signaling, calcium oscillation, enzyme activities, and membrane potential. Despite the large diverse group of fluorescent-protein-based probes, a few basic principles have been established and are shared by most of these probes. In this article, the focus is on these general principles and strategies that guide the development of fluorescent-protein-based probes. A few examples are provided in each category to illustrate the corresponding principles. Since these principles are quite straightforward, others may adapt them to create fluorescent probes for their own interest. Hopefully, the development of the ever-growing family of fluorescent-protein-based probes will no longer be limited to a small number of laboratories specialized in senor development, leading to the situation that biological studies will be bettered assisted by genetically encoded sensors.

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

PubMed Central

Chen, Wei-Tsung; Mahmood, Umar; Weissleder, Ralph; Tung, Ching-Hsuan

2005-01-01

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases. PMID:15743478

Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

PubMed

Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

2017-10-02

Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity.

PubMed

Tang, Yunqiang; Jin, Longyi; Yin, Bingzhu

2017-11-15

A novel fluorescent probe 1 based on acridine orange was developed for the selective detection and bioimaging of biothiols. The probe exhibits higher selectivity and turn-on fluorescence response to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) than to other amino acids. Importantly, the probe responds to GSH and Cys/Hcy with distinct fluorescence emissions in PBS buffer at pH of 7.4. The Cys/Hcy-triggered tandem S N Ar-rearrangement reaction and GSH-induced S N Ar reaction with the probe led to the corresponding amino-acridinium and thio-acridinium dyes, respectively, which can discriminate GSH from Cys/Hcy through different emission channels. Interestingly, Cys finishes the tandem reaction with the probe and subsequently forms amino-acridinium and Hcy/GSH induces S N Ar reaction with the probe to form thio-acridiniums at weakly acidic conditions (pH 6.0), enabling Cys to be discriminated from Hcy/GSH at different emissions. Finally, we demonstrated that probe 1 can selectively probe GSH over Cys and Hcy or Cys over GSH and Hcy in HeLa cells through multicolor imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

A flavone-based turn-on fluorescent probe for intracellular cysteine/homocysteine sensing with high selectivity.

PubMed

Zhang, Jian; Lv, Yanlin; Zhang, Wei; Ding, Hui; Liu, Rongji; Zhao, Yongsheng; Zhang, Guangjin; Tian, Zhiyuan

2016-01-01

A new type of flavone-based fluorescent probe (DMAF) capable of cysteine (Cys)/homocysteine (Hcy) sensing with high selectivity over other amino acids was developed. Such type of probe undergoes Cys/Hcy-mediated cyclization reaction with the involvement of its aldehyde group, which suppresses of the photoinduced electron transfer (PET) process of the probe molecule and consequently leads to the enhancement of fluorescence emission upon excitation using visible light. The formation of product of the Cys/Hcy-mediated cyclization reaction was confirmed and the preliminary fluorescence imaging experiments revealed the biocompatibility of the as-prepared probe and validated its practicability for intracellular Cys/Hcy sensing. Copyright © 2015 Elsevier B.V. All rights reserved.

Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

DOEpatents

Ramsey, J. Michael; Foote, Robert S.

2003-12-09

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

DOEpatents

Ramsey, J. Michael; Foote, Robert S.

2002-01-01

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

"Off-on" red-emitting fluorescent probes with large Stokes shifts for nitric oxide imaging in living cells.

PubMed

Chen, Jian-Bo; Zhang, Hui-Xian; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan

2013-09-01

Fluorescent probes with larger Stokes shifts in the far-visible and near-infrared spectral region (600-900 nm) are more superior for cellular imaging and biological analysis due to avoiding light scattering interference, reducing autofluorescence from biological sample and encouraging deeper tissue penetration in vivo imaging. In this work, two bis-methoxyphenyl-BODIPY fluorescent probes for the detection of nitric oxide (NO) have been firstly synthesized. Under physiological conditions, these probes can react with NO to form the corresponding triazoles with 250- and 70-fold turn-on fluorescence emitting at 590 and 620 nm, respectively. Moreover, the triazole forms of these probes have large Stokes shifts of 38 nm, in contrast to 10 nm of existing BODIPY probes for NO. Excellent selectivity has been observed against other reactive oxygen/nitrogen species, ascorbic acid and biological matrix. After the evaluation of MTT assay, new fluorescent probes have been successfully applied to fluorescence imaging of NO released from RAW 264.7 macrophages by co-stimulation of lipopolysaccharide and interferon-γ. The experimental results indicate that our fluorescent probes can be powerful candidates for fluorescence imaging of NO due to the low background interference and high detection sensitivity.

Redox-Responsive Fluorescent Probes with Different Design Strategies.

PubMed

Lou, Zhangrong; Li, Peng; Han, Keli

2015-05-19

In an aerobic organism, reactive oxygen species (ROS) are an inevitable metabolic byproduct. Endogenously produced ROS have a significant role in physiological processes, but excess ROS can cause oxidative stress and can damage tissue. Cells possess elaborate mechanisms to regulate their internal redox status. The intracellular redox homeostasis plays an essential role in maintaining cellular function. However, moderate alterations in redox balance can accompany major transitions in a cell's life cycle. Because of the role of ROS in physiology and in pathology, researchers need new tools to study redox chemistry in biological systems.In recent years, researchers have made remarkable progress in developing new, highly sensitive and selective fluorescent probes that respond to redox changes, and in this Account we highlight related research, primarily from our own group. We present an overview of the design, photophysical properties, and fluorescence transduction mechanisms of reported molecules that probe redox changes. We have designed and synthesized a series of fluorescent probes for redox cycles in biological systems relying on the active center of glutathione peroxidase (GPx). We have also constructed probes based on the oxidation and reduction of hydroquinone and of 2,2,6,6-tetramethylpiperidinooxy (TEMPO). Most of these probes exhibit high sensitivity and good selectivity, absorb in the near-infrared, and respond rapidly. Such probes are useful for confocal fluorescence microscopy, a dynamic imaging technique that could allow researchers to observe biologically important ROS and antioxidants in real time. This technique and these probes provide potentially useful tools for exploring the generation, transport, physiological function, and pathogenic mechanisms of ROS and antioxidants.We also describe features that could improve the properties of redox-responsive fluorescent probes: greater photostability; rapid, dynamic, cyclic and ratiometric responses; and

Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

PubMed

Zhang, Shaojuan

2016-01-01

Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

The interaction of amino acids with macrocyclic pH probes of pseudopeptidic nature.

PubMed

Izquierdo, M Angeles; Wadhavane, Prashant D; Vigara, Laura; Burguete, M Isabel; Galindo, Francisco; Luis, Santiago V

2017-08-09

The fluorescence quenching, by a series of amino acids, of pseudopeptidic compounds acting as probes for cellular acidity has been investigated. It has been found that amino acids containing electron-rich aromatic side chains like Trp or Tyr, as well as Met quench the emission of the probes mainly via a collisional mechanism, with Stern-Volmer constants in the 7-43 M -1 range, while other amino acids such as His, Val or Phe did not cause deactivation of the fluorescence. Only a minor contribution of a static quenching due to the formation of ground-state complexes has been found for Trp and Tyr, with association constants in the 9-24 M -1 range. For these ground-state complexes, a comparison between the macrocyclic probes and an open chain analogue reveals the existence of a moderate macrocyclic effect due to the preorganization of the probes in the more rigid structure.

Study of protein-probe interaction and protective action of surfactant sodium dodecyl sulphate in urea-denatured HSA using charge transfer fluorescence probe methyl ester of N,N-dimethylamino naphthyl acrylic acid.

PubMed

Mahanta, Subrata; Singh, Rupashree Balia; Guchhait, Nikhil

2009-03-01

We have demonstrated that the intramolecular charge transfer (ICT) probe Methyl ester of N,N-dimethylamino naphthyl acrylic acid (MDMANA) serves as an efficient reporter of the proteinous microenvironment of Human Serum Albumin (HSA). This work reports the binding phenomenon of MDMANA with HSA and spectral modulation thereupon. The extent of binding and free energy change for complexation reaction along with efficient fluorescence resonance energy transfer from Trp-214 of HSA to MDMANA indicates strong binding between probe and protein. Fluorescence anisotropy, red edge excitation shift, acrylamide quenching and time resolved measurements corroborate the binding nature of the probe with protein and predicts that the probe molecule is located at the hydrophobic site of the protein HSA. Due to the strong binding ability of MDMANA with HSA, it is successfully utilized for the study of stabilizing action of anionic surfactant Sodium Dodecyl Sulphate to the unfolding and folding of protein with denaturant urea in concentration range 1M to 9M.

Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.

PubMed

Roland, Felicia M; Peck, Evan M; Rice, Douglas R; Smith, Bradley D

2017-04-19

A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

Chromenoquinoline-based thiol probes: a study on the quencher position for controlling fluorescent Off-On characteristics.

PubMed

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Talukdar, Pinaki

2013-02-13

The design, synthesis and thiol sensing ability of chromenoquinoline-based fluorescent probes 4, 5 and 6 and are reported here. The relative position of the maleimide moiety was varied along the chromenoquinoline fluorophore to decrease the background fluorescence. Lower background fluorescence in probes 4 and 6 was rationalized by the smaller k(r)/k(nr) values compared to that of probe 5. An intramolecular charge transfer (ICT) mechanism was proposed for quenching and the extent was dependent on the position of the maleimide quencher. Fluorescent Off-On characteristics were evaluated by theoretical calculations. All probes were selective only towards thiol containing amino acids. Thiol sensing by probes 4 and 6 were much better compared to 5. Probe 4 displayed a better fluorescence response for less hindered thiol (185-, 223- and 156-fold for Hcy, Cys and GSH, respectively), while for probe 6, a higher enhancement in fluorescence was observed with more hindered thiols (180-, 205- and 245-fold for Hcy, Cys and GSH, respectively). The better response to bulkier thiol, GSH by probe 6 was attributed to the steric crowding at the C-4 position and bulkiness of the GSH group which force the succinimide unit to be in a nearly orthogonal conformation. This spatial arrangement was important in reducing the fluorescence quenching ability of the succinimide moiety. The application of probes 4, 5 and 6 was demonstrated by naked eye detection thiols using a 96-well plate system as well as by live-cell imaging.

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

PubMed Central

Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

2010-01-01

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

Determination of ethambutol by a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wu, Wen-Ying; Yang, Ji-Yuan; Du, Li-Ming; Wu, Hao; Li, Chang-Feng

2011-08-01

The competitive reaction between ethambutol and two fluorescent probes (i.e., berberine and palmatine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry. The CB[7] reacts with these probes to form stable complexes, and the fluorescence intensity of the complexes is greatly enhanced. In addition, the excitation and emission wavelengths of their complexes moved to wavelengths of 343 nm and 495 nm, respectively. However, the addition of ethambutol dramatically quenches the fluorescence intensity of the two complexes. Accordingly, a couple of new fluorescence quenching methods for the determination of ethambutol were established. The methods can be applied for quantifying ethambutol. A linear relationship between the fluorescence quenching values (Δ F) and ethambutol concentration exists in the range of 5.0-1000.0 ng mL -1, with a correlation coefficient ( r) of 0.9997. The detection limit is 1.7 ng mL -1. The fluorescent probe of berberine has higher sensitivity than palmatine. This paper also discusses the mechanism of fluorescence indicator probes.

Fluorescent Probes for Sensing and Imaging within Specific Cellular Organelles.

PubMed

Zhu, Hao; Fan, Jiangli; Du, Jianjun; Peng, Xiaojun

2016-10-18

long fluorescence lifetimes, which are crucial for long-time tracking of biological processes, tissue and body imaging with deep penetration and low autofluorescence, and time-resolved fluorescence imaging. Research on fluorescent probes with both analyte responsiveness and organelle targetability is a new and emerging area that has attracted increasing attention over the past few years. We have extended the diversity by developing organelle-specific responsive probes capable of detecting changes in biomolecular levels (reactive oxygen species, fluoride ion, hydrogen sulfide, zinc cation, thiol-containing amino acids, and cyclooxygenase-2) and the microenvironment (viscosity, polarity, and pH). Future research should give more considerations of the "low-concern" organelles, such as the Golgi apparatus, the endoplasmic reticulum, and ribosomes. In addition, given the tiny sizes of subcellular organelles (20-1000 nm), we anticipate that clearer visulization of the cellular events within specific organelles will rely on super-resolution optical microscopy with nanoscopic-scale resolution.

A rhodamine 6G derived Schiff base as a fluorescent and colorimetric probe for pH detection and its crystal structure

NASA Astrophysics Data System (ADS)

Guo, Ping; Liu, Lijuan; Shi, Qian; Yin, Chunyan; Shi, Xuefang

2017-02-01

A fluorescent and colorimetric pH probe based on a rhodamine 6G derivative, RP1, was designed and synthesized. The probe was based on the pH induced change in the structure between the spirocyclic (non-fluorescent, colorless) and quinoid (fluorescent, pink) forms of rhodamine 6G. The effect of the acid concentration on the fluorescence "off-on" behaviors of RP1 was investigated. RP1 was fluorescent in the pH range of 1.1-3.1 and has a pKa value of 2.08 (±0.07). Thus RP1 should be useful for studies in strongly acidic environments. Possible interferences from fourteen common metal ions were tested and excluded showing the excellent selectivity of the probe. Finally, the probe exhibits an intense color change at pH values lower than 3.1 which makes it useful for naked-eye pH detection.

Fluorescent Probes for Sensitive and Selective Detection of pH Changes in Live Cells in Visible and Near-infrared Channels.

PubMed

Fang, Mingxi; Adhikari, Rashmi; Bi, Jianheng; Mazi, Wafa; Dorh, Nethaniah; Wang, Jianbo; Conner, Nathan; Ainsley, Jon; Karabencheva-Christova, Tatyana G; Luo, Fen-Tair; Tiwari, Ashutosh; Liu, Haiying

2017-12-28

We report five fluorescent probes based on coumarin-hybridized fluorescent dyes with spirolactam ring structures (A-E) to detect pH changes in live cell by monitoring visible and near-infrared fluorescence changes. Under physiological or basic conditions, the fluorescent probes A, B, C, D and E preserve their spirolactam ring-closed forms and only display fluorescent peaks in the visible region corresponding to coumarin moieties at 497, 483, 498, 497 and 482 nm, respectively. However, at acidic pH, the rings of the spirolactam forms of the fluorescent probes A, B, C, D and E open up, generating new near-infrared fluorescence peaks at 711, 696, 707, 715, and 697 nm, respectively, through significantly extended π-conjugation to coumarin moieties of the fluorophores. The fluorescent probes B and E can be applied to visualize pH changes by monitoring visible as well as near-infrared fluorescence changes. This helps avoid fluorescence imaging blind spots at neutral or basic pH, which typical pH fluorescent probes encounter. The probes exhibit high sensitivity to pH changes, excellent photostability, low auto-fluorescence background and good cell membrane permeability.

In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

NASA Astrophysics Data System (ADS)

Wang, Haolu; Jayachandran, Aparna; Gravot, Germain; Liang, Xiaowen; Thorling, Camilla A.; Zhang, Run; Liu, Xin; Roberts, Michael S.

2016-11-01

Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

Two-Photon Fluorescent Probe for Monitoring Autophagy via Fluorescence Lifetime Imaging.

PubMed

Hou, Liling; Ning, Peng; Feng, Yan; Ding, Yaqi; Bai, Lei; Li, Lin; Yu, Haizhu; Meng, Xiangming

2018-06-19

We reported the first lysosome targeted two-photon fluorescent probe (Lyso-NP) as a viscosity probe for monitoring autophagy. The fluorescence lifetime of Lyso-NP exhibited an excellent linear relationship with viscosity value ( R 2 = 0.99, x = 0.39). Lyso-NP also showed the specific capability for imaging lysosomal viscosity under two-photon excitation at 860 nm along with good biocompatibility. More importantly, Lyso-NP could be used to monitor the autophagy process in living cells by quantitatively detecting lysosomal viscosity changes during the membrane fusion process via two-photon fluorescence lifetime imaging.

Sensitive detection of strong acidic condition by a novel rhodamine-based fluorescent pH chemosensor.

PubMed

Tan, Jia-Lian; Yang, Ting-Ting; Liu, Yu; Zhang, Xue; Cheng, Shu-Jin; Zuo, Hua; He, Huawei

2016-05-01

A novel rhodamine-based fluorescent pH probe responding to extremely low pH values has been synthesized and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic structure under basic conditions opened to a colored and highly fluorescent form under extreme acidity. The quantitative relationship between fluorescence intensity and pH value (1.75-2.62) was consistent with the equilibrium equation pH = pKa + log[(Imax - I)/(I - Imin)]. This sensitive pH probe was also characterized with good reversibility and no interaction with interfering metal ions, and was successfully applied to image Escherichia coli under strong acidity. Copyright © 2015 John Wiley & Sons, Ltd.

A NBD-based simple but effective fluorescent pH probe for imaging of lysosomes in living cells.

PubMed

Cao, Xiang-Jian; Chen, Li-Na; Zhang, Xuan; Liu, Jin-Ting; Chen, Ming-Yu; Wu, Qiu-Rong; Miao, Jun-Ying; Zhao, Bao-Xiang

2016-05-12

NBDlyso with lysosome-locating morpholine moiety has been developed as a high selective and sensitive fluorescent pH probe. This probe can respond to acidic pH (2.0-7.0) in a short time (less than 1 min) and not almost change after continuously illuminated for an extended period by ultraviolet light. The fluorescence intensity of NBDlyso enhanced 100-fold in acidic solution, with very good linear relationship (R(2) = 0.996). The pKa of probe NBDlyso is 4.10. Therefore, NBDlyso was used to detect lysosomal pH changes successfully. Besides, X-ray crystallography was used to verify the structure of NBDlyso, and the recognition mechanism involving photo-induced electron transfer was interpreted theoretically by means of DFT and TDDFT calculations skillfully when NBDlyso comes into play under the acidic condition. This probe showed good ability to sense pH change in living cell image. Copyright © 2016 Elsevier B.V. All rights reserved.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.

PubMed

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X

2017-03-22

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.

Combination of hand-held probe and microscopy for fluorescence guided surgery in the brain tumor marginal zone.

PubMed

Richter, Johan C O; Haj-Hosseini, Neda; Hallbeck, Martin; Wårdell, Karin

2017-06-01

Visualization of the tumor is crucial for differentiating malignant tissue from healthy brain during surgery, especially in the tumor marginal zone. The aim of the study was to introduce a fluorescence spectroscopy-based hand-held probe (HHF-probe) for tumor identification in combination with the fluorescence guided resection surgical microscope (FGR-microscope), and evaluate them in terms of diagnostic performance and practical aspects of fluorescence detection. Eighteen operations were performed on 16 patients with suspected high-grade glioma. The HHF-probe and the FGR-microscope were used for detection of protoporphyrin (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) and evaluated against histopathological analysis and visual grading done through the FGR-microscope by the surgeon. A ratio of PpIX fluorescence intensity to the autofluorescence intensity (fluorescence ratio) was used to quantify the spectra detected by the probe. Fluorescence ratio medians (range 0 - 40) measured by the probe were related to the intensity of the fluorescence in the FGR-microscope, categorized as "none" (0.3, n=131), "weak" (1.6, n=34) and "strong" (5.4, n=28). Of 131 "none" points in the FGR-microscope, 88 (67%) exhibited fluorescence with the HHF-probe. For the tumor marginal zone, the area under the receiver operator characteristics (ROC) curve was 0.49 for the FGR-microscope and 0.65 for the HHF-probe. The probe was integrated in the established routine of tumor resection using the FGR-microscope. The HHF-probe was superior to the FGR-microscope in sensitivity; it detected tumor remnants after debulking under the FGR-microscope. The combination of the HHF-probe and the FGR-microscope was beneficial especially in the tumor marginal zone. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A turn-on coordination nanoparticle-based fluorescent probe for phosphate in human serum

NASA Astrophysics Data System (ADS)

Lin, Na; Li, Jian; Lu, Zhixiang; Bian, Longchun; Zheng, Liyan; Cao, Qiue; Ding, Zhongtao

2015-03-01

Coordination nanoparticles (CNPs) are becoming attractive platforms for chemical sensing applications because their unique adjustable properties offer the opportunity to design various luminescent nanoprobes. Here, we present a CNP-based fluorescent nanoprobe, in which fluorophores (rhodamine B, RB) and quenchers (methylene blue, MB) were spontaneously enfolded by coordination networks self-assembled of adenine, biphenyl-4,4'-dicarboxylic acid (BDA) and zinc ions. The aggregation of fluorophores and quenchers in CNPs resulted in a quenched state fluorescence of RB. RB and MB could be released from CNPs in the presence of phosphate, which triggered the fluorescence of RB. On the basis of recognition-driven disassembly principle, a novel turn-on fluorescent probe for the determination of PO43- with a wide response range (0.5-50 μM) has been successfully applied in the detection of phosphate in human serum samples. This work not only develops a probe for phosphate but also provides a general strategy for designing nanoprobes or nanocarriers towards various targets by altering organic linkers or metal ions.Coordination nanoparticles (CNPs) are becoming attractive platforms for chemical sensing applications because their unique adjustable properties offer the opportunity to design various luminescent nanoprobes. Here, we present a CNP-based fluorescent nanoprobe, in which fluorophores (rhodamine B, RB) and quenchers (methylene blue, MB) were spontaneously enfolded by coordination networks self-assembled of adenine, biphenyl-4,4'-dicarboxylic acid (BDA) and zinc ions. The aggregation of fluorophores and quenchers in CNPs resulted in a quenched state fluorescence of RB. RB and MB could be released from CNPs in the presence of phosphate, which triggered the fluorescence of RB. On the basis of recognition-driven disassembly principle, a novel turn-on fluorescent probe for the determination of PO43- with a wide response range (0.5-50 μM) has been successfully applied in

Design Mechanism and Property of the Novel Fluorescent Probes for the Identification of Microthrix parvicella In Situ

PubMed Central

Jiao, Xiumei; Fei, Xuening; Li, Songya; Lin, Dayong; Ma, Huaji; Zhang, Baolian

2017-01-01

In this study, two novel fluorescent probes, probe A and probe B were designed, synthesized and characterized, based on Microthrix parvicella (M. parvicella) preferring to utilize long-chain fatty acid (LCFA), for the labeling of M. parvicella in activated sludge. The molecular structure of probe A and probe B include long-chain alkane and LCFA, respectively. The results indicated that probe A and probe B had a large stokes shift of 118 nm and 120 nm and high quantum yield of 0.1043 and 0.1058, respectively, which were significantly helpful for the fluorescent labeling. As probe A was more stable than probe B in activated sludge, and the fluorescence intensity keep stable during 24 h, probe A was more suitable for labeling M. parvicella in situ. In addition, through the Image Pro Plus 6 (IPP 6) analysis, a quantitative relationship was established between sludge volume index (SVI) and integral optical density (IOD) of the labeled M. parvicella in activated sludge samples. The relationship between IOD and SVI conforms to Logistic curve (R2 = 0.94). PMID:28773166

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential

NASA Astrophysics Data System (ADS)

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X.

2017-03-01

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry—xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe’s limitations.

Microlensed dual-fiber probe for depth-resolved fluorescence measurements

NASA Astrophysics Data System (ADS)

Choi, Hae Young; Ryu, Seon Young; Kim, Jae Young; Kim, Geon Hee; Park, Seong Jun; Lee, Byeong Ha; Chang, Ki Soo

2011-07-01

We propose and demonstrate a compact microlensed dual-fiber probe that has a good collection efficiency and a high depth-resolution ability for fluorescence measurements. The probe is formed with a conventional fusion splicer creating a common focusing lens on two fibers placed side by side. The collection efficiency of the fabricated probe was evaluated by measuring the fluorescence signal of a fresh ginkgo leaf. It was shown experimentally that the proposed probe could effectively collect the fluorescence signal with a six-fold increase compared to that of a general flat-tipped probe. The beam propagation method was used to design a probe with an optimized working distance and an improved resolving depth. It was found that the working distance depends mainly on the radius of curvature of the lens, whereas the resolving depth is determined by the core diameters of the illumination and collection fibers. The depth-resolved ability of probes with working distances of ~100 μm and 300 μm was validated by using a two-layer tissue phantom. The experimental results demonstrate that the microlensed dual-fiber probe has the potential to facilitate depth-resolved fluorescence detection of epithelial tissue.

Fluorenone based fluorescent probe for selective "turn-on" detection of pyrophosphate and alanine

NASA Astrophysics Data System (ADS)

Daniel Thangadurai, T.; Nithya, I.; Manjubaashini, N.; Bhuvanesh, N.; Bharathi, G.; Nandhakumar, R.; Nataraj, D.

2018-06-01

To sense biologically important entities with different size and dimensions, a fluorenone based fluorescent receptor was designed and synthesized. Probe 1 displayed a distinct fluorescence enhancement emission at 565 nm for pyrophosphate and 530 nm for alanine in polar solvent. The fluorescence titration experiments confirm 1:1 stoichiometric ratio with high-binding constant and very low limit of detection (LoD) values. Receptor 1 showed a highly selective and sensitive recognition to HP2O73 - and to alanine over other competitive anions and amino acids. In addition, the fluorescence lifetime measurement and reversible binding study results support the practical importance of 1.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

PubMed

Aliberti, A; Cusano, A M; Battista, E; Causa, F; Netti, P A

2016-02-21

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

2016-03-01

Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (<114nm), high two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

Curcumin-cysteine and curcumin-tryptophan conjugate as fluorescence turn on sensors for picric Acid in aqueous media.

PubMed

Gogoi, Bedanta; Sen Sarma, Neelotpal

2015-06-03

Rapid detection of picric acid in real sample is of outmost importance from the perspective of health, safety, and environment. In this study, a very simple and cost-effective detection of picric acid is accomplished by developing a couple of biobased conjugates curcumin-cysteine (CC) and curcumin-tryptophan (CT), which undergo efficient fluorescence turn on toward picric acid in aqueous media. Both the probes experience about 26.5-fold fluorescence enhancements at 70 nM concentration of the analyte. Here, the fluorescence turn on process is governed by the aggregation induced emission, which is induced from the electrostatic interaction between the conjugates with picric acid. The detection limit of CC and CT are about 13.51 and 13.54 nM of picric acid, respectively. Importantly, both the probes exhibit high selectivity and low interference of other analogues toward the detection of picric acid. In addition, the probes are highly photostable, show low response time and are practically applicable for sensing picric acid in real environmental samples, which is the ultimate goal of this work.

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

PubMed

Wu, Luling; Li, Xiaolin; Ling, Yifei; Huang, Chusen; Jia, Nengqin

2017-08-30

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

PubMed Central

Wilkinson, T C; Wilton, D C

1986-01-01

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

PubMed Central

Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

Readily Available Fluorescent Probe for Carbon Monoxide Imaging in Living Cells.

PubMed

Feng, Weiyong; Liu, Dandan; Feng, Shumin; Feng, Guoqiang

2016-11-01

Carbon monoxide (CO) is an important gasotransmitter in living systems and its fluorescent detection is of particular interest. However, fluorescent detection of CO in living cells is still challenging due to lack of effective probes. In this paper, a readily available fluorescein-based fluorescent probe was developed for rapid detection of CO. This probe can be used to detect CO in almost wholly aqueous solution under mild conditions and shows high selectivity and sensitivity for CO with colorimetric and remarkable fluorescent turn-on signal changes. The detection limit of this probe for CO is as low as 37 nM with a linear range of 0-30 μM. More importantly, this probe (1 μM dose) can be conveniently used for fluorescent imaging CO in living cells.

Strategies in the design of small-molecule fluorescent probes for peptidases.

PubMed

Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

2014-11-01

Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for diverse types of peptidases. © 2014 Wiley Periodicals, Inc.

Protein recognition by a pattern-generating fluorescent molecular probe.

PubMed

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Protein recognition by a pattern-generating fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Novel DNA probes with low background and high hybridization-triggered fluorescence.

PubMed

Lukhtanov, Eugeny A; Lokhov, Sergey G; Gorn, Vladimir V; Podyminogin, Mikhail A; Mahoney, Walt

2007-01-01

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher at the 3'-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2-4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB-quencher interaction and concealment of the MGB moiety inside the minor groove.

Novel DNA probes with low background and high hybridization-triggered fluorescence

PubMed Central

Lukhtanov, Eugeny A.; Lokhov, Sergey G.; Gorn, Vladimir V.; Podyminogin, Mikhail A.; Mahoney, Walt

2007-01-01

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5′-end and a non-fluorescent quencher at the 3′-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove. PMID:17259212

A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

PubMed

Dong, Jiang Xue; Song, Xiao Fang; Shi, Yan; Gao, Zhong Feng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

2016-07-15

Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

Design strategies of fluorescent probes for selective detection among biothiols.

PubMed

Niu, Li-Ya; Chen, Yu-Zhe; Zheng, Hai-Rong; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng

2015-10-07

Simple thiol derivatives, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play key roles in biological processes, and the fluorescent probes to detect such thiols in vivo selectively with high sensitivity and fast response times are critical for understanding their numerous functions. However, the similar structures and reactivities of these thiols pose considerable challenges to the development of such probes. This review focuses on various strategies for the design of fluorescent probes for the selective detection of biothiols. We classify the fluorescent probes for discrimination among biothiols according to reaction types between the probes and thiols such as cyclization with aldehydes, conjugate addition-cyclization with acrylates, native chemical ligation, and aromatic substitution-rearrangement.

Nanostructure and Corresponding Quenching Efficiency of Fluorescent DNA Probes.

PubMed

Guo, Wenjuan; Wei, Yanhong; Dai, Zhao; Chen, Guangping; Chu, Yuanyuan; Zhao, Yifei

2018-02-09

Based on the fluorescence resonance energy transfer (FRET) mechanism, fluorescent DNA probes were prepared with a novel DNA hairpin template method, with SiO₂ coated CdTe (CdTe/SiO₂) core/shell nanoparticles used as the fluorescence energy donors and gold (Au) nanoparticles (AuNPs) as the energy acceptors. The nanostructure and energy donor/acceptor ratio in a probe were controlled with this method. The relationship between the nanostructure of the probes and FRET efficiency (quenching efficiency) were investigated. The results indicated that when the donor/acceptor ratios were 2:1, 1:1, and 1:2; the corresponding FRET efficiencies were about 33.6%, 57.5%, and 74.2%, respectively. The detection results indicated that the fluorescent recovery efficiency of the detecting system was linear when the concentration of the target DNA was about 0.0446-2.230 nmol/L. Moreover, the probes showed good sensitivity and stability in different buffer conditions with a low detection limit of about 0.106 nmol/L.

A mitochondria-targeted turn-on fluorescent probe for the detection of glutathione in living cells.

PubMed

Zhang, Jian; Bao, Xiaolong; Zhou, Junliang; Peng, Fangfang; Ren, Hang; Dong, Xiaochun; Zhao, Weili

2016-11-15

A novel turn-on red fluorescent BODIPY-based probe (Probe 1) for the detection of glutathione was developed. Such a probe carries a para-dinitrophenoxy benzyl pyridinium moiety at the meso position of a BODIPY dye as self-immolative linker. Probe 1 responds selectively to glutathione with the detection limit of 109nM over other amino acids, common metal ions, reactive oxygen species, reactive nitrogen species, and reactive sulfur species. A novel electrostatic interaction to modulate the SNAr attack of glutathione was believed to play significant role for the observed selective response to glutathione. The cleavage of dinitrophenyl ether by glutathione leads to the production of para-hydroxybenzyl moiety which is able to self-immolate through an intramolecular 1,4-elimination reaction to release the fluorescent BODIPY dye. The low toxic probe has been successfully used to detect mitochondrial glutathione in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel Water-soluble Ratiometric Fluorescent Probe Based on FRET for Sensing Lysosomal pH.

PubMed

Song, Guang-Jie; Bai, Su-Yun; Luo, Jing; Cao, Xiao-Qun; Zhao, Bao-Xiang

2016-11-01

A new ratiometric fluorescent probe based on Förster resonance energy transfer (FRET) for sensing lysosomal pH has been developed. The probe (RMPM) was composed of imidazo[1,5-α]pyridine quaternary ammonium salt fluorophore as the FRET donor and the rhodamine moiety as the FRET acceptor. It's the first time to report that imidazo[1,5-α]pyridine quaternary ammonium salt acts as the FRET donor. The ratio of fluorescence intensity of the probe at two wavelengths (I 424 /I 581 ) changed significantly and responded linearly toward minor pH changes in the range of 5.4-6.6. It should be noted that it's rare to report that a ratiometric pH probe could detect so weak acidic pH with pKa = 6.31. In addition, probe RMPM exhibited excellent water-solubility, fast-response, all-right selectivity and brilliant reversibility. Moreover, RMPM has been successfully applied to sensing lysosomal pH in HeLa cells and has low cytotoxicity.

Gold nanocluster-based ratiometric fluorescent probes for hydrogen peroxide and enzymatic sensing of uric acid.

PubMed

Yang, Dongqin; Luo, Minchuan; Di, Junwei; Tu, Yifeng; Yan, Jilin

2018-05-18

A method is described for ratiometric fluorometric assays of H 2 O 2  by using two probes that have distinct response profiles. Under the catalytic action of ferrous ion, the 615 nm emission of protein-stabilized gold nanoclusters (under 365 nm photoexcitation) is quenched by H 2 O 2 , while an increased signal is generated with a peak at 450 nm by oxidizing coumarin with the H 2 O 2 /Fe(II) system to form a blue emitting fluorophore. These decrease/increase responses give a ratiometric signal. The ratio of the fluorescences at the two peaks are linearly related to the concentration of H 2 O 2 in the range from 0.05 to 10 μM, with a 7.7 nM limit of detection. The detection scheme was further coupled to the urate oxidase catalyzed oxidation of uric acid which proceeds under the formation of H 2 O 2 . This method provides an simple and effective means for the construction of ratiometric fluorometric (enzymatic) assays that involve the detection of H 2 O 2 . Graphical abstract Under catalysis by ferrous ion, hydrogen peroxide quenches the luminescence of gold nanoclusters (AuNCs) and oxidizes coumarin into a fluorescent derivative, which rendered fluorescence ON and OFF at two distinct wavelengths for ratiometric measurements.

A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene for the detection of bisulfite anions and its practical application

NASA Astrophysics Data System (ADS)

Chao, Jianbin; Liu, Yuhong; Zhang, Yan; Zhang, Yongbin; Huo, Fangjun; Yin, Caixia; Wang, Yu; Qin, Liping

2015-07-01

A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene is developed, which shows high selectivity and sensitivity for the detection of bisulfite anions at Na2HPO4 citric acid buffer solutions (pH 5.0). When addition of HSO3-, the fluorescence intensity is significantly enhanced and the probe displays apparent fluorescence color changes from non-fluorescence to blue under a UV lamp illumination, the solution color also changes from yellow to colorless. The detection limit is determined to be as low as 6.30 μM. This offers another specific colorimetric and fluorescent probe for bisulfite anions detection, furthermore it is applied in detecting the level of bisulfite in sugar samples.

Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

PubMed

Wu, Luling; Li, Xiaolin; Huang, Chusen; Jia, Nengqin

2016-08-16

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

BODIPY-Based Fluorescent Probes for Sensing Protein Surface-Hydrophobicity.

PubMed

Dorh, Nethaniah; Zhu, Shilei; Dhungana, Kamal B; Pati, Ranjit; Luo, Fen-Tair; Liu, Haiying; Tiwari, Ashutosh

2015-12-18

Mapping surface hydrophobic interactions in proteins is key to understanding molecular recognition, biological functions, and is central to many protein misfolding diseases. Herein, we report synthesis and application of new BODIPY-based hydrophobic sensors (HPsensors) that are stable and highly fluorescent for pH values ranging from 7.0 to 9.0. Surface hydrophobic measurements of proteins (BSA, apomyoglobin, and myoglobin) by these HPsensors display much stronger signal compared to 8-anilino-1-naphthalene sulfonic acid (ANS), a commonly used hydrophobic probe; HPsensors show a 10- to 60-fold increase in signal strength for the BSA protein with affinity in the nanomolar range. This suggests that these HPsensors can be used as a sensitive indicator of protein surface hydrophobicity. A first principle approach is used to identify the molecular level mechanism for the substantial increase in the fluorescence signal strength. Our results show that conformational change and increased molecular rigidity of the dye due to its hydrophobic interaction with protein lead to fluorescence enhancement.

A theoretical investigation of two typical two-photon pH fluorescent probes.

PubMed

Xu, Zhong; Ren, Ai-Min; Guo, Jing-Fu; Liu, Xiao-Ting; Huang, Shuang; Feng, Ji-Kang

2013-01-01

Intracellular pH plays an important role in many cellular events, such as cell growth, endocytosis, cell adhesion and so on. Some pH fluorescent probes have been reported, but most of them are one-photon fluorescent probes, studies about two-photon fluorescent probes are very rare. In this work, the geometrical structure, electronic structure and one-photon properties of a series of two-photon pH fluorescent probes have been theoretically studied by using density functional theory (DFT) method. Their two-photon absorption (TPA) properties are calculated using the method of ZINDO/sum-over-states method. Two types of two-photon pH fluorescent probes have been investigated by theoretical methods. The mechanisms of the Photoinduced Charge Transfer (PCT) probes and the Photoinduced Electron Transfer (PET) probes are verified specifically. Some designed strategies of good two-photon pH fluorescent probes are suggested on the basis of the investigated results of two mechanisms. For the PCT probes, substituting a stronger electron-donating group for the terminal methoxyl group is an advisable choice to increase the TPA cross section. For the PET probes, the TPA cross sections increase upon protonation. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Near-Infrared Fluorescent Turn-on Probe with a Remarkable Large Stokes Shift for Imaging Selenocysteine in Living Cells and Animals.

PubMed

Feng, Weiyong; Li, Meixing; Sun, Yao; Feng, Guoqiang

2017-06-06

Selenocysteine (Sec) is the 21st naturally occurring amino acid and has emerged as an important sensing target in recent years. However, fluorescent detection of Sec in living systems is challenging. To date, very few fluorescent Sec probes have been reported and most of them respond fluorescence to Sec in the visible region. In this paper, a very promising near-infrared fluorescent probe for Sec was developed. This probe works in aqueous solution over a wide pH range under mild conditions and can be used for rapid, highly selective and sensitive detection of Sec with significant near-infrared fluorescent turn-on signal changes. In addition, it features a remarkable large Stokes shift (192 nm) and a low detection limit (60 nM) for Sec with a wide linear range (0-70 μM). Moreover, this probe can be conveniently used to detect Sec in serum samples, living cells, and animals, indicating it holds great promise for biological applications.

Diketopyrrolopyrrole: brilliant red pigment dye-based fluorescent probes and their applications.

PubMed

Kaur, Matinder; Choi, Dong Hoon

2015-01-07

The development of fluorescent probes for the detection of biologically relevant species is a burgeoning topic in the field of supramolecular chemistry. A number of available dyes such as rhodamine, coumarin, fluorescein, and cyanine have been employed in the design and synthesis of new fluorescent probes. However, diketopyrrolopyrrole (DPP) and its derivatives have a distinguished role in supramolecular chemistry for the design of fluorescent dyes. DPP dyes offer distinctive advantages relative to other organic dyes, including high fluorescence quantum yields and good light and thermal stability. Significant advancements have been made in the development of new fluorescent probes based on DPP in recent years as a result of tireless research efforts by the chemistry scientific community. In this tutorial review, we highlight the recent progress in the development of DPP-based fluorescent probes for the period spanning 2009 to the present time and the applications of these probes to recognition of biologically relevant species including anions, cations, reactive oxygen species, thiols, gases and other miscellaneous applications. This review is targeted toward providing the readers with deeper understanding for the future design of DPP-based fluorogenic probes for chemical and biological applications.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

PubMed

Xie, Yusheng; Ge, Jingyan; Lei, Haipeng; Peng, Bo; Zhang, Huatang; Wang, Danyang; Pan, Sijun; Chen, Ganchao; Chen, Lanfang; Wang, Yi; Hao, Quan; Yao, Shao Q; Sun, Hongyan

2016-12-07

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Fluorescent probes for lipid rafts: from model membranes to living cells.

PubMed

Klymchenko, Andrey S; Kreder, Rémy

2014-01-16

Membrane microdomains (rafts) remain one of the controversial issues in biophysics. Fluorescent molecular probes, which make these lipid nanostructures visible through optical techniques, are one of the tools currently used to study lipid rafts. The most common are lipophilic fluorescent probes that partition specifically into liquid ordered or liquid disordered phase. Their partition depends on the lipid composition of a given phase, which complicates their use in cellular membranes. A second class of probes is based on environment-sensitive dyes, which partition into both phases, but stain them by different fluorescence color, intensity, or lifetime. These probes can directly address the properties of each separate phase, but their cellular applications are still limited. The present review focuses on summarizing the current state in the field of developing and applying fluorescent molecular probes to study lipid rafts. We highlight an urgent need to develop new probes, specifically adapted for cell plasma membranes and compatible with modern fluorescence microscopy techniques to push the understanding of membrane microdomains forward. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular Probe Fluorescence Monitoring of Polymerization

NASA Technical Reports Server (NTRS)

Bunton, Patrick

2002-01-01

This project investigated the feasibility of using fluorescence spectroscopy to determine viscosity of polymer/monomer in support of Transient Interfacial Phenomena in Miscible Polymer Systems (TIPMPS). This project will attempt to measure gradient induced flow at a miscible interface during and / or after in-flight polymerization of dodecyl acrylate (lauryl acrylate). Concentration and temperature gradients will be intentionally introduced during polymerization and the resultant fluid flow determined by Particle Imaging Velocimetry (PIV). This report describes an investigation of the feasibility of using fluorescence of a probe molecule to monitor viscosity and/or concentration during and after polymerization. The probe used was pyrene which has been shown to be sensitive to its local environment in methyl methacrylate.

Fluorescence properties of Schiff base - N,N‧-bis(salicylidene) - 1,2-Phenylenediamine in presence of bile acid host

NASA Astrophysics Data System (ADS)

Roy, Nayan; Paul, Pradip C.; Singh, T. Sanjoy

2015-05-01

Fluorescence properties of Schiff base - N,N‧-bis(salicylidene) - 1,2-phenylenediamine (LH2) is used to study the micelles formed by aggregation of different important bile acids like cholic acid, deoxycholic acid, chenodeoxycholic acid and glycocholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence band intensity was found out to increase with concomitant red shift with gradual addition of different bile acids. Binding constant of the probe with different bile acids as well as critical micelle concentration was obtained from the variation of fluorescence intensity on increasing concentration of bile acids in the medium. The increase in fluorescence quantum yields, fluorescence decay times and substantial decrease in nonradiative decay rate constants in bile acids micellar environment points to the restricted motion of the fluorophore inside the micellar subdomains.

Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand

NASA Astrophysics Data System (ADS)

Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao

2017-05-01

A novel adenine (Ad) fluorescence probe (EuIII-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the EuIII-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the EuIII-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the EuIII-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using EuIII-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of EuIII-dtpa-bis(guanine) at 570 nm as a function of adenine (Ad) concentration in the range of 0.00-5.00 × 10- 5 mol L- 1 was observed. The detection limit is about 4.70 × 10- 7 mol L- 1.

Nanostructure and Corresponding Quenching Efficiency of Fluorescent DNA Probes

PubMed Central

Guo, Wenjuan; Wei, Yanhong; Dai, Zhao; Chen, Guangping; Chu, Yuanyuan; Zhao, Yifei

2018-01-01

Based on the fluorescence resonance energy transfer (FRET) mechanism, fluorescent DNA probes were prepared with a novel DNA hairpin template method, with SiO2 coated CdTe (CdTe/SiO2) core/shell nanoparticles used as the fluorescence energy donors and gold (Au) nanoparticles (AuNPs) as the energy acceptors. The nanostructure and energy donor/acceptor ratio in a probe were controlled with this method. The relationship between the nanostructure of the probes and FRET efficiency (quenching efficiency) were investigated. The results indicated that when the donor/acceptor ratios were 2:1, 1:1, and 1:2; the corresponding FRET efficiencies were about 33.6%, 57.5%, and 74.2%, respectively. The detection results indicated that the fluorescent recovery efficiency of the detecting system was linear when the concentration of the target DNA was about 0.0446–2.230 nmol/L. Moreover, the probes showed good sensitivity and stability in different buffer conditions with a low detection limit of about 0.106 nmol/L. PMID:29425163

Probe diffusion of labeled polymers inside polyacrylic acid solutions: A polyelectrolyte effect

NASA Astrophysics Data System (ADS)

Mishra, Banani; Mithra, K.; Khandai, Santripti; Jena, Sidhartha S.

2018-05-01

Probe diffusion of fluorescently labeled Dextran 40 inside polyelectrolyte solution of polyacrylic acid (PAA) was investigated using Fluorescence Recovery After Photobleaching technique. The crowding and interaction effects on probe diffusion were controlled by tuning background polymer and added external electrolyte concentration. For all the salt concentration, an overall decrease in diffusion coefficient is observed with rise in polymer concentration. The diffusion coefficient decreases with decrease in salt concentration whereas the solution viscosity increases, indicating a competition between viscous drag and electrostatic interaction. A large positive deviation from the ideal Stokes-Einstein relation is observed for high polymer and low salt concentration, which reduces markedly with addition of salt confirming polyelectrolyte effects, plays a major role in deciding the probe diffusion.

Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

NASA Astrophysics Data System (ADS)

Kujala, Naresh Gandhi

Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

PubMed Central

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

PubMed

Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

2016-05-18

A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

Fluorescent probes and nanoparticles for intracellular sensing of pH values

NASA Astrophysics Data System (ADS)

Shi, Wen; Li, Xiaohua; Ma, Huimin

2014-12-01

Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 - 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail.

Room temperature synthesis of pH-switchable polyaniline quantum dots as a turn-on fluorescent probe for acidic biotarget labeling.

PubMed

Liu, Yanfeng; Ding, Yin; Gou, Huilin; Huang, Xin; Zhang, Guiyang; Zhang, Qi; Liu, Yunzhong; Meng, Zhen; Xi, Kai; Jia, Xudong

2018-04-05

The synthesis of well-defined light-element-derived quantum dots (LEQDs) with advanced optical properties under mild conditions is highly desirable yet challenging. Here, a polyaniline (PANI) structure is introduced into carbon-rich LEQDs to yield well-defined, fluorescent polyaniline quantum dots (PAQDs), PAQD24, through a one-pot room temperature reaction. The mild synthetic conditions effectively minimize the defects introduced during the conventional synthesis and endow PAQD24 with desirable optical properties, including a narrow emission band (full width at half maximum = 55 nm), an optimal quantum yield of 32.5% and two-photon fluorescence. Furthermore, the bandgap of PAQD24 is highly sensitive toward pH variations in the near-neutral region, due to the proton doping and dedoping of the PANI structure. Such unique properties together with its fine bio-compatibility enable the application of this material as a turn-on fluorescent probe for the labeling of acidic biotargets from sub-cellular to organ levels, providing potential applications in diagnosis and surgery guidance for certain diseases.

Discerning the Chemistry in Individual Organelles with Small-Molecule Fluorescent Probes.

PubMed

Xu, Wang; Zeng, Zebing; Jiang, Jian-Hui; Chang, Young-Tae; Yuan, Lin

2016-10-24

Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing Zeolite Crystal Architecture and Structural Imperfections using Differently Sized Fluorescent Organic Probe Molecules.

PubMed

Hendriks, Frank C; Schmidt, Joel E; Rombouts, Jeroen A; Lammertsma, Koop; Bruijnincx, Pieter C A; Weckhuysen, Bert M

2017-05-05

A micro-spectroscopic method has been developed to probe the accessibility of zeolite crystals using a series of fluorescent 4-(4-diethylaminostyryl)-1-methylpyridinium iodide (DAMPI) probes of increasing molecular size. Staining large zeolite crystals with MFI (ZSM-5) topology and subsequent mapping of the resulting fluorescence using confocal fluorescence microscopy reveal differences in structural integrity: the 90° intergrowth sections of MFI crystals are prone to develop structural imperfections, which act as entrance routes for the probes into the zeolite crystal. Polarization-dependent measurements provide evidence for the probe molecule's alignment within the MFI zeolite pore system. The developed method was extended to BEA (Beta) crystals, showing that the previously observed hourglass pattern is a general feature of BEA crystals with this morphology. Furthermore, the probes can accurately identify at which crystal faces of BEA straight or sinusoidal pores open to the surface. The results show this method can spatially resolve the architecture-dependent internal pore structure of microporous materials, which is difficult to assess using other characterization techniques such as X-ray diffraction. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

A simple rhodamine hydrazide-based turn-on fluorescent probe for HOCl detection.

PubMed

Zhang, Zhen; Zou, Yuan; Deng, Chengquan; Meng, Liesu

2016-06-01

Hypochlorous acid (HOCl) plays a crucial role in daily life and mediates a variety of physiological processes, however, abnormal levels of HOCl have been associated with numerous human diseases. It is therefore of significant interest to establish a simple, selective, rapid and sensitive fluorogenic method for the detection of HOCl in environmental and biological samples. A hydrazide-containing fluorescent probe based on a rhodamine scaffold was facilely developed that could selectively detect HOCl over other biologically relevant reactive oxygen species, reactive nitrogen species and most common metal ions in vitro. Via an irreversible oxidation-hydrolysis mechanism, and upon HOCl-triggered opening of the intramolecular spirocyclic ring during detection, the rhodamine hydrazide-based probe exhibited large fluorescence enhancement in the emission spectra with a fast response, low detection limit and comparatively wide pH detection range in aqueous media. The probe was further successfully applied to monitoring trace HOCl in tap water and imaging both exogenous and endogenous HOCl within living cells. It is anticipated that this simple and useful probe might be an efficient tool with which to facilitate more HOCl-related chemical and biological research. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

PubMed

Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

2018-06-01

We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems.

PubMed

Aron, Allegra T; Ramos-Torres, Karla M; Cotruvo, Joseph A; Chang, Christopher J

2015-08-18

Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed "recognition" and "reactivity". Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent

Fluorescent coumarin-based probe for cysteine and homocysteine with live cell application

NASA Astrophysics Data System (ADS)

Wei, Ling-Fang; Thirumalaivasan, Natesan; Liao, Yu-Cheng; Wu, Shu-Pao

2017-08-01

Cysteine (Cys) and homocysteine (Hcy) are two of important biological thiols and function as important roles in several biological processes. The development of Cys and Hcy probes will help to explore the functions of biothiols in biological systems. In this work, a new coumarin-based probe AC, containing an acryloyl moiety, was developed for Cys and Hcy detection in cells. Cys and Hcy undergo a nucleophilic addition and subsequent cyclization reaction to remove to the acryloyl group and yield a fluorescent product, 7-hydroxylcomuarin. The probe AC showed good selectivity for cysteine and homocysteine over glutathione and other amino acids and had low detection limits of 65 nM for Cys and 79 nM for Hcy, respectively. Additionally, confocal imaging experiments demonstrated that the probe AC can be applied to visualize Cys and Hcy in living cells.

FRET-based small-molecule fluorescent probes: rational design and bioimaging applications.

PubMed

Yuan, Lin; Lin, Weiying; Zheng, Kaibo; Zhu, Sasa

2013-07-16

Fluorescence imaging has emerged as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution. Researchers have constructed a large number of synthetic intensity-based fluorescent probes for bio-imaging. However, intensity-based fluorescent probes have some limitations: variations in probe concentration, probe environment, and excitation intensity may influence the fluorescence intensity measurements. In principle, the use of ratiometric fluorescent probes can alleviate this shortcoming. Förster resonance energy transfer (FRET) is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. However, the development of synthetic FRET probes with favorable photophysical properties that are also suitable for biological imaging applications remains challenging. In this Account, we review the rational design and biological applications of synthetic FRET probes, focusing primarily on studies from our laboratory. To construct useful FRET probes, it is a pre-requisite to develop a FRET platform with favorable photophysical properties. The design criteria of a FRET platform include (1) well-resolved absorption spectra of the donor and acceptor, (2) well-separated emission spectra of the donor and acceptor, (3) donors and acceptors with comparable brightness, (4) rigid linkers, and (5) near-perfect efficiency in energy transfer. With an efficient FRET platform in hand, it is then necessary to modulate the donor-acceptor distance or spectral overlap integral in an analyte-dependent fashion for development of FRET probes. Herein, we emphasize our most recent progress on the development of FRET probes by spectral overlap integral, in particular by changing the molar absorption coefficient of the donor dyes such as rhodamine dyes, which undergo unique changes in the absorption profiles during the ring-opening and -closing processes. Although partial success has been obtained in design of

Monitoring Membrane Hydration with 2-(Dimethylamino)-6-Acylnaphtalenes Fluorescent Probes.

PubMed

Bagatolli, Luis A

2015-01-01

A family of polarity sensitive fluorescent probes (2-(dimethylamino)-6-acylnaphtalenes, i.e. LAURDAN, PRODAN, ACDAN) was introduced by Gregorio Weber in 1979, with the aim to monitor solvent relaxation phenomena on protein matrices. In the following years, however, PRODAN and particularly LAURDAN, were used to study membrane lateral structure and associated dynamics. Once incorporated into membranes, the (nanosecond) fluorescent decay of these probes is strongly affected by changes in the local polarity and relaxation dynamics of restricted water molecules existing at the membrane/water interface. For instance, when glycerophospholipid containing membranes undertake a solid ordered (gel) to liquid disordered phase transition the fluorescence emission maximum of these probes shift ~ 50 nm with a significant change in their fluorescence lifetime. Furthermore, the fluorescence parameters of LAURDAN and PRODAN are exquisitely sensitive to cholesterol effects, allowing interpretations that correlate changes in membrane packing with membrane hydration. Different membrane model systems as well as innate biological membranes have been studied with this family of probes allowing interesting comparative studies. This chapter presents a short historical overview about these fluorescent reporters, discusses on different models proposed to explain their sensitivity to membrane hydration, and includes relevant examples from experiments performed in artificial and biological membranes.

Recent Advances in Macrocyclic Fluorescent Probes for Ion Sensing.

PubMed

Wong, Joseph K-H; Todd, Matthew H; Rutledge, Peter J

2017-01-25

Small-molecule fluorescent probes play a myriad of important roles in chemical sensing. Many such systems incorporating a receptor component designed to recognise and bind a specific analyte, and a reporter or transducer component which signals the binding event with a change in fluorescence output have been developed. Fluorescent probes use a variety of mechanisms to transmit the binding event to the reporter unit, including photoinduced electron transfer (PET), charge transfer (CT), Förster resonance energy transfer (FRET), excimer formation, and aggregation induced emission (AIE) or aggregation caused quenching (ACQ). These systems respond to a wide array of potential analytes including protons, metal cations, anions, carbohydrates, and other biomolecules. This review surveys important new fluorescence-based probes for these and other analytes that have been reported over the past five years, focusing on the most widely exploited macrocyclic recognition components, those based on cyclam, calixarenes, cyclodextrins and crown ethers; other macrocyclic and non-macrocyclic receptors are also discussed.

Allyl Fluorescein Ethers as Promising Fluorescent Probes for Carbon Monoxide Imaging in Living Cells.

PubMed

Feng, Shumin; Liu, Dandan; Feng, Weiyong; Feng, Guoqiang

2017-03-21

Recently, the fluorescent detection of carbon monoxide (CO) in living cells has attracted great attention. However, due to the lack of effective ways to construct fluorescent CO probes, fluorescent detection of CO in living cells is still in its infancy. In this paper, we report for the first time the use of allyl ether as a reaction site for construction of fluorescent CO probes. By this way, two readily available allyl fluorescein ethers were prepared, which were found to be highly selective and sensitive probes for CO in the presence of PdCl 2 . These probes have the merits of good stability, good water-solubility, and rapid and distinct colorimetric and remarkable fluorescent turn-on signal changes. Moreover, a very low dose of these two probes can be used to detect and track CO in living cells, indicating that these two probes could be very promising biological tools for CO detection in living systems. Overall, this work provided not only two new promising fluorescent CO probes but also a new way to devise fluorescent CO probes.

Estimation of the binding ability of main transport proteins of blood plasma with liver cirrhosis by the fluorescent probe method

NASA Astrophysics Data System (ADS)

Korolenko, E. A.; Korolik, E. V.; Korolik, A. K.; Kirkovskii, V. V.

2007-07-01

We present results from an investigation of the binding ability of the main transport proteins (albumin, lipoproteins, and α-1-acid glycoprotein) of blood plasma from patients at different stages of liver cirrhosis by the fluorescent probe method. We used the hydrophobic fluorescent probes anionic 8-anilinonaphthalene-1-sulfonate, which interacts in blood plasma mainly with albumin; cationic Quinaldine red, which interacts with α-1-acid glycoprotein; and neutral Nile red, which redistributes between lipoproteins and albumin in whole blood plasma. We show that the binding ability of albumin and α-1-acid glycoprotein to negatively charged and positively charged hydrophobic metabolites, respectively, increases in the compensation stage of liver cirrhosis. As the pathology process deepens and transitions into the decompensation stage, the transport abilities of albumin and α-1-acid glycoprotein decrease whereas the binding ability of lipoproteins remains high.

“Turn-on” fluorescence probe integrated polymer nanoparticles for sensing biological thiol molecules

NASA Astrophysics Data System (ADS)

Ang, Chung Yen; Tan, Si Yu; Lu, Yunpeng; Bai, Linyi; Li, Menghuan; Li, Peizhou; Zhang, Quan; Selvan, Subramanian Tamil; Zhao, Yanli

2014-11-01

A ``turn-on'' thiol-responsive fluorescence probe was synthesized and integrated into polymeric nanoparticles for sensing intracellular thiols. There is a photo-induced electron transfer process in the off state of the probe, and this process is terminated upon the reaction with thiol compounds. Configuration interaction singles (CIS) calculation was performed to confirm the mechanism of this process. A series of sensing studies were carried out, showing that the probe-integrated nanoparticles were highly selective towards biological thiol compounds over non-thiolated amino acids. Kinetic studies were also performed to investigate the relative reaction rate between the probe and the thiolated amino acids. Subsequently, the Gibbs free energy of the reactions was explored by means of the electrochemical method. Finally, the detection system was employed for sensing intracellular thiols in cancer cells, and the sensing selectivity could be further enhanced with the use of a cancer cell-targeting ligand in the nanoparticles. This development paves a path for the sensing and detection of biological thiols, serving as a potential diagnostic tool in the future.

Zinc complexes as fluorescent chemosensors for nucleic acids: new perspectives for a "boring" element.

PubMed

Terenzi, Alessio; Lauria, Antonino; Almerico, Anna Maria; Barone, Giampaolo

2015-02-28

Zinc(II) complexes are effective and selective nucleic acid-binders and strongly fluorescent molecules in the low energy range, from the visible to the near infrared. These two properties have often been exploited to quantitatively detect nucleic acids in biological samples, in both in vitro and in vivo models. In particular, the fluorescent emission of several zinc(II) complexes is drastically enhanced or quenched by the binding to nucleic acids and/or upon visible light exposure, in a different fashion in bulk solution and when bound to DNA. The twofold objective of this perspective is (1) to review recent utilisations of zinc(II) complexes as selective fluorescent probes for nucleic acids and (2) to highlight their novel potential applications as diagnostic tools based on their photophysical properties.

Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells.

PubMed

Wang, Xuzhe; Zhou, Li; Qiang, Fei; Wang, Feiyi; Wang, Rui; Zhao, Chunchang

2016-03-10

A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4-10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells.

PubMed

Winterbourn, Christine C

2014-02-01

Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells. The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed. Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells. Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe.

PubMed

Liu, Hua; Na, Weidan; Liu, Ziping; Chen, Xueqian; Su, Xingguang

2017-06-15

In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H 2 O 2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H 2 O 2 in the range of 3.33-500µM with a detection limit of 1.2µM. The linear detection for AA was in the range from 1.11 to 300µM with a detection limit of 0.32µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Copyright © 2017. Published by Elsevier B.V.

Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations

PubMed Central

Kalyanaraman, Balaraman; Darley-Usmar, Victor; Davies, Kelvin J.A.; Dennery, Phyllis A.; Forman, Henry Jay; Grisham, Matthew B.; Mann, Giovanni E.; Moore, Kevin; Roberts, L. Jackson; Ischiropoulos, Harry

2013-01-01

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results. PMID:22027063

A fluorescent probe for ecstasy.

PubMed

Masseroni, D; Biavardi, E; Genovese, D; Rampazzo, E; Prodi, L; Dalcanale, E

2015-08-18

A nanostructure formed by the insertion in silica nanoparticles of a pyrene-derivatized cavitand, which is able to specifically recognize ecstasy in water, is presented. The absence of effects from interferents and an efficient electron transfer process occurring after complexation of ecstasy, makes this system an efficient fluorescent probe for this popular drug.

Single Cell Fluorescence Imaging Using Metal Plasmon-Coupled Probe

PubMed Central

Zhang, Jian; Fu, Yi; Lakowicz, Joseph R.

2009-01-01

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A. PMID:17375898

A new class of homogeneous nucleic acid probes based on specific displacement hybridization

PubMed Central

Li, Qingge; Luan, Guoyan; Guo, Qiuping; Liang, Jixuan

2002-01-01

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant. PMID:11788731

Carbon dots-based fluorescent probe for "off-on" sensing of Hg(II) and I⁻.

PubMed

He, Jiangling; Zhang, Haoran; Zou, Jinliang; Liu, Yingliang; Zhuang, Jianle; Xiao, Yong; Lei, Bingfu

2016-05-15

Herein, we report a simple, one-step reflux method for synthesis of photoluminescent carbon dots (CDs) using citric acid as the carbon source and diethylenetriamine (DETA) as the surface passivation reagent along with a high quantum yield (82.40%), the fluorescence intensity of the CDs was found to be effectively quenched by Hg(II) ions. Upon addition of I(-) to the CDs/Hg(II) complex dispersion, the fluorescence intensity of the CDs was significantly recovered. Furthermore, we developed an "off-on" fluorescence assay for the detection of I(-) using CDs/Hg(II) as a fluorescence probe. This probe enables the selective detection of Hg(II) with a linear range of 0-80 μM and a limit of detection is 0.201 µM and a limit of detection about I(-) is 0.234 µM with a linear range of 0-70 μM. Most importantly, the sensors can be successfully applied to the determination of Hg(II) and I(-) in real lake water and urine of cattles, the "off-on" sensor demonstrates high selectivity, repeatability, stability, which offer this CDs-based "off-on" fluorescent sensor a promising platform for environmental and biological sensing applications. Copyright © 2015 Elsevier B.V. All rights reserved.

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods.

PubMed

Westergaard Mulberg, Mads; Taskova, Maria; Thomsen, Rasmus P; Okholm, Anders H; Kjems, Jørgen; Astakhova, Kira

2017-08-17

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence detection of glutathione and oxidized glutathione in blood with a NIR-excitable cyanine probe

NASA Astrophysics Data System (ADS)

Liu, Chang-hui; Qi, Feng-pei; Wen, Fu-bin; Long, Li-ping; Liu, Ai-juan; Yang, Rong-hua

2018-04-01

Cyanine has been widely utilized as a near infrared (NIR) fluorophore for detection of glutathione (GSH). However, the excitation of most of the reported cyanine-based probes was less than 800 nm, which inevitably induce biological background absorption and lower the sensitivity, limiting their use for detection of GSH in blood samples. To address this issue, here, a heptamethine cyanine probe (DNIR), with a NIR excitation wavelength at 804 nm and a NIR emission wavelength at 832 nm, is employed for the detection of GSH and its oxidized form (GSSG) in blood. The probe displays excellent selectivity for GSH over GSSG and other amino acids, and rapid response to GSH, in particular a good property for indirect detection of GSSG in the presence of enzyme glutathione reductase and the reducing agent nicotinamideadenine dinucleotide phosphate, without further separation prior to fluorescent measurement. To the best of our knowledge, this is the first attempt to explore NIR fluorescent approach for the simultaneous assay of GSH and GSSG in blood. As such, we expect that our fluorescence sensors with both NIR excitation and NIR emission make this strategy suitable for the application in complex physiological systems.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids.

PubMed

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-06-04

Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

Recognition- and Reactivity-Based Fluorescent Probes for Studying Transition Metal Signaling in Living Systems

PubMed Central

2015-01-01

Conspectus Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed “recognition” and “reactivity”. Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give

A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

PubMed

Ning, Dianhua; He, Changtian; Liu, Zhengjie; Liu, Cui; Wu, Qilong; Zhao, TingTing; Liu, Renyong

2017-05-21

Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

PubMed

Smertenko, Andrei; Moschou, Panagiotis; Zhang, Laining; Fahy, Deirdre; Bozhkov, Peter

2016-01-01

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging.

PubMed

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Varma, Sreejith Jayasree; Talukdar, Pinaki

2012-03-11

A new chromenoquinoline-based fluorescent off-on thiol probe 2 is reported. In aqueous buffer solutions at physiological pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addition of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated. This journal is © The Royal Society of Chemistry 2012

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

PubMed

Dang, Dung Thanh; Phan, Anh Tuân

2016-01-01

We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine

NASA Astrophysics Data System (ADS)

Shang, Xuefang; Li, Jie; Guo, Kerong; Ti, Tongyu; Wang, Tianyun; Zhang, Jinlian

2017-04-01

Inspired from biological counter parts, chemical modification of Schiff base derivatives with function groups may provide a highly efficient method to detect amino acids. Therefore, a fluorescent probe involving Schiff base and hydroxyl group has been designed and prepared, which showed high response and specificity for Arginine (Arg) among normal eighteen standard kinds of amino acids (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, the synthesized fluorescent probe exhibited high binding ability for Arg and low cytotoxicity to MCF-7 cells over a concentration range of 0-200 μg mL-1 which can be also used as a biosensor for the Arg detection in vivo.

Exploring accessibility of pretreated poplar cell walls by measuring dynamics of fluorescent probes.

PubMed

Paës, Gabriel; Habrant, Anouck; Ossemond, Jordane; Chabbert, Brigitte

2017-01-01

The lignocellulosic cell wall network is resistant to enzymatic degradation due to the complex chemical and structural features. Pretreatments are thus commonly used to overcome natural recalcitrance of lignocellulose. Characterization of their impact on architecture requires combinatory approaches. However, the accessibility of the lignocellulosic cell walls still needs further insights to provide relevant information. Poplar specimens were pretreated using different conditions. Chemical, spectral, microscopic and immunolabeling analysis revealed that poplar cell walls were more altered by sodium chlorite-acetic acid and hydrothermal pretreatments but weakly modified by soaking in aqueous ammonium. In order to evaluate the accessibility of the pretreated poplar samples, two fluorescent probes (rhodamine B-isothiocyanate-dextrans of 20 and 70 kDa) were selected, and their mobility was measured by using the fluorescence recovery after photobleaching (FRAP) technique in a full factorial experiment. The mobility of the probes was dependent on the pretreatment type, the cell wall localization (secondary cell wall and cell corner middle lamella) and the probe size. Overall, combinatory analysis of pretreated poplar samples showed that even the partial removal of hemicellulose contributed to facilitate the accessibility to the fluorescent probes. On the contrary, nearly complete removal of lignin was detrimental to accessibility due to the possible cellulose-hemicellulose collapse. Evaluation of plant cell wall accessibility through FRAP measurement brings further insights into the impact of physicochemical pretreatments on lignocellulosic samples in combination with chemical and histochemical analysis. This technique thus represents a relevant approach to better understand the effect of pretreatments on lignocellulose architecture, while considering different limitations as non-specific interactions and enzyme efficiency.

Fluorescent carbohydrate probes for cell lectins

NASA Astrophysics Data System (ADS)

Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

2001-09-01

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe.

PubMed

Xu, Yujuan; Zhang, Peng; Wang, Zhen; Lv, Shaoping; Ding, Caifeng

2018-02-27

Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity. Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

An Environmentally Sensitive Fluorescent Dye as a Multidimensional Probe of Amyloid Formation

PubMed Central

2016-01-01

We have explored amyloid formation using poly(amino acid) model systems in which differences in peptide secondary structure and hydrophobicity can be introduced in a controlled manner. We show that an environmentally sensitive fluorescent dye, dapoxyl, is able to identify β-sheet structure and hydrophobic surfaces, structural features likely to be related to toxicity, as a result of changes in its excitation and emission profiles and its relative quantum yield. These results show that dapoxyl is a multidimensional probe of the time dependence of amyloid aggregation, which provides information about the presence and nature of metastable aggregation intermediates that is inaccessible to the conventional probes that rely on changes in quantum yield alone. PMID:26865546

Nitrogen-doped carbon quantum dots as a fluorescence probe combined with magnetic solid-phase extraction purification for analysis of folic acid in human serum.

PubMed

Wang, Meng; Jiao, Yang; Cheng, Chunsheng; Hua, Jianhao; Yang, Yaling

2017-12-01

A novel and sensitive method based on nitrogen-doped carbon quantum dots as a fluorescence probe coupled with magnetic solid-phase extraction (MSPE) purification for analysis of folic acid (FA) in human serum samples has been established for the first time. In the developed system, magnetic nanoparticles coated with hexanoic acid (Fe 3 O 4 @C 6 ) were synthesized by a one-step chemical co-precipitation method with good magnetic properties and dispersibility for sample purification, and it is better to be separated from the sample. High fluorescence nitrogen-doped carbon quantum dots (N-CQDs), simply prepared using a one-step hydrothermal method with nitrilotriacetic acid, could be selectively quenched by FA. Based on this phenomenon, a fluorescence assay was proposed for specific determination of FA. Various operational experiment parameters have been studied and optimized in detail. Under the optimum experimental conditions, the detection limit of the proposed method for FA was evaluated to be 0.5 nM (S/N = 3), while the relative standard deviation (RSD) was 1.2% (n = 6). Finally, the proposed method was applied for determination of trace levels of FA from human serum samples and quantitative recoveries were achieved within the range of 95.7-103.5%. All of the results showed that the proposed method had significant application in further research. Graphical abstract Schematic of synthesis of N-CQDs and schematic of suggested mode for analysis of folic acid (FA).

[In situ visual imaging of oral squamous cell carcinoma in mice by using near-infrared quantum dots conjugated with arginine-glycine-aspartic acid peptide fluorescent probes].

PubMed

Yunlong, Bai; Hao, Huang; Kai, Yang; Hong, Tang

2014-10-01

To investigate in situ visualization using near-infrared quantum dots (QDs) conjugated with arginine- glycine-aspartic acid (ROD) peptide fluorescent probes in oral squamous cell carcinoma (08CC). QDs with emission wavelength of 800 nm (QD800) were conjugated with RGD peptides to produce QD800-RGD fluorescent probes. Human OSCC cell line BcaCD885 was inoculated in nude mice cheeks to establish OSCC mouse models. Frozen BcaCD885 tumor slices were immunofluorescence double stained by using QD800-RGD and CD105 monoclonal antibody and were observed using a laser scanning confocal microscope. QD800-RGD was injected into the OSCC models through the tail veins, and the in situ visualization was analyzed at different time points. The mice were sacrificed 12 h after injection to isolate tumors for the ex vivo analysis of probe localization in the tumors. QD800-RGD specifically targeted the integrin avβ3 expressed in the endothelial cells of tumor angiogenic vessels in vitro and in vivo, producing clear tumor fluorescence images after intravenous injection. The most complete tumor images with maximal signal-to-noise ratios were observed 0.5 h to 6 h after injection of the probe and significantly reduced 9 h after the injection. However, the tumor image was still clearly visible at 12 h. Using intravenously injected QD800-RGD generates high quality OSCC images when integrin avβ3, which is expressed in the endothelial cells of tumor angiogenic vessels, is used as the target. The technique offers great potential in the diagnosis and individual treatment of OSCC.

CdTe/ZnS quantum dots as fluorescent probes for ammonium determination.

PubMed

Yi, Kui-Yu

2016-06-01

Novel CdTe/ZnS quantum dot (QD) probes based on the quenching effect were proposed for the simple, rapid, and specific determination of ammonium in aqueous solutions. The QDs were modified using 3-mercaptopropionic acid, and the fluorescence responses of the CdTe/ZnS QD probes to ammonium were detected through regularity quenching. The quenching levels of the CdTe/ZnS QDs and ammonium concentration showed a good linear relationship between 4.0 × 10(-6) and 5.0 × 10(-4) mol/L; the detection limit was 3.0 × 10(-7) mol/L. Ammonium contents in synthetic explosion soil samples were measured to determine the practical applications of the QD probes and a probable quenching mechanism was described. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

PubMed Central

Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

PubMed

Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

2012-07-01

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

PubMed Central

2013-01-01

Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

Fluorescent probes for real-time measurement of nitric oxide in living cells.

PubMed

Li, Huili; Wan, Ajun

2015-11-07

Nitric oxide (NO) is an important signaling molecule in biology. Both NO excess and insufficiency have been implicated in numerous physiological and pathological conditions. In order to study the diverse biological roles of NO in cells and tissues, many techniques have been developed for assaying NO. Recently, new generations of fluorescent probes have become indispensible tools for the study of NO biology because of their sensitivity, selectivity, spatiotemporal resolution, and experimental feasibility. Rational application of these probes in the study requires the understanding of the molecular mechanism that the probes are involved in. In this review, we will present an arsenal of fluorescent probes used to detect NO in living cells and animal tissues. We will also discuss the molecular mechanisms, actualities and prospects of fluorescent probes in detecting NO in cell biology.

New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging.

PubMed

Pauli, Jutta; Licha, Kai; Berkemeyer, Janis; Grabolle, Markus; Spieles, Monika; Wegner, Nicole; Welker, Pia; Resch-Genger, Ute

2013-07-17

The rational design of bright optical probes and dye-biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M(-1) cm(-1) and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of

Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer.

PubMed

Koide, Yuichiro; Urano, Yasuteru; Hanaoka, Kenjiro; Terai, Takuya; Nagano, Tetsuo

2011-06-17

The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ*-π* conjugation between group 14 atom-C (methyl) σ* orbitals and a π* orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (Φ(fl) = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (Φ(fl)) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn(2+) sensor. We synthesized these probes and confirmed that they work well.

Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species

NASA Astrophysics Data System (ADS)

Needham, Lisa-Maria; Weber, Judith; Fyfe, James W. B.; Kabia, Omaru M.; Do, Dung T.; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M.; Ghandi, Sonia; Bohndiek, Sarah E.; Snaddon, Thomas N.; Lee, Steven F.

2018-02-01

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species.

PubMed

Needham, Lisa-Maria; Weber, Judith; Fyfe, James W B; Kabia, Omaru M; Do, Dung T; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M; Ghandi, Sonia; Bohndiek, Sarah E; Snaddon, Thomas N; Lee, Steven F

2018-02-01

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H 2 O 2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H 2 O 2 . We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H 2 O 2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Congying; Gu, Yueqing

2014-09-01

Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

PubMed Central

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-01-01

Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. PMID:18522756

Piezoresistor-equipped fluorescence-based cantilever probe for near-field scanning.

PubMed

Kan, Tetsuo; Matsumoto, Kiyoshi; Shimoyama, Isao

2007-08-01

Scanning near-field optical microscopes (SNOMs) with fluorescence-based probes are promising tools for evaluating the optical characteristics of nanoaperture devices used for biological investigations, and this article reports on the development of a microfabricated fluorescence-based SNOM probe with a piezoresistor. The piezoresistor was built into a two-legged root of a 160-microm-long cantilever. To improve the displacement sensitivity of the cantilever, the piezoresistor's doped area was shallowly formed on the cantilever surface. A fluorescent bead, 500 nm in diameter, was attached to the bottom of the cantilever end as a light-intensity-sensitive material in the visible-light range. The surface of the scanned sample was simply detected by the probe's end being displaced by contact with the sample. Measuring displacements piezoresistively is advantageous because it eliminates the noise arising from the use of the optical-lever method and is free of any disturbance in the absorption or the emission spectrum of the fluorescent material at the probe tip. The displacement sensitivity was estimated to be 6.1 x 10(-6) nm(-1), and the minimum measurable displacement was small enough for near-field measurement. This probe enabled clear scanning images of the light field near a 300 x 300 nm(2) aperture to be obtained in the near-field region where the tip-sample distance is much shorter than the light wavelength. This scanning result indicates that the piezoresistive way of tip-sample distance regulation is effective for characterizing nanoaperture optical devices.

Fluorescence in vivo imaging of live tumor cells with pH-activatable targeted probes via receptor-mediated endocytosis

NASA Astrophysics Data System (ADS)

Asanuma, Daisuke; Urano, Yasuteru; Nagano, Tetsuo; Hama, Yukihiro; Koyama, Yoshinori; Kobayashi, Hisataka

2009-02-01

One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background. Here, we achieve specific in vivo tumor visualization with a newly-designed "activatable" targeted fluorescence probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody, Trastuzumab, or galactosyl serum albumin (GSA). As proof of concept, ex and in vivo imaging of two different tumor mouse models was performed: HER2-overexpressed lung metastasis tumor with Trastuzumab-pH probe conjugates and lectin-overexpressed i.p. disseminated tumor with GSA-pH probe conjugates. These pH-activatable targeted probes were highly specific for tumors with minimal background signal. Because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. Furthermore, this strategy was also applied to fluorescence endoscopy in tumor mouse models, resulting in specific visualization of tumors as small as submillimeter in size that could hardly detected by naked eyes because of their poor contrast against normal tissues. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.

A fluorescent pH probe for acidic organelles in living cells.

PubMed

Chen, Jyun-Wei; Chen, Chih-Ming; Chang, Cheng-Chung

2017-09-26

A water-soluble pH sensor, 2-(6-(4-aminostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N, N-dimethylethanamine (ADA), was synthesized based on the molecular design of photoinduced electron transfer (PET) and intramolecular charge transfer (ICT). The fluorescence emission response against a pH value is in the range 3-6, which is suitable for labelling intracellular pH-dependent microenvironments. After biological evolution, ADA is more than a pH biosensor because it is also an endocytosis pathway tracking biosensor that labels endosomes, late endosomes, and lysosome pH gradients. From this, the emissive aggregates of ADA and protonated-ADA in these organs were evaluated to explore how this probe stresses emission colour change to cause these unique cellular images.

;Turn-on; fluorescent probe detection of Ca2 + ions and applications to bioimaging

NASA Astrophysics Data System (ADS)

Zhang, Huifang; Yin, Caixia; Liu, Tao; Zhang, Yongbin; Huo, Fangjun

2017-06-01

Ca2 + is intracellular divalent cation with the largest concentration variations and involved in many biological phenomena and often acted as a second messenger in signaling pathway. Therefore, the development of probes for specific Ca2 + detection is of great importance. Herein, a novel turn-on fluorescent probe for the detection of Ca2 + in MeCN-aqueous medium was designed and synthesized. The probe displayed responses to Ca2 + with a fluorescence enhancement at 525 nm, accompanying with a distinct fluorescence change from nearly colorless to bright yellow-green. Besides, the probe exhibited a rapid signal response time (within 25 s), a good linearity range and a lower detection limit (2.70 × 10- 7 M). In addition, the ability of the probe to detect Ca2 + in living cells (HeLa cells) via an enhancement of the fluorescence has also been demonstrated.

Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species

PubMed Central

Needham, Lisa-Maria; Weber, Judith; Fyfe, James W. B.; Kabia, Omaru M.; Do, Dung T.; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M.; Ghandi, Sonia; Bohndiek, Sarah E.; Snaddon, Thomas N.

2018-01-01

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease. PMID:29515860

Fluorescent and colorimetric ion probes based on conjugated oligopyrroles.

PubMed

Ding, Yubin; Tang, Yunyu; Zhu, Weihong; Xie, Yongshu

2015-03-07

Metal ions and anions play important roles in many industrial and biochemical processes, and thus it is highly desired to detect them in the relevant systems. Small organic molecule based sensors for selective and sensitive detection of target ions show the advantages of low cost, high sensitivity and convenient implementation. In this area, pyrrole has incomparable advantages. It can be easily incorporated into linear and macrocyclic conjugated structures such as dipyrrins, porphyrins, and N-confused porphyrins, which may utilize the imino N and amino NH moieties for binding metal ions and anions, respectively. In this tutorial review, we focus on representative examples to describe the design, syntheses, sensing mechanisms, and applications of the conjugated oligopyrroles. These compounds could be used as colorimetric or fluorescent ion probes, with the advantages of vivid colour and fluorescence changes, easy structural modification and functionalization, and tunable emission wavelengths. Compared with normal porphyrins, simple di- and tripyrrins, as well as some porphyrinoids are more suitable for designing fluorescence "turn-on" metal probes, because they may exhibit flexible confirmations, and metal coordination will improve the rigidity, resulting in vivid fluorescence enhancement. It is noteworthy that the oligopyrrolic moieties may simultaneously act as the binding unit as well as the reporting moiety, which simplifies the design and syntheses of the probes.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes.

PubMed

Harrington, Walter N; Haji, Mwafaq R; Galanzha, Ekaterina I; Nedosekin, Dmitry A; Nima, Zeid A; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S; Zharov, Vladimir P

2016-11-08

Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

NASA Astrophysics Data System (ADS)

Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

2016-11-01

Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

PubMed Central

Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

2016-01-01

Photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light–dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy. PMID:27824110

FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface.

PubMed

Swiatkowska, Angelika; Kosman, Joanna; Juskowiak, Bernard

2016-01-05

Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na(+) and K(+)). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

PubMed

Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

2017-08-17

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery In Vivo

PubMed Central

2015-01-01

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications. PMID:25184440

Ultrasensitive near-infrared fluorescence-enhanced probe for in vivo nitroreductase imaging.

PubMed

Li, Yuhao; Sun, Yun; Li, Jiachang; Su, Qianqian; Yuan, Wei; Dai, Yu; Han, Chunmiao; Wang, Qiuhong; Feng, Wei; Li, Fuyou

2015-05-20

Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.

Active-site-matched fluorescent probes for rapid and direct detection of vicinal-sulfydryl-containing peptides/proteins in living cells.

PubMed

Pan, Xiaohong; Liang, Ziye; Li, Jing; Wang, Shanshan; Kong, Fanpeng; Xu, Kehua; Tang, Bo

2015-01-26

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hybridization-based biosensor containing hairpin probes and use thereof

DOEpatents

Miller, Benjamin L.; Strohsahl, Christopher M.

2010-10-12

A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

Direct probing of chromatography columns by laser-induced fluorescence

NASA Astrophysics Data System (ADS)

McGuffin, V. L.

1992-12-01

This report summarizes the progress and accomplishments of this research project from 1 Sep. 1989 to 28 Feb. 1993. During this period, we have accomplished all of the primary scientific objectives of the research proposal: (1) constructed and evaluated a laser-induced fluorescence detection system that allows direct examination of the chromatographic column, (2) examined nonequilibrium processes that occur upon solute injection and elution, (3) examined solute retention in liquid chromatography as a function of temperature and pressure, (4) examined solute zone dispersion in liquid chromatography as a function of temperature and pressure, and (5) developed appropriate theoretical models to describe these phenomena. In each of these studies, substantial knowledge has been gained of the fundamental processes that are responsible for chromatographic separations. In addition to these primary research objectives, we have made significant progress in three related areas: (1) examined pyrene as a fluorescent polarity probe in supercritical fluids and liquids as a function of temperature and pressure, (2) developed methods for the class-selective identification of polynuclear aromatic hydrocarbons in coal-derived fluids by microcolumn liquid chromatography with fluorescence quenching detection, and (3) developed methods for the determination of saturated and unsaturated (including omega-3) fatty acids in fish oil extracts by microcolumn liquid chromatography with laser-induced fluorescence detection. In these studies, the advanced separation and detection techniques developed in our laboratory are applied to practical problems of environmental and biomedical significance.

A turn-on supramolecular fluorescent probe for sensing benzimidazole fungicides and its application in living cell imaging

NASA Astrophysics Data System (ADS)

Tang, Qing; Zhang, Jing; Sun, Tao; Wang, Cheng-Hui; Huang, Ying; Zhou, Qingdi; Wei, Gang

2018-02-01

A cucurbit[8]uril-based turn-on supramolecular fluorescent probe between cucurbit[8]uril (Q[8]) and pyronine Y (PyY) (designated 2PyY@Q[8]) in acidic aqueous solution showed a remarkable fluorescence 'turn-on' response to benzimidazole fungicides such as thiabendazole, fuberidazole and carbendazim. The 2PyY@Q[8] fluorescent probe can be used to detect benzimidazole fungicides with high sensitivity and selectivity with a detection limit of 10- 8 mol/L. A good linear relationship of emission intensity at 580 nm for benzimidazole fungicides at concentrations of 0.4-5.0 μmol/L was observed. The proposed sensing mechanism was investigated using 1H NMR spectroscopy combined with density functional theory calculations at the B3LYP/6-31G(d) level. The cell imaging study showed that the 2PyY@Q[8] complex could be used to image benzimidazole fungicide in prostate cancer (PC3) cells, which may help to elucidate relevant biological processes at the molecular level.

Fast and Selective Two-Stage Ratiometric Fluorescent Probes for Imaging of Glutathione in Living Cells.

PubMed

Gong, Deyan; Han, Shi-Chong; Iqbal, Anam; Qian, Jing; Cao, Ting; Liu, Wei; Liu, Weisheng; Qin, Wenwu; Guo, Huichen

2017-12-19

Two fluorescent, m-nitrophenol-substituted difluoroboron dipyrromethene dyes have been designed by nucleophilic substitution reaction of 3,5-dichloro-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). Nonsymmetric and symmetric probes, that is. BODIPY 1 (with one nitrophenol group at the position 3) and BODIPY 2 (with two nitrophenol groups at the positions 3 and 5) were applied to ratiometric fluorescent glutathione detection. The detection is based on the two-step nucleophilic aromatic substitution of the nitrophenol groups of the probes by glutathione in buffer solution containing CTAB. In the first stage, probe 1 showed ratiometric fluorescent color change from green (λ em = 530 nm) to yellow (λ em = 561 nm) because of monosubstitution with glutathione (I 561nm /I 530nm ). Addition of excess glutathione caused the second stage of ratiometric fluorescent color change from yellow to reddish orange (λ em = 596 nm, I 596nm /I 561nm ) due to disubstitution with glutathione. Therefore, different concentration ranges of glutathione (from less to excess) could be rapidly detected by the two-stage ratiometric fluorescent probe 1 in 5 min. While, probe 2 shows single-stage ratiometric fluorescent detection to GSH (from green to reddish orange, I 596nm /I 535nm ). Probes 1 and 2 exhibit excellent properties with sensitive, specific colorimetric response and ratiometric fluorescent response to glutathione over other sulfur nucleophiles. Application to cellular ratiometric fluorescence imaging indicated that the probes were highly responsive to intracellular glutathione.

A near-infrared fluorescent probe for rapid detection of carbon monoxide in living cells.

PubMed

Yan, Liqiang; Nan, Ding; Lin, Cheng; Wan, Yi; Pan, Qiang; Qi, Zhengjian

2018-09-05

A near-infrared (NIR) and colorimetric fluorescent probe system was developed for Carbon Monoxide (CO) via a Pd 0 -mediated Tsuji-Trost reaction. In this probe, phenoxide anion formation (DPCO - ) was acted as the signal unit and an allyl carbonate group was used as the recognition unit. This non-fluorescent probe molecule can release the relevant fluorophore after conversion of Pd 2+ to Pd 0 by CO. The probe system including probe 1 and Pd 2+ can be used for "naked-eye" detection of CO, and exhibited high selectivity to CO over various other sensing objects. More importantly, the probe system has great potential for fluorescence imaging of intracellular CO in living cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Sprayable enzyme-activatable fluorescent probes: kinetic mapping using dynamic fluorescence imaging can help detecting tiny cancer foci (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka

2017-02-01

Optical fluorescence-guided imaging is increasingly used to guide surgery and endoscopic procedures. Sprayable enzyme-activatable probes are particularly useful because of high target-to-background ratios that increase sensitivity for tiny cancer foci. However, green fluorescent activatable probes suffers from interference from autofluorescence found in biological tissue. Dynamic imaging followed by the kinetic analysis could be detected local enzyme activity and used to differentiate specific fluorescence arising from an activated probe in a tumor from autofluorescence in background tissues especially when low concentrations of the dye are applied to detect tiny cancer foci. Serial fluorescence imaging was performed using various concentrations of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) which was sprayed on the peritoneal surface with tiny implants of SHIN3-dsRed ovarian cancer tumors. Temporal differences in signal between specific green fluorescence in cancer foci and non-specific autofluorescence in background tissue was measured and processed into three kinetic maps reflecting maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC), respectively. Especially at lower concentrations, kinetic maps derived from dynamic fluorescence imaging were clearly superior to unprocessed images for detection small cancer foci.

Advances in development of fluorescent probes for detecting amyloid-β aggregates.

PubMed

Xu, Ming-Ming; Ren, Wen-Ming; Tang, Xi-Can; Hu, You-Hong; Zhang, Hai-Yan

2016-06-01

With accumulating evidence suggesting that amyloid-β (Aβ) deposition is a good diagnostic biomarker for Alzheimer's disease (AD), the discovery of active Aβ probes has become an active area of research. Among the existing imaging methods, optical imaging targeting Aβ aggregates (fibrils or oligomers), especially using near-infrared (NIR) fluorescent probes, is increasingly recognized as a promising approach for the early diagnosis of AD due to its real time detection, low cost, lack of radioactive exposure and high-resolution. In the past decade, a variety of fluorescent probes have been developed and tested for efficiency in vitro, and several probes have shown efficacy in AD transgenic mice. This review classifies these representative probes based on their chemical structures and functional modes (dominant solvent-dependent mode and a novel solvent-independent mode). Moreover, the pharmaceutical characteristics of these representative probes are summarized and discussed. This review provides important perspectives for the future development of novel NIR Aβ diagnostic probes.

Advances in development of fluorescent probes for detecting amyloid-β aggregates

PubMed Central

Xu, Ming-ming; Ren, Wen-ming; Tang, Xi-can; Hu, You-hong; Zhang, Hai-yan

2016-01-01

With accumulating evidence suggesting that amyloid-β (Aβ) deposition is a good diagnostic biomarker for Alzheimer's disease (AD), the discovery of active Aβ probes has become an active area of research. Among the existing imaging methods, optical imaging targeting Aβ aggregates (fibrils or oligomers), especially using near-infrared (NIR) fluorescent probes, is increasingly recognized as a promising approach for the early diagnosis of AD due to its real time detection, low cost, lack of radioactive exposure and high-resolution. In the past decade, a variety of fluorescent probes have been developed and tested for efficiency in vitro, and several probes have shown efficacy in AD transgenic mice. This review classifies these representative probes based on their chemical structures and functional modes (dominant solvent-dependent mode and a novel solvent-independent mode). Moreover, the pharmaceutical characteristics of these representative probes are summarized and discussed. This review provides important perspectives for the future development of novel NIR Aβ diagnostic probes. PMID:26997567

A new azine derivative colorimetric and fluorescent dual-channel probe for cyanide detection

NASA Astrophysics Data System (ADS)

Yu, Bin; Li, Chun-Yu; Sun, Yin-Xia; Jia, Hao-Ran; Guo, Jian-Qiang; Li, Jing

2017-09-01

A novel azine derivative colorimetric and fluorescent dual-channel probe salicylaldehyde hydrazine-3,5-dibromosalicylaldehyde (1) has been designed, synthesized and characterized. The probe 1 is confirmed to have especial selectivity and good sensitivity on detecting CN- via UV-vis absorption and fluorescence spectrum in aqueous solution (H2O/DMSO, 1:4, v/v). This colorimetric and fluorescent dual-channel probe response to CN- owed to the deprotonation process and established the mechanism by using 1H NMR spectroscopy. Further researches showed that the detection limit of the probe 1 to CN- anions is 8.01 × 10- 9 M, significantly lower than the maximum level 1.9 × 10- 6 M in potable water from WHO guidelines.

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging.

PubMed

Hudson, Devin A; Caplan, Jeffrey L; Thorpe, Colin

2018-02-20

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD + /NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD + /NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

An active fluorescent probe based on aggregation-induced emission for intracellular bioimaging of Zn2+ and tracking of interactions with single-stranded DNA.

PubMed

Wen, Xiaoye; Wang, Qi; Fan, Zhefeng

2018-07-12

A novel dual-sensing fluorescence probe L was designed and synthesized for highly selective and sensitive detection of Zn 2+ and DNA. The probe L achieved a detection limit of 3.8 nM for Zn 2+ , which is lower than the acceptable level of Zn 2+ in living cells. The probe L displayed high selectivity toward Zn 2+ over other interference metal ions and amino acids. Moreover, the probe L displayed low cytotoxicity and good cell permeability, indicating its potential for detecting and bio-imaging of Zn 2+ . In addition, the probe L-Zn 2+ exhibited enhanced fluorescence signal for DNA detection through the metal-coordination interaction between Zn 2+ and DNA. The enhanced signal is higher than that of the classical ethidium bromide probe. The experiments in aqueous media verified the feasibility of applying probe L in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Egawa, Takahiro; Kobayashi, Chiaki; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Ikegaya, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-10-01

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Cu-free clickable fluorescent probe for intracellular targeting of small biomolecules.

PubMed

Yamagishi, Kento; Sawaki, Kazuaki; Murata, Atsushi; Takeoka, Shinji

2015-05-07

We synthesized a novel cyclooctyne-based clickable fluorescent probe with versatile properties such as high cell-membrane permeability and free diffusibility in the cell. Our probe "FC-DBCO" was conjugated to an azide-modified mannose via a Cu-free click reaction in living HeLa cells and displayed intracellular specific fluorescence imaging with low background signals.

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

PubMed Central

Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

2015-01-01

Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

Development of tumor-targeted near infrared probes for fluorescence guided surgery.

PubMed

Kelderhouse, Lindsay E; Chelvam, Venkatesh; Wayua, Charity; Mahalingam, Sakkarapalayam; Poh, Scott; Kularatne, Sumith A; Low, Philip S

2013-06-19

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications.

PubMed

Jiang, Xiqian; Wang, Lingfei; Carroll, Shaina L; Chen, Jianwei; Wang, Meng C; Wang, Jin

2018-02-16

The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 00, 000-000.

Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements

PubMed Central

Lim, Liang; Nichols, Brandon; Rajaram, Narasimhan; Tunnell, James W.

2011-01-01

Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0 < 9 mN∕mm2) effects are within 0 ± 10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0 ± 5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects. Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra. PMID:21280899

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

PubMed Central

2011-01-01

Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of

Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M. Uljana; Cao, Haishi

2013-01-15

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Copper nanoclusters as probes for turn-on fluorescence sensing of L-lysine.

PubMed

Zhang, Mingming; Qiao, Juan; Zhang, Shufeng; Qi, Li

2018-05-15

Herein, a unique protocol based on copper nanoclusters (CuNCs) probe for turn-on fluorescence sensing of L-lysine was developed. The fluorescent CuNCs with ovalbumin as the stabilizer was prepared by a simple, one-step and green method. When 370 nm was used as the excitation wavelength, the resultant CuNCs exhibited a pale blue fluorescence with the maximum emission at 440 nm. Interestingly, existence of L-lysine evoked the obvious fluorescence intensity increase of CuNCs. The detection limit of the proposed method for L-lysine was 5.5 μM, with a good linear range from 10.0 μM to 1.0 mM (r 2 = 0.999). Moreover, the possible mechanism for enhanced fluorescence intensity of CuNCs by addition of L-lysine was explored and discussed briefly. Further, the as-prepared fluorescent CuNCs was successfully applied in detection of L-lysine in urine. Our results demonstrated that L-lysine could be monitored by the probe, providing new path for construction of CuNCs as fluorescent probes and showing great potential in quantification of L-lysine in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultrahigh resolution multicolor colocalization of single fluorescent probes

DOEpatents

Weiss, Shimon; Michalet, Xavier; Lacoste, Thilo D.

2005-01-18

A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

Fluorescent probes for the simultaneous detection of multiple analytes in biology.

PubMed

Kolanowski, Jacek L; Liu, Fei; New, Elizabeth J

2018-01-02

Many of the key questions facing cellular biology concern the location and concentration of chemical species, from signalling molecules to metabolites to exogenous toxins. Fluorescent sensors (probes) have revolutionised the understanding of biological systems through their exquisite sensitivity to specific analytes. Probe design has focussed on selective sensors for individual analytes, but many of the most pertinent biological questions are related to the interaction of more than one chemical species. While it is possible to simultaneously use multiple sensors for such applications, data interpretation will be confounded by the fact that sensors will have different uptake, localisation and metabolism profiles. An alternative solution is to instead use a single probe that responds to two analytes, termed a dual-responsive probe. Recent progress in this field has yielded exciting probes, some of which have demonstrated biological application. Here we review work that has been carried out to date, and suggest future research directions that will harness the considerable potential of dual-responsive fluorescent probes.

A ratiometric fluorescent probe for rapid and sensitive detection of biothiols in fetal bovine serum.

PubMed

Wang, Fengyang; Feng, Chongchong; Lu, Linlin; Xu, Zhiai; Zhang, Wen

2017-07-01

Herein, a ratiometric turn-on fluorescent probe for sensitive detection of biothiols was designed. The probe consisted of two parts: one was rhodamine B serving as a fluorescence reference, and the other was coumarin derivative as the responsive fluorophore with an acrylate group for biothiols recognition. The response was based on the mechanism of Michael addition and intramolecular cyclization reaction, and the probe showed ratiometric and sensitive response to biothiols. Especially, the detection limit of this probe for cysteine was found to be 0.13μΜ. More importantly, the probe showed the advantage of fast response, of which the fluorescence intensity can reach the maximum within 10min. The ratiometric fluorescent probe has been successfully applied for the determination of biothiols in fetal bovine serum samples and the result was in good agreement with that tested by Ellman method. Copyright © 2017. Published by Elsevier B.V.

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

PubMed

Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

2015-02-11

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging

PubMed Central

2008-01-01

With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially “lighting up” after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated. PMID:19090700

Near-field fluorescence thermometry using highly efficient triple-tapered near-field optical fiber probe.

PubMed

Fujii, T; Taguchi, Y; Saiki, T; Nagasaka, Y

2012-12-01

A novel local temperature measurement method using fluorescence near-field optics thermal nanoscopy (Fluor-NOTN) has been developed. Fluor-NOTN enables nanoscale temperature measurement in situ by detecting the temperature-dependent fluorescence lifetime of CdSe quantum dots (QDs). In this paper, we report a novel triple-tapered near-field optical fiber probe that can increase the temperature measurement sensitivity of Fluor-NOTN. The performance of the proposed probe was numerically evaluated by the finite difference time domain method. Due to improvements in both the throughput and collection efficiency of near-field light, the sensitivity of the proposed probe was 1.9 times greater than that of typical double-tapered probe. The proposed shape of the triple-tapered core was successfully fabricated utilizing a geometrical model. The detected signal intensity of dried layers of QDs was greater by more than two orders than that of auto-fluorescence from the fiber core. In addition, the near-field fluorescence lifetime of the QDs and its temperature dependence were successfully measured by the fabricated triple-tapered near-field optical fiber probe. These measurement results verified the capability of the proposed triple-tapered near-field optical fiber probe to improve the collection efficiency of near-field fluorescence.

Analysis of chemical equilibrium of silicon-substituted fluorescein and its application to develop a scaffold for red fluorescent probes.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Takayanagi, Toshio; Toki, Yuko; Egawa, Takahiro; Kamiya, Mako; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Yoshida, Kengo; Uchiyama, Masanobu; Nagano, Tetsuo; Urano, Yasuteru

2015-09-01

Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10' position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pKa inversion, i.e., pKa1 > pKa2. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4' and 5' positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase.

Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation.

PubMed

He, Huaizhen; Zhan, Yingzhuan; Zhang, Yanmin; Zhang, Jie; He, Langchong

2012-01-01

Two novel taspine diphenyl derivatives (Ta-dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta-dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF-7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.

A red-emitting indolium fluorescence probe for membranes - flavonoids interactions.

PubMed

Gao, Qingyun; Liu, Han; Ding, Qiongjie; Du, Jinya; Liu, Chunlin; Yang, Wei; Shen, Ping; Yang, Changying

2018-05-01

The red-emitting indolium derivative compound (E)-2-(4-(diphenylamino)styryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (H3) was demonstrated as a sensitive membrane fluorescence probe. The probe located at the interface of liposomes when mixed showed much fluorescence enhancement by inhibiting the twisted intramolecular charge transfer state. After ultrasonic treatment, it penetrated into lipid bilayers with the emissions leveling off and a rather large encapsulation efficiency (71.4%) in liposomes. The ζ-potential and particle size measurement confirmed that the charged indolium group was embedded deeply into lipid bilayers. The probe was then used to monitor the affinities of antioxidant flavonoids for membranes. It was verified that quercetin easily interacted with liposomes and dissociated the probe from the internal lipid within 60 s under the condition of simply mixing. The assessment of binding affinities of six flavonoids and the coincident results with their antioxidation activities indicated that it was a promising membrane probe for the study of drug bio-affinities. Copyright © 2018 John Wiley & Sons, Ltd.

Detection of nitrite based on fluorescent carbon dots by the hydrothermal method with folic acid

NASA Astrophysics Data System (ADS)

Lin, Haitao; Ding, Liyun; Zhang, Bingyu; Huang, Jun

2018-05-01

A fluorescent carbon dots probe for the detection of aqueous nitrite was fabricated by a one-pot hydrothermal method, and the transmission electron microscope, X-ray diffractometer, UV-Vis absorption spectrometer and fluorescence spectrophotometer were used to study the property of carbon dots. The fluorescent property of carbon dots influenced by the concentration of aqueous nitrite was studied. The interaction between the electron-donating functional groups and the electron-accepting nitrous acid could account for the quenching effect on carbon dots by adding aqueous nitrite. The products of the hydrolysis of aqueous nitrite performed a stronger quenching effect at lower pH. The relationship between the relative fluorescence intensity of carbon dots and the concentration of nitrite was described by the Stern-Volmer equation (I0/I - 1 = 0.046[Q]) with a fine linearity (R2 = 0.99). The carbon dots-based probe provides a convenient method for the detection of nitrite concentration.

Truxene-cored π-expanded triarylborane dyes as single- and two-photon fluorescent probes for fluoride.

PubMed

Yuan, Mao-Sen; Wang, Qi; Wang, Wenji; Wang, Dong-En; Wang, Junru; Wang, Jinyi

2014-03-21

Fluoride anion (F(-)) significantly affects chemical, biological, and environmental processes. Fluoride recognition and detection have received increasing attention. Convenient, effective, and sensitive fluorescent probes for F(-) should urgently be designed and synthesized. In this study, we describe a strategy for constructing two triarylborane-based fluoride fluorescent probes: 2,7,12-tri(2-(5-(dimesitylboryl)thiophen-2-yl)ethynyl)-5,5',10,10',15,15'-hexaethyltruxene (C3B3) with π-3A (acceptor) configuration and 2,7-di(N,N-diphenylamino)-12-(5-(dimesitylboryl)thiophen-2-yl)-5,5',10,10',15,15'-hexaethyltruxene (N2SB) with 2D (donor)-π-A configuration. The loss of color of the tetrahydrofuran solution of these probes from greenish yellow suggests that they can conveniently monitor F(-) at a low concentration (10 μM) free of apparatus. The different structural features of these probes varied their fluorescent responses to F(-). The single-photon fluorescence intensity of C3B3 declined to 90% upon the addition of 4.5 equivalents of F(-) to its tetrahydrofuran solution. However, the single-photon fluorescence intensity of N2SB was enhanced six-fold upon addition of 2.5 equivalents of the F(-). Under the experimental conditions, the detection limits of the two probes for F(-) can reach 12-13 μM (C3B3) and 3-5 μM (N2SB). The ability of the two probes in detecting F(-) in their toluene solutions in the two-photon mode was also investigated. The sensitive two-photon fluorescence responses of both probes make them excellent two-photon fluorescence probes.

Sensitive determination of endogenous hydroxyl radical in live cell by a BODIPY based fluorescent probe.

PubMed

Lei, Kepeng; Sun, Mingtai; Du, Libo; Zhang, Xiaojie; Yu, Huan; Wang, Suhua; Hayat, Tasawar; Alsaedi, Ahmed

2017-08-01

The sensitive and selective fluorescence probe for hydroxyl radical analysis is of significance because hydroxyl radical plays key roles in many physiological and pathological processes. In this work, a novel organic fluorescence molecular probe OHP for hydroxyl radical is synthesized by a two-step route. The probe employs 4-bora-3a,4a-diaza-s-indacene (difluoroboron dipyrromethene, BODIPY) as the fluorophore and possesses relatively high fluorescence quantum yields (77.14%). Hydroxyl radical can rapidly react with the probe and quench the fluorescence in a good linear relationship (R 2 =0.9967). The limit of detection is determined to be as low as 11nM. In addition, it has been demonstrated that the probe has a good stability against pH and light illumination, low cytotoxicity and high biocompatibility. Cell culture experimental results show that the probe OHP is sensitive and selective for imaging and tracking endogenous hydroxyl radical in live cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly selective and rapidly responsive fluorescent probe for hydrogen sulfide detection in wine.

PubMed

Wang, Hao; Wang, Jialin; Yang, Shaoxiang; Tian, Hongyu; Liu, Yongguo; Sun, Baoguo

2018-08-15

A new fluorescent probe 6-(2, 4-dinitrophenoxy)-2-naphthonitrile (probe 1) was designed and synthesized for the selective detection of hydrogen sulfide (H 2 S). The addition of H 2 S to a solution of probe 1 resulted in a marked fluorescence turn-on alongside a visual color change from colorless to light yellow. Importantly, this distinct color response indicated that probe 1 could be used as a visual sensor for H 2 S. Moreover, probe 1 was successfully used as a signal tool to determine the H 2 S levels in beer and red wine. Copyright © 2018 Elsevier Ltd. All rights reserved.

BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme.

PubMed

Raut, Sangram L; Rich, Ryan; Fudala, Rafal; Kokate, R; Kimball, J D; Borejdo, Julian; Vishwanatha, Jamboor K; Gryczynski, Zygmunt; Gryczynski, Ignacy

2014-01-01

Although BSA Au clusters fluoresce in red region (λmax: 650 nm), they are of limited use due to low fluorescence quantum yield (~6%). Here we report an enhanced fluorescence imaging application of fluorescent bio-nano probe BSA Au clusters using multipulse excitation scheme. Multipulse excitation takes advantage of long fluorescence lifetime (> 1 µs) of BSA Au clusters and enhances its fluorescence intensity 15 times over short lived cellular auto-fluorescence. Moreover we have also shown that by using time gated detection strategy signal (fluorescence of BSA Au clusters) to noise (auto-fluorescence) ratio can be increased by 30 fold. Thereby with multipulse excitation long lifetime probes can be used to develop biochemical assays and perform optical imaging with zero background.

Spectroscopic study of fluorescent probes based on G-quadruplex oligonucleotides labeled with ethynylpyrenyldeoxyuridine.

PubMed

Switalska, Angelika; Kierzek, Ryszard; Dembska, Anna; Juskowiak, Bernard

2017-12-01

The design, synthesis, and spectral properties of four pyrene labeled oligonucleotide probes with G-quadruplex structure (Tel22-Tpy, Tel22-Upy, Tel22-6Upy, Tel22-18Upy) based on the 22-mer human telomeric sequence (Tel22) have been reported. Pyrene labels in the form of ethynylpyrenyldeoxyuridine have been inserted efficiently into oligodeoxynucleotides probes using phosphoramidite chemistry. The probes exhibited abilities to fold into G-quadruplex structures and to bind metal cations (Na + and K + ). Folding properties of probes and their spectral behavior were examined by recording the UV-vis, fluorescence, and CD spectra as well as by analyzing melting profiles. Fluorescence characteristics and G-quadruplex folding of probes were also studied at the interface of cationic dioctadecyldimethylammonium bromide (DODAB) monolayer. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. Copyright © 2017 Elsevier B.V. All rights reserved.

Hemoglobin detection using carbon dots as a fluorescence probe.

PubMed

Barati, Ali; Shamsipur, Mojtaba; Abdollahi, Hamid

2015-09-15

Herein, we have described the application of high fluorescent carbon dots (CDs) without any surface modification as a simple and fast responding fluorescence probe for sensitive and selective determination of hemoglobin (Hb) in the presence of H2O2. Although Hb itself was able to quench the fluorescence of CDs, based on the inner filter effect (IFE) of the protein that affects both excitation and emission spectra of CDs, the presence of H2O2 resulted in further improvement of the sensitivity of Hb detection. The assay is based on the reaction of Hb with H2O2 that generates reactive oxygen species including hydroxyl (OH•) and superoxide (O2(•-)) radicals under heme degradation and/or iron release from Hb and the subsequent reaction of hydroxyl radicals, as strong oxidizing agents, with CDs resulting in high fluorescence quenching. The proposed probe was used for determination of Hb in concentration range of 1-100 nM with a detection limit of 0.4 nM. The method was successfully applied to the determination of Hb in human blood samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Femtomolar level sensing of inorganic arsenic(III) in water and in living-systems using a non-toxic fluorescent probe.

PubMed

Dey, Biswajit; Mukherjee, Priyanka; Mondal, Ranjan Kumar; Chattopadhyay, Asoke Prasun; Hauli, Ipsit; Mukhopadhyay, Subhra Kanti; Fleck, Michel

2014-12-14

A highly selective femtomolar level sensing of inorganic arsenic(III) as arsenious acid has been accomplished in water medium and in living-systems (on pollen grains of Tecoma stans; Candida albicans cells (IMTECH No. 3018) and Peperomia pellucida stem section) using a non-toxic fluorescent probe of a Cu(II)-complex.

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

PubMed Central

González-Vera, Juan A.; Morris, May C.

2015-01-01

Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes. PMID:28248276

A rhodamine chromene-based turn-on fluorescence probe for selectively imaging Cu2+ in living cell

NASA Astrophysics Data System (ADS)

Liu, Wei-Yong; Li, Hai-Ying; Lv, Hong-Shui; Zhao, Bao-Xiang; Miao, Jun-Ying

We describe the development of a rhodamine chromene-based turn-on fluorescence probe to monitor the intracellular Cu2+ level in living cells. The new fluorescent probe with a chlorine group in chromene moiety exhibits good membrane-permeable property than previous reported because the predicted lipophilicity of present probe 4 is stronger than that of methoxyl substituted probe in our previous work (CLogP of 4: 8.313, CLogP of methoxyl substituted probe: 7.706), and a fluorescence response toward Cu2+ under physiological conditions with high sensitivity and selectivity, and facilitates naked-eye detection of Cu2+. The fluorescence intensity was remarkably increased upon the addition of Cu2+ within 1 or 2 min, while the other sixteen metal ions caused no significant effect.

Dual-Reactable Fluorescent Probes for Highly Selective and Sensitive Detection of Biological H2 S.

PubMed

Wei, Chao; Wang, Runyu; Zhang, Changyu; Xu, Guoce; Li, Yanyan; Zhang, Qiang-Zhe; Li, Lu-Yuan; Yi, Long; Xi, Zhen

2016-05-06

Hydrogen sulfide (H2 S) is an important endogenous signaling molecule with a variety of biological functions. Development of fluorescent probes for highly selective and sensitive detection of H2 S is necessary. We show here that dual-reactable fluorescent H2 S probes could react with higher selectivity than single-reactable probes. One of the dual-reactable probes gives more than 4000-fold turn-on response when reacting with H2 S, the largest response among fluorescent H2 S probes reported thus far. In addition, the probe could be used for high-throughput enzymatic assays and for the detection of Cys-induced H2 S in cells and in zebrafish. These dual-reactable probes hold potential for highly selective and sensitive detection of H2 S in biological systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

PubMed

Okamura, Yukio; Watanabe, Yuichiro

2006-01-01

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

A rhodamine-based fluorescent probe for colorimetric and fluorescence lighting-up determination of toxic thiophenols in environmental water and living cells.

PubMed

Wu, Juanjuan; Ye, Zhuo; Wu, Feng; Wang, Hongying; Zeng, Lintao; Bao, Guang-Ming

2018-05-01

Thiophenols are a class of highly toxic environmental pollutant, hence it is very necessary to monitor thiophenols in environment and living cells with an efficient and reliable method. Herein, a novel fluorescent probe for thiophenols has been developed, which exhibited a colorimetric and fluorescence turn-on dual response towards thiophenols with good selectivity and fast response. The sensing mechanism for thiophenols was attributed to nucleophilic substitution reaction, which was confirmed by HPLC. The probe exhibited good recovery (from 90% to 107%) and low limit of detection for thiophenols (37nM) in industrial wastewater. Moreover, the probe has been successfully employed to visualize thiophenol in living cells. Therefore, the fluorescent probe has good capability for monitoring thiophenols in environmental samples and biological systems. Copyright © 2018 Elsevier B.V. All rights reserved.

The Mechanisms and Biomedical Applications of an NIR BODIPY-Based Switchable Fluorescent Probe

PubMed Central

Cheng, Bingbing; Bandi, Venugopal; Yu, Shuai; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Tang, Liping; Yuan, Baohong

2017-01-01

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing. PMID:28208666

A cancer cell-specific fluorescent probe for imaging Cu2 + in living cancer cells

NASA Astrophysics Data System (ADS)

Wang, Chao; Dong, Baoli; Kong, Xiuqi; Song, Xuezhen; Zhang, Nan; Lin, Weiying

2017-07-01

Monitoring copper level in cancer cells is important for the further understanding of its roles in the cell proliferation, and also could afford novel copper-based strategy for the cancer therapy. Herein, we have developed a novel cancer cell-specific fluorescent probe for the detecting Cu2 + in living cancer cells. The probe employed biotin as the cancer cell-specific group. Before the treatment of Cu2 +, the probe showed nearly no fluorescence. However, the probe can display strong fluorescence at 581 nm in response to Cu2 +. The probe exhibited excellent sensitivity and high selectivity for Cu2 + over the other relative species. Under the guidance of biotin group, could be successfully used for detecting Cu2 + in living cancer cells. We expect that this design strategy could be further applied for detection of the other important biomolecules in living cancer cells.

Preparation of a Nile Red-Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo.

PubMed

Liu, Keyin; Kong, Xiuqi; Ma, Yanyan; Lin, Weiying

2018-05-01

Carbon monoxide (CO) is a key gaseous signaling molecule in living cells and organisms. This protocol illustrates the synthesis of a highly sensitive Nile Red (NR)-Pd-based fluorescent probe, NR-PdA, and its applications for detecting endogenous CO in tissue culture cells, ex vivo organs, and zebrafish embryos. In the NR-PdA synthesis process, 3-diethylamine phenol reacts with sodium nitrite in the acidic condition to afford 5-(diethylamino)-2-nitrosophenol hydrochloride (compound 1), which is further treated with 1-naphthalenol at a high temperature to provide the NR dye via a cyclization reaction. Finally, NR is reacted with palladium acetate to obtain the desired Pd-based fluorescent probe NR-PdA. NR-PdA possesses excellent two-photon excitation and near-IR emission properties, high stability, low background fluorescence, and a low detection limit. In addition to the chemical synthesis procedures, we provide step-by-step procedures for imaging endogenous CO in RAW 264.7 cells, mouse organs ex vivo, and live zebrafish embryos. The synthesis process for the probe requires ∼4 d, and the biological imaging experiments take ∼14 d.

Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

2010-08-17

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes

PubMed Central

Klymchenko, Andrey S.; Duportail, Guy; Mély, Yves; Demchenko, Alexander P.

2003-01-01

The principle of electrochromic modulation of excited-state intramolecular proton-transfer reaction was applied for the design of fluorescence probes with high two-color sensitivity to dipole potential, Ψd, in phospholipid bilayers. We report on the effect of Ψd variation on excitation and fluorescence spectra of two new 3-hydroxyflavone probes, which possess opposite orientations of the fluorescent moiety in the lipid bilayer. The dipole potential in the bilayer was modulated by the addition of 6-ketocholestanol or phloretin and by substitution of dimyristoyl phosphatidylcholine lipid with its ether analog 1,2-di-o-tetradecyl-sn-glycero-3-phosphocholine, and its value was estimated by the reference styryl dye 1-(3-sulfonatopropyl)-4-{β[2-(di-n-octylamino)-6-naphthyl]vinyl}pyridinium betaine. We demonstrate that after Ψd changes, the probe orienting in the bilayer similarly to the reference dye shows similar shifts in the excitation spectra, whereas the probe with the opposite orientation shows the opposite shifts. The new observation is that the response of 3-hydroxyflavone probes to Ψd in excitation spectra is accompanied by and quantitatively correlated with dramatic changes of relative intensities of the two well separated emission bands that belong to the initial normal and the product tautomer forms of the excited-state intramolecular proton-transfer reaction. This provides a strong response to Ψd by change in emission color. PMID:12972636

A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography

NASA Astrophysics Data System (ADS)

Wu, Changfeng; Barnhill, Hannah; Liang, Xiaoping; Wang, Qian; Jiang, Huabei

2005-11-01

A fluorescent probe based on bionanoparticle cowpea mosaic virus has been developed for near-infrared fluorescence tomography. A unique advantage of this probe is that over 30 dye molecules can be loaded onto each viral nanoparticle with an average diameter of 30 nm, making high local dye concentration (∼1.8 mM) possible without significant fluorescence quenching. This ability of high loading of local dye concentration would increase the signal-to-noise ratio considerably, thus sensitivity for detection. We demonstrate successful tomographic fluorescence imaging of a target containing the virus-dye nanoparticles embedded in a tissue-like phantom. Tomographic fluorescence data were obtained through a multi-channel frequency-domain system and the spatial maps of fluorescence quantum yield were recovered with a finite-element-based reconstruction algorithm.

Fluorescent probes for nanoscopy: four categories and multiple possibilities.

PubMed

Ni, Ming; Zhuo, Shuangmu; So, Peter T C; Yu, Hanry

2017-01-01

Nanoscopy enables breaking down the light diffraction limit and reveals the nanostructures of objects being studied using light. In 2014, three scientists pioneered the development of nanoscopy and won the Nobel Prize in Chemistry. This recognized the achievement of the past twenty years in the field of nanoscopy. However, fluorescent probes used in the field of nanoscopy are still numbered. Here, we review the currently available four categories of probes and existing methods to improve the performance of probes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes.

PubMed

Jin, Xilang; Hao, Likai; She, Mengyao; Obst, Martin; Kappler, Andreas; Yin, Bing; Liu, Ping; Li, Jianli; Wang, Lanying; Shi, Zhen

2015-01-01

Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study

NASA Astrophysics Data System (ADS)

Singh, T. Sanjoy; Mitra, Sivaprasad

2011-03-01

Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern-Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant ( κq), Stern-Volmer constant ( KSV) and also the ligand-protein association constant ( Ka). The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 10 4 dm 3 mol -1. In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure.

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells.

PubMed

Zhang, Jingtuo; Li, Cong; Dutta, Colina; Fang, Mingxi; Zhang, Shuwei; Tiwari, Ashutosh; Werner, Thomas; Luo, Fen-Tair; Liu, Haiying

2017-05-22

A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a d-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with K m  = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrates that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescent probes for understanding soil water repellency: the novel application of a chemist's tool to soil science

NASA Astrophysics Data System (ADS)

Balshaw, Helen M.; Davies, Matthew L.; Doerr, Stefan H.; Douglas, Peter

2015-04-01

Food security and production is one of the key global issues faced by society. It has become essential to work the land efficiently, through better soil management and agronomy whilst protecting the environment from air and water pollution. The failure of soil to absorb water - soil water repellency can lead to major environmental problems such as increased overland flow and soil erosion, poor uptake of agricultural chemicals, and increased risk of groundwater pollution due to the rapid transfer of contaminants and nutrient leaching through uneven wetting and preferential flow pathways. Understanding the causes of soil hydrophobicity is essential for the development of effective methods for its amelioration, supporting environmental stability and food security. Organic compounds deposited on soil mineral or aggregate surfaces have long been recognised as a major factor in causing soil water repellency. It is widely accepted that the main groups of compounds responsible are long-chain acids, alkanes and other organic compounds with hydrophobic properties. However, when reapplied to sands and soils, the degree of water repellency induced by these compounds and mixtures varied widely with compound type, amount, and mixture, in a seemingly unpredictable way. Fluorescent and phosphorescent probes are widely used in chemistry and biochemistry due to their sensitive response to their physical and chemical environment, such as polarity, and viscosity. However, they have to-date not been used to study soil water repellency. Here we present preliminary work on the evaluation of fluorescent probes as tools to study two poorly understood features that determine the degree of wettability for water repellent soils: (i) the distribution of organics on soils; (ii) the changes in polarity at soil surfaces required for water drops to infiltrate. In our initial work we have examined probes adsorbed onto model soils, prepared by adsorption of specific organics onto acid washed sand

Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection.

PubMed

Li, Cheng-Hung; Kuo, Tsung-Rong; Su, Hsin-Jan; Lai, Wei-Yun; Yang, Pan-Chyr; Chen, Jinn-Shiun; Wang, Di-Yan; Wu, Yi-Chun; Chen, Chia-Chun

2015-10-28

Recent development of molecular imaging probes for fluorescence-guided surgery has shown great progresses for determining tumor margin to execute the tissue resection. Here we synthesize the fluorescent gold nanoparticles conjugated with diatrizoic acid and nucleolin-targeted AS1411 aptamer. The nanoparticle conjugates exhibit high water-solubility, good biocompatibility, visible fluorescence and strong X-ray attenuation for computed tomography (CT) contrast enhancement. The fluorescent nanoparticle conjugates are applied as a molecular contrast agent to reveal the tumor location in CL1-5 tumor-bearing mice by CT imaging. Furthermore, the orange-red fluorescence emitting from the conjugates in the CL1-5 tumor can be easily visualized by the naked eyes. After the resection, the IVIS measurements show that the fluorescence signal of the nanoparticle conjugates in the tumor is greatly enhanced in comparison to that in the controlled experiment. Our work has shown potential application of functionalized nanoparticles as a dual-function imaging agent in clinical fluorescence-guided surgery.

Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection

PubMed Central

Li, Cheng-Hung; Kuo, Tsung-Rong; Su, Hsin-Jan; Lai, Wei-Yun; Yang, Pan-Chyr; Chen, Jinn-Shiun; Wang, Di-Yan; Wu, Yi-Chun; Chen, Chia-Chun

2015-01-01

Recent development of molecular imaging probes for fluorescence-guided surgery has shown great progresses for determining tumor margin to execute the tissue resection. Here we synthesize the fluorescent gold nanoparticles conjugated with diatrizoic acid and nucleolin-targeted AS1411 aptamer. The nanoparticle conjugates exhibit high water-solubility, good biocompatibility, visible fluorescence and strong X-ray attenuation for computed tomography (CT) contrast enhancement. The fluorescent nanoparticle conjugates are applied as a molecular contrast agent to reveal the tumor location in CL1-5 tumor-bearing mice by CT imaging. Furthermore, the orange-red fluorescence emitting from the conjugates in the CL1-5 tumor can be easily visualized by the naked eyes. After the resection, the IVIS measurements show that the fluorescence signal of the nanoparticle conjugates in the tumor is greatly enhanced in comparison to that in the controlled experiment. Our work has shown potential application of functionalized nanoparticles as a dual-function imaging agent in clinical fluorescence-guided surgery. PMID:26507179

A novel polymer probe for Zn(II) detection with ratiometric fluorescence signal

NASA Astrophysics Data System (ADS)

Diao, Haipeng; Guo, Lixia; Liu, Wen; Feng, Liheng

2018-05-01

A conjugated polymer probe comprised of fluorene, quinolone and benzothiazole units was designed and synthesized by the Suzuki coupling reaction. Through the studies of photophysical and thermal properties, the polymer displays blue-emitting feature and good thermal stability. A ratiometric fluorescence signal of the probe for Zn(II) was observed in ethanol with a new emission peak at 555 nm. The probe possesses a high selectivity and sensitivity for Zn(II) during familiar metal ions in ethanol. The detection limit of the probe for Zn (II) is up to 10-8 mol/L. The electron distributions of the polymer before and after bonding with Zn (II) were investigated by the Gaussian 09 software, which agreed with the experimental results. Noticeably, based on the color property of the probe with Zn(II), a series of color test paper were developed for visual detecting Zn(II) ions. This work helps to provide a platform or pattern for the development of polymer fluorescence probe in the chemosensor field.

Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and π-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells.

PubMed

Wang, Jianbo; Xia, Shuai; Bi, Jianheng; Fang, Mingxi; Mazi, Wafa; Zhang, Yibin; Conner, Nathan; Luo, Fen-Tair; Lu, H Peter; Liu, Haiying

2018-04-18

In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp 2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

PubMed Central

Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

2012-01-01

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541

Boronate-Based Fluorescent Probes: Imaging Hydrogen Peroxide in Living Systems

PubMed Central

Lin, Vivian S.; Dickinson, Bryan C.; Chang, Christopher J.

2014-01-01

Hydrogen peroxide, a reactive oxygen species with unique chemical properties, is produced endogenously in living systems as a destructive oxidant to ward off pathogens or as a finely tuned second messenger in dynamic cellular signaling pathways. In order to understand the complex roles that hydrogen peroxide can play in biological systems, new tools to monitor hydrogen peroxide in its native settings, with high selectivity and sensitivity, are needed. Knowledge of organic synthetic reactivity provides the foundation for the molecular design of selective, functional hydrogen peroxide probes. A palette of fluorescent and luminescent probes that react chemoselectively with hydrogen peroxide has been developed, utilizing a boronate oxidation trigger. These indicators offer a variety of colors and in cellulo characteristics and have been used to examine hydrogen peroxide in a number of experimental setups, including in vitro fluorometry, confocal fluorescence microscopy, and flow cytometry. In this chapter, we provide an overview of the chemical features of these probes and information on their behavior to help researchers select the optimal probe and application. PMID:23791092

Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe.

PubMed

Zhang, Ruilong; Zhao, Jun; Han, Guangmei; Liu, Zhengjie; Liu, Cui; Zhang, Cheng; Liu, Bianhua; Jiang, Changlong; Liu, Renyong; Zhao, Tingting; Han, Ming-Yong; Zhang, Zhongping

2016-03-23

Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.

A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

PubMed

Ma, Junbao; Li, Wenqi; Li, Juanjuan; Shi, Rongguang; Yin, Gui; Wang, Ruiyong

2018-05-15

A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

PubMed

Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

2012-07-03

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective

On the Development, Characterization, and Use of Protein Fluorescence and Infrared Spectroscopic Probes

NASA Astrophysics Data System (ADS)

Hilaire, Mary Rose

Proteins possess unique physical and chemical properties that allow them to carry out a wide variety of biological activities and functions. While it is generally understood that a protein's function is dictated by its structure and dynamics, arriving at a molecule-level understanding of the underlying structure-dynamics-function relationship still poses a challenging task in many cases. This is due, at least in part, to the fact that we lack the ability to take snapshots along the reaction coordinate of proteins with sufficient temporal and structural resolution. Therefore, to improve one's ability to acquire site-specific structural and/or environmental information of proteins via either infrared (IR) or fluorescence spectroscopy, the main focus of this thesis is to develop and characterize amino acid-based spectroscopic probes as well as to use such probes to study important biological questions. Specifically, we show that (1) p-cyanophenylalanine and selenomethionine constitute an efficient fluorophore-quencher pair, useful for characterizing protein conformational changes that occur on a short distance; (2) 4-cyanotryptophan is a novel blue fluorescent amino acid, applicable for biological imaging due to its unique photophysical properties; (3) the dielectric constant inside the hydrophobic interior of staphylococcal nuclease is about 10-15, significantly larger than previously assumed; and (4) a single mutation in a short segment of the protein transthyretin (i.e., 110-115) induces formation of amyloid fibrils consisting of both beta- and alpha-sheets, where the latter is a proposed structure in proteins, but has never been observed previously.

Fluorescent sensors based on boronic acids

NASA Astrophysics Data System (ADS)

Cooper, Christopher R.; James, Tony D.

1999-05-01

Sensor systems have long been needed for detecting the presence in solution of certain chemically or biologically important species. Sensors are used in a wide range of applications from simple litmus paper that shows a single color change in acidic or basic environments to complex biological assays that use enzymes, antibodies and antigens to display binding events. With this work the use of boronic acids in the design and synthesis of sensors for saccharides (diols) will be presented. The fluorescent sensory systems rely on photoinduced electron transfer (PET) to modulate the observed fluorescence. When saccharides form cyclic boronate esters with boronic acids, the Lewis acidity of the boronic acid is enhanced and therefore the Lewis acid-base interaction between the boronic acid and a neighboring amine is strengthened. The strength of this acid-base interaction modulates the PET from the amine (acting as a quencher) to anthracene (acting as a fluorophore). These compounds show increased fluorescence at neutral pH through suppression of the PET from nitrogen to anthracene on saccharide binding. The general strategy for the development of saccharide selective systems will be discussed. The potential of the boronic acid based systems will be illustrated using the development of glucose and glucosamine selective fluorescent sensors as examples.

Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems.

PubMed

Yan, Yuling; Marriott, M Emma; Petchprayoon, Chutima; Marriott, Gerard

2011-02-01

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

Development of fluorescent probes based on protection-deprotection of the key functional groups for biological imaging.

PubMed

Tang, Yonghe; Lee, Dayoung; Wang, Jiaoliang; Li, Guanhan; Yu, Jinghua; Lin, Weiying; Yoon, Juyoung

2015-08-07

Recently, the strategy of protection-deprotection of functional groups has been widely employed to design fluorescent probes, as the protection-deprotection of functional groups often induces a marked change in electronic properties. Significant advances have been made in the development of analyte-responsive fluorescent probes based on the protection-deprotection strategy. In this tutorial review, we highlight the representative examples of small-molecule based fluorescent probes for bioimaging, which are operated via the protection-deprotection of key functional groups such as aldehyde, hydroxyl, and amino functional groups reported from 2010 to 2014. The discussion includes the general protection-deprotection methods for aldehyde, hydroxyl, or amino groups, as well as the design strategies, sensing mechanisms, and deprotection modes of the representative fluorescent imaging probes applied to bio-imaging.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

2012-01-01

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

Highly sensitive low-background fluorescent probes for imaging of nitric oxide in cells and tissues.

PubMed

Zhang, Hui-Xian; Chen, Jian-Bo; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan

2014-03-18

Small-molecule fluorescent probes in combination with fluorescent microscopy can be a powerful tool to provide real-time detection and high spatiotemporal resolution of transient molecules in cells and bodies. For the design of fluorescent probes for transient molecule imaging, high detection sensitivity is crucial. In this report, two new fluorescent probes, 8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-H) and 8-(3,4-diaminophenyl)-1,7-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-M), have been developed for nitric oxide (NO) imaging. The detection sensitivity has been efficiently improved by use of these probes through increasing NO detection signals and decreasing background fluorescence. Fluorescence in the far-red region is enhanced by 400- and 550-fold after reaction with NO is achieved and remains stable for at least 24 h under the irradiation of xenon lamp. Excitation and emission wavelengths longer than 600 nm and excellent intracellular retention of these probes and their NO products create dark background inside and outside cells and tissues. What is more, the excellent intracellular retention of these compounds is obtained by their strong lipophilicity, which is a novel design concept diametrically opposite to the traditional approaches. The high sensitivity and dark background make DANPBO-H and DANPBO-M competitive for NO imaging in cells and tissues. The lipophilicity-based intracellular retention mechanism as a design strategy has great potential in the development of fluorescent probes for bioimaging.

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor.

PubMed

Zhou, Zhixue; Du, Yan; Dong, Shaojun

2011-07-01

Double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) at low concentration of CuSO(4), and the formed Cu NPs have excellent fluorescence, whereas a single-strand DNA (ssDNA) template does not support Cu NPs' formation. This property of dsDNA-Cu NPs makes it suitable for DNA sensing. However, exploration of dsDNA-Cu NPs applied in biological analysis is still at an early stage. In this regard, we report herein for the first time a sensitive, cost-effective, and simple aptamer sensor (aptasensor) using dsDNA-Cu NPs as fluorescent probe. The design consists of a dsDNA with reporter DNA (here, aptamer) as template for the formation of Cu NPs, and the formed dsDNA-Cu NPs show high fluorescence. Using adenosine triphosphate (ATP) as a model analyte, the introduction of ATP triggers the structure switching of reporter DNA to form aptamer-ATP complex, causing the destruction of the double helix and thus no formation of the Cu NPs, resulting in low fluorescence. The preferable linear range (0.05-500 μM), sensitivity (LOD 28 nM), and simplicity for the detection of ATP indicate that dsDNA-Cu NPs may have great prospects in the field of biological analysis. We also use this novel fluorescent probe to determine ATP in 1% human serum and human adenocarcinoma HeLa cells. The dsDNA-Cu NPs probes provide recovery of 104-108% in 1% human serum and a prominent fluorescent signal is obtained in cellular ATP assay, revealing the practicality of using dsDNA-Cu NPs for the determination of ATP in real samples. Besides, this design is simply based on nucleic acid hybridization, so it can be generally applied to other aptamers for label-free detection of a broad range of analytes. Successful detection of cocaine with detection limit of 0.1 μM demonstrates its potential to be a general method.

Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

PubMed

Liu, Liwei; Lin, Guimiao; Yin, Feng; Law, Wing-Cheung; Yong, Ken-Tye

2016-04-01

Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells. © 2016 Wiley Periodicals, Inc.

Fluorescent probes for exploring plant cell wall deconstruction: a review.

PubMed

Paës, Gabriel

2014-07-03

Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

A large stokes-shifted fluorescent dye synthesized as a new probe for the determination of protein.

PubMed

Lin, Dayong; Fei, Xuening; Li, Ran; Gu, Yingchun; Tang, Yalin; Zhou, Jianguo; Zhang, Baolian

2016-07-01

A novel fluorescent dye, 1-(2-hydroxyethyl)-4-((E)-2-(3-benzothiazol-2yl-9-ethyl-carbazole-3yl)vinyl) pyridinium bromide, was synthesized for determination of protein and its structure was characterized by (1)H NMR. Photophysics of the new probe in different solvents has been delineated in this paper, the new fluorescent molecular dye exhibited a large stokes-shifted and fluorescence quantum yields in organic solvent. The photostability and thermostability of the new dye were also studied and the results suggested the stable was excellent. The interactions of the dye with bovine serum albumin (BSA) , Human serumal bumin (HSA) and calf thymus deoxyribonucleic acid (ctDNA) were studied by fluorescence and absorption spectroscopy. The binding constant for BSA, HSA and DNA were calculated to be 8.91 × 10(7), 1.86 × 10(6) and 2.9 × 10(4), respectively. The experimental results indicated a potential value of the new dye for biomarker.

Detection of reactive oxygen species in mainstream cigarette smoke by a fluorescent probe

NASA Astrophysics Data System (ADS)

Liu, Li; Xu, Shi-jie; Li, Song-zhan

2009-07-01

A mass of reactive oxygen species(ROS) are produced in the process of smoking. Superfluous ROS can induce the oxidative stress in organism, which will cause irreversible damage to cells. Fluorescent probe is taken as a marker of oxidative stress in biology and has been applied to ROS detection in the field of biology and chemistry for high sensitivity, high simplicity of data collection and high resolution. As one type of fluorescent probe, dihydrorhodamine 6G (dR6G) will be oxidized to the fluorescent rhodamine 6G, which could be used to detect ROS in mainstream cigarette smoke. We investigated the action mechanism of ROS on dR6G, built up the standard curve of R6G fluorescence intensity with its content, achieved the variation pattern of R6G fluorescence intensity with ROS content in mainstream cigarette smoke and detected the contents of ROS from the 4 types of cigarettes purchased in market. The result shows that the amount of ROS has close relationship with the types of tobacco and cigarette production technology. Compared with other detecting methods such as electronic spin resonance(ESR), chromatography and mass spectrometry, this detection method by the fluorescent probe has higher efficiency and sensitivity and will have wide applications in the ROS detection field.

Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid

PubMed Central

Kalstrup, Tanja; Blunck, Rikard

2013-01-01

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions. PMID:23630265

Fluorescent Probes and Selective Inhibitors for Biological Studies of Hydrogen Sulfide- and Polysulfide-Mediated Signaling.

PubMed

Takano, Yoko; Echizen, Honami; Hanaoka, Kenjiro

2017-10-01

Hydrogen sulfide (H 2 S) plays roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. Also, hydropersulfide (R-S-SH) and polysulfide (-S-S n -S-) have recently been identified as reactive sulfur species (RSS) that regulate the bioactivities of multiple proteins via S-sulfhydration of cysteine residues (protein Cys-SSH) and show cytoprotection. Chemical tools such as fluorescent probes and selective inhibitors are needed to establish in detail the physiological roles of H 2 S and polysulfide. Recent Advances: Although many fluorescent probes for H 2 S are available, fluorescent probes for hydropersulfide and polysulfide have only recently been developed and used to detect these sulfur species in living cells. In this review, we summarize recent progress in developing chemical tools for the study of H 2 S, hydropersulfide, and polysulfide, covering fluorescent probes based on various design strategies and selective inhibitors of H 2 S- and polysulfide-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase), and we summarize their applications in biological studies. Despite recent progress, the precise biological functions of H 2 S, hydropersulfide, and polysulfide remain to be fully established. Fluorescent probes and selective inhibitors are effective chemical tools to study the physiological roles of these sulfur molecules in living cells and tissues. Therefore, further development of a broad range of practical fluorescent probes and selective inhibitors as tools for studies of RSS biology is currently attracting great interest. Antioxid. Redox Signal. 27, 669-683.

Blood interference in fiber-optical based fluorescence guided resection of glioma using 5-aminolevulinic acid

NASA Astrophysics Data System (ADS)

Haj-Hosseini, Neda; Lowndes, Shannely; Salerud, Göran; Wårdell, Karin

2011-03-01

Fluorescence guidance in brain tumor resection is performed intra-operatively where bleeding is included. When using fiber-optical probes, the transmission of light to and from the tissue is totally or partially blocked if a small amount of blood appears in front of the probe. Sometimes even after rinsing with saline, the remnant blood cells on the optical probe head, disturb the measurements. In such a case, the corresponding spectrum cannot be reliably quantified and is therefore discarded. The optimal case would be to calculate and take out the blood effect systematically from the collected signals. However, the first step is to study the pattern of blood interference in the fluorescence spectrum. In this study, a fiber-optical based fluorescence spectroscopy system with a laser excitation light of 405 nm (1.4 J/cm2) was used during fluorescence guided brain tumor resection using 5-aminolevulinic acid (5-ALA). The blood interference pattern in the fluorescence spectrum collected from the brain was studied in two patients. The operation situation was modeled in the laboratory by placing blood drops from the finger tip on the skin of forearm and the data was compared to the brain in vivo measurements. Additionally, a theoretical model was developed to simulate the blood interference pattern on the skin autofluorescence. The blood affects the collected fluorescence intensity and leaves traces of oxy and deoxy-hemoglobin absorption peaks. According to the developed theoretical model, the autofluorescence signal is considered to be totally blocked by an approximately 500 μm thick blood layer.

Single Probe for Imaging and Biosensing of pH, Cu(2+) Ions, and pH/Cu(2+) in Live Cells with Ratiometric Fluorescence Signals.

PubMed

Han, Yingying; Ding, Changqin; Zhou, Jie; Tian, Yang

2015-01-01

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu(2+), to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu(2+), and pH/Cu(2+) with different ratiometric fluorescent signals. First, 2,2',2″-(2,2',2″-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-yl-methyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu(2+), as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu(2+), and pH/Cu(2+) excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu(2+) against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu(2+) and in understanding the interplaying roles of Cu(2+) and pH in live cells by ratiometric multicolor fluorescent imaging.

The Development of Fluorescent Probes for Visualizing Intracellular Hydrogen Polysulfides.

PubMed

Chen, Wei; Rosser, Ethan W; Matsunaga, Tetsuro; Pacheco, Armando; Akaike, Takaaki; Xian, Ming

2015-11-16

Endogenous hydrogen polysulfides (H2Sn; n>1) have been recognized as important regulators in sulfur-related redox biology. H2Sn can activate tumor suppressors, ion channels, and transcription factors with higher potency than H2S. Although H2Sn are drawing increasing attention, their exact mechanisms of action are still poorly understood. A major hurdle in this field is the lack of reliable and convenient methods for H2Sn detection. Herein we report a H2Sn-mediated benzodithiolone formation under mild conditions. This method takes advantage of the unique dual reactivity of H2Sn as both a nucleophile and an electrophile. Based on this reaction, three fluorescent probes (PSP-1, PSP-2, and PSP-3) were synthesized and evaluated. Among the probes prepared, PSP-3 showed a desirable off/on fluorescence response to H2Sn and high specificity. The probe was successfully applied in visualizing intracellular H2Sn. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

PubMed

Rocha, Danilo D; Espejo, Vinson R; Rainier, Jon D; La Clair, James J; Costa-Lotufo, Letícia V

2015-09-01

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus. Copyright © 2015 Elsevier Inc. All rights reserved.

Determination of phenformin hydrochloride employing a sensitive fluorescent probe.

PubMed

Shi, Lin; Xie, Jian-Hong; Du, Li-Ming; Chang, Yin-xia; Wu, Hao

2016-06-05

A complexation of non-fluorescent phenformin hydrochloride (PFH) with cucurbit [7]uril (CB [7]) in aqueous solution was investigated using the fluorescent probe of palmatine (PAL) coupled with CB [7]. The fluorescent probe of CB [7]-PAL exhibited strong fluorescence in aqueous solution, which was quenched gradually with the increase of PFH. This effect is observed because when PFH was added to the host-guest system of CB [7]-PAL, PFH and PAL competed to occupy the CB [7] cavity. Portions of the PAL molecule were expelled from the CB [7] cavity owing to the introduction of PFH. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescence method of high sensitivity and selectivity was developed to determine PFH with good precision and accuracy for the first time. The linear range of the method was 0.005-1.9 μg mL(-1) with a detection limit of 0.003 μg mL(-1). In this work, association constants (K) of PFH with CB [7] were also determined. KCB [7]-PFH=(2.52±0.05)×10(5) L mol(-1). The ability of PFH to bind with CB [7] is stronger than that of PAL. The results of a density functional theory calculation authenticated that the moiety of PFH was embedded in the hydrophobic cavity of CB [7] tightly, and the nitrogen atom is located in the vicinity of a carbonyl-laced portal in the energy-minimized structure. The molecular modelling of the interaction between PFH and CB [7] was also confirmed by (1)H NMR spectra (Bruker 600 MHz). Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of phenformin hydrochloride employing a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Shi, Lin; Xie, Jian-Hong; Du, Li-Ming; Chang, Yin-xia; Wu, Hao

2016-06-01

A complexation of non-fluorescent phenformin hydrochloride (PFH) with cucurbit [7]uril (CB [7]) in aqueous solution was investigated using the fluorescent probe of palmatine (PAL) coupled with CB [7]. The fluorescent probe of CB [7]-PAL exhibited strong fluorescence in aqueous solution, which was quenched gradually with the increase of PFH. This effect is observed because when PFH was added to the host-guest system of CB [7]-PAL, PFH and PAL competed to occupy the CB [7] cavity. Portions of the PAL molecule were expelled from the CB [7] cavity owing to the introduction of PFH. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescence method of high sensitivity and selectivity was developed to determine PFH with good precision and accuracy for the first time. The linear range of the method was 0.005-1.9 μg mL- 1 with a detection limit of 0.003 μg mL- 1. In this work, association constants (K) of PFH with CB [7] were also determined. KCB [7]-PFH = (2.52 ± 0.05) × 105 L mol- 1. The ability of PFH to bind with CB [7] is stronger than that of PAL. The results of a density functional theory calculation authenticated that the moiety of PFH was embedded in the hydrophobic cavity of CB [7] tightly, and the nitrogen atom is located in the vicinity of a carbonyl-laced portal in the energy-minimized structure. The molecular modelling of the interaction between PFH and CB [7] was also confirmed by 1H NMR spectra (Bruker 600 MHz).

A novel polymer probe for Zn(II) detection with ratiometric fluorescence signal.

PubMed

Diao, Haipeng; Guo, Lixia; Liu, Wen; Feng, Liheng

2018-05-05

A conjugated polymer probe comprised of fluorene, quinolone and benzothiazole units was designed and synthesized by the Suzuki coupling reaction. Through the studies of photophysical and thermal properties, the polymer displays blue-emitting feature and good thermal stability. A ratiometric fluorescence signal of the probe for Zn(II) was observed in ethanol with a new emission peak at 555 nm. The probe possesses a high selectivity and sensitivity for Zn(II) during familiar metal ions in ethanol. The detection limit of the probe for Zn (II) is up to 10 -8  mol/L. The electron distributions of the polymer before and after bonding with Zn (II) were investigated by the Gaussian 09 software, which agreed with the experimental results. Noticeably, based on the color property of the probe with Zn(II), a series of color test paper were developed for visual detecting Zn(II) ions. This work helps to provide a platform or pattern for the development of polymer fluorescence probe in the chemosensor field. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide.

PubMed

Nian, Linge; Hu, Yue; Fu, Caihong; Song, Chen; Wang, Jie; Xiao, Jianxi

2018-01-01

The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG) 8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Facile synthesis of a two-photon fluorescent probe based on pyrimidine 2-isothiocyanate and its application in bioimaging.

PubMed

Yang, Jie; Hu, Wei; Li, Huirong; Hou, Hanna; Tu, Yi; Liu, Bo

2018-04-18

Two-photon microscopy imaging has been widely applied in biological imaging, but the development of two-photon absorption probes is obviously lagging behind in the development of imaging technology. In this paper, a two-photon fluorescent probe (1) based on pyrimidine 2-isothiocyanate has been designed and synthesized through a simple method for two-photon biological imaging. Probe 1 was able to couple effectively with the amino groups on biomolecules. To verify the reactivity of the isothiocyanate group on probe 1 and the amine groups on the biomolecules, d-glucosamine was chosen as a model biomolecule to conjugate with probe 1. The result showed that probe 1 could effectively conjugate with d-glucosamine to synthesize probe 2, and the yield of probe 2 was 83%. After conjugating with d-glucosamine, linear absorption spectra, single-photon fluorescence spectra, and two-photon fluorescence spectra of probes 1 and 2 did not present significant changes. Probes 1 and 2 exhibited high fluorescence quantum yields (0.71-0.79) in toluene and chloroform. They also exhibited different photo-physical properties in solvents with different polarities. The two-photon absorption cross-section of probe 1 was 953 GM in toluene. In addition, probe 1 could be effectively conjugated with transferrin, and the conjugated probe (Tf-1) could be transported into Hep G2 cells through a receptor-mediated process for biological imaging. These results demonstrate that such probes are expected to have great potential applications in two-photon fluorescence bioimaging.

Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

PubMed Central

Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

2016-01-01

Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

ICT-Isomerization-Induced Turn-On Fluorescence Probe with a Large Emission Shift for Mercury Ion: Application in Combinational Molecular Logic.

PubMed

Bhatta, Sushil Ranjan; Mondal, Bijan; Vijaykumar, Gonela; Thakur, Arunabha

2017-10-02

A unique turn-on fluorescent device based on a ferrocene-aminonaphtholate derivative specific for Hg 2+ cation was developed. Upon binding with Hg 2+ ion, the probe shows a dramatic fluorescence enhancement (the fluorescence quantum yield increases 58-fold) along with a large red shift of 68 nm in the emission spectrum. The fluorescence enhancement with a red shift may be ascribed to the combinational effect of C═N isomerization and an extended intramolecular charge transfer (ICT) mechanism. The response was instantaneous with a detection limit of 2.7 × 10 -9 M. Upon Hg 2+ recognition, the ferrocene/ferrocenium redox peak was anodically shifted by ΔE 1/2 = 72 mV along with a "naked eye" color change from faint yellow to pale orange for this metal cation. Further, upon protonation of the imine nitrogen, the present probe displays a high fluorescence output due to suppression of the C═N isomerization process. Upon deprotonation using strong base, the fluorescence steadily decreases, which indicates that H + and OH - can be used to regulate the off-on-off fluorescence switching of the present probe. Density functional theory studies revealed that the addition of acid leads to protonation of the imine N (according to natural bond orbital analysis), and the resulting iminium proton forms a strong H-bond (2.307 Å) with one of the triazole N atoms to form a five-membered ring, which makes the molecule rigid; hence, enhancement of the ICT process takes place, thereby leading to a fluorescence enhancement with a red shift. The unprecedented combination of H + , OH - , and Hg 2+ ions has been used to generate a molecular system exhibiting the INHIBIT-OR combinational logic operation.

A novel ''donor-π-acceptor'' type fluorescence probe for sensing pH: mechanism and application in vivo.

PubMed

Chao, Jianbin; Wang, Huijuan; Zhang, Yongbin; Yin, Caixia; Huo, Fangjun; Song, Kailun; Li, Zhiqing; Zhang, Ting; Zhao, Yaqin

2017-11-01

A novel pH fluorescent probe 1-(pyren-1-yl)-3-(6-methoxypridin-3-yl)-acrylketone, (PMPA), which had a pyrene structure attached to methoxypyridine, was synthesized for monitoring extremely acidic and alkaline pH. The pH titrations indicated that PMPA displayed a remarkable emission enhancement with a pK a of 2.70 and responded linearly to minor pH fluctuations within the extremely acidic range of 1.26-3.97. Interestingly, PMPA also exhibited strong pH-dependent characteristics with pK a 9.32 and linear response to extreme-alkalinity range of 8.54-10.36. In addition, PMPA displayed a good selectivity, excellent photostability and large Stokes shift (167nm). Furthermore, the probe PMPA had excellent cell membrane permeability and was applied successfully to rapidly detect pH in living cells. pH value in these organs was closely related to many diseases, so these findings suggested that the probe had potential application in pH detecting for disease diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly water-soluble BODIPY-based fluorescent probe for sensitive and selective detection of nitric oxide in living cells.

PubMed

Vegesna, Giri K; Sripathi, Srinivas R; Zhang, Jingtuo; Zhu, Shilei; He, Weilue; Luo, Fen-Tair; Jahng, Wan Jin; Frost, Megan; Liu, Haiying

2013-05-22

A highly water-soluble BODIPY dye bearing electron-rich o-diaminophenyl groups at 2,6-positions was prepared as a highly sensitive and selective fluorescent probe for detection of nitric oxide (NO) in living cells. The fluorescent probe displays an extremely weak fluorescence with fluorescence quantum yield of 0.001 in 10 mM phosphate buffer (pH 7.0) in the absence of NO as two electron-rich o-diaminophenyl groups at 2,6-positions significantly quench the fluorescence of the BODIPY dye via photoinduced electron transfer mechanism. The presence of NO in cells enhances the dye fluorescence dramatically. The fluorescent probe demonstrates excellent water solubility, membrane permeability, and compatibility with living cells for sensitive detection of NO.

Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

PubMed

Patra, Digambara

2010-01-15

A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

A fluorescent probe for the efficient discrimination of Cys, Hcy and GSH based on different cascade reactions.

PubMed

Li, Ying; Liu, Weimin; Zhang, Panpan; Zhang, Hongyan; Wu, Jiasheng; Ge, Jiechao; Wang, Pengfei

2017-04-15

A fluorescent probe (1) for distinguishing amongst biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), is developed based on different cascade reactions. The key design feature of fluorescent probe 1 is the integration of two potential reaction groups for the thiol and amino groups of biothiols in one molecule. By reacting with the halogen atom and α, β-unsaturated malonitrile in probe 1, Cys, Hcy and GSH can generate a total of three main products with distinct photophysical properties. Probe 1 shows a strong fluorescence turn-on response to Cys with blue-green emission by using an excitation wavelength of 390nm. At an excitation wavelength of 500nm, probe 1 responds to GSH over Cys and Hcy and emits strong orange fluorescence. The discrimination of biothiols can be demonstrated by cell imaging experiments, indicating that probe 1 can be a useful tool for the selective imaging of Cys and GSH in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Location of fluorescent probes (2'-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) in lipid membrane studied by fluorescence spectroscopy and molecular dynamics simulation.

PubMed

Posokhov, Yevgen; Kyrychenko, Alexander

2018-04-01

2'-Hydroxy derivatives of 2,5-diaryl-1,3-oxazole are known as environment-sensitive ratiometric excited-state intramolecular proton transfer (ESIPT) fluorescent probes, which are used to monitor physicochemical properties of lipid membranes. However, because of their heterogeneous membrane distribution, accurate experimental determination of the probe position is difficult. To estimate the location of the ESIPT probes in lipid membranes we have performed fluorescence measurements and molecular dynamics (MD) simulations. In the series composed of 2-(2'-hydroxy-phenyl)-5-phenyl-1,3-oxazole (1), 2-(2'-hydroxy-phenyl)-5-(4'-biphenyl)-1,3-oxazole (2), and 2-(2'-hydroxy-phenyl)-phenanthro[9,10-d]-1,3-oxazole (3), the structure of the ESIPT-moiety of 2-(2'-hydroxy-phenyl)-oxazole was varied by either aromatic ring substitution or annealing, leading to the systematical increase in the hydrophobic character of the probes. The comparison of the fluorescence behavior of probes 1-3 in a wide variety of solvents with those in phospholipid vesicles revealed that all three probes prefer to reside inside a membrane. Our MD results demonstrate that the probes locate from the glycerol residues and the polar carbonyl groups of phospholipids up to hydrophobic acyl chain units. It has been found that the probe location correlates well with the size of the aromatic moiety, being gradually shifted from 11.1 Å to 7.6 Å from the bilayer center for probes 1 to 3, respectively. Our results may be useful for the design of novel fluorescent probes for fluorescence sensing of specific regions within a lipid membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Ionic calcium determination in skim milk with molecular probes and front-face fluorescence spectroscopy: simple linear regression.

PubMed

Gangidi, R R; Metzger, L E

2006-11-01

The purpose of this study was to determine if the ionic calcium content of skim milk could be determined using molecular probes and front-face fluorescence spectroscopy. Current methods for determining ionic calcium are not sensitive, overestimate ionic calcium, or require complex procedures. Molecular probes designed specifically for measuring ionic calcium could potentially be used to determine the ionic calcium content of skim milk. The goal of the current study was to develop foundation methods for future studies to determine ionic calcium directly in skim milk and other dairy products with molecular probes and fluorescence spectroscopy. In this study, the effect of pH on calcium-sensitive fluorescent probe (Rhod-5N and Fluo-5N) performance using various concentrations of skim milk was determined. The pH of diluted skim milk (1.9 to 8.9% skim milk), was adjusted to either 6.2 or 7.0, after which the samples were analyzed with fluorescent probes (1 microM) and front-face fluorescence spectroscopy. The ionic calcium content of each sample was also determined using a calcium ion-selective electrode. The results demonstrated that the ionic calcium content of each sample was highly correlated (R2 > 0.989) with the fluorescence intensities of the probe-calcium adduct using simple linear regression. Higher than suggested ionic calcium contents of 1,207 and 1,973 microM were determined with the probes (Fluo-5N and Rhod-5N) in diluted skim milk with pH 7.0 and 6.2, respectively. The fluorescence intensity of the probe-calcium adduct decreased with a decrease in pH for the same ionic calcium concentration. This study demonstrates that Fluo-5N and Rhod-5N can be used to determine the ionic-calcium content of diluted milk with front-face fluorescence spectroscopy. Furthermore, these probes may also have the potential to determine the ionic calcium content of undiluted skim milk.

Hydrophobic interactions in donor-disulphide-acceptor (DSSA) probes looking beyond fluorescence resonance energy transfer theory.

PubMed

Sanjeeva, Shilpa Kammaradi; Korrapati, Swathi; Nair, Chandrasekhar B; Rao, P V Subba; Pullela, Phani Kumar; Vijayalakshmi, U; Siva, Ramamoorthy

2014-07-01

Donor-linker-acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of -0.60 V, much lower than the best thiol reductant reported in literature, the DTT (-0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo' has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.

Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications.

PubMed

Kobayashi, Hisataka; Choyke, Peter L

2011-02-15

Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance

Fluorescent Probes and Selective Inhibitors for Biological Studies of Hydrogen Sulfide- and Polysulfide-Mediated Signaling

PubMed Central

Takano, Yoko; Echizen, Honami

2017-01-01

Abstract Significance: Hydrogen sulfide (H2S) plays roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. Also, hydropersulfide (R−S−SH) and polysulfide (−S−Sn−S−) have recently been identified as reactive sulfur species (RSS) that regulate the bioactivities of multiple proteins via S-sulfhydration of cysteine residues (protein Cys−SSH) and show cytoprotection. Chemical tools such as fluorescent probes and selective inhibitors are needed to establish in detail the physiological roles of H2S and polysulfide. Recent Advances: Although many fluorescent probes for H2S are available, fluorescent probes for hydropersulfide and polysulfide have only recently been developed and used to detect these sulfur species in living cells. Critical Issues: In this review, we summarize recent progress in developing chemical tools for the study of H2S, hydropersulfide, and polysulfide, covering fluorescent probes based on various design strategies and selective inhibitors of H2S- and polysulfide-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase), and we summarize their applications in biological studies. Future Directions: Despite recent progress, the precise biological functions of H2S, hydropersulfide, and polysulfide remain to be fully established. Fluorescent probes and selective inhibitors are effective chemical tools to study the physiological roles of these sulfur molecules in living cells and tissues. Therefore, further development of a broad range of practical fluorescent probes and selective inhibitors as tools for studies of RSS biology is currently attracting great interest. Antioxid. Redox Signal. 27, 669–683. PMID:28443673

A Reaction-Based Novel Fluorescent Probe for Detection of Hydrogen Sulfide and Its Application in Wine.

PubMed

Wang, Hao; Wang, Jialin; Yang, Shaoxiang; Tian, Hongyu; Sun, Baoguo; Liu, Yongguo

2018-01-01

A new reaction-based fluorescent probe 6-cyanonaphthalen-2-yl-2,4- dinitrobenzenesulfonate (probe 1) was designed and synthesized for detection of hydrogen sulfide (H 2 S). The addition of H 2 S to a solution of probe 1 resulted in a marked fluorescence increased accompanied by a visual color change from colorless to yellow. Importantly, this distinct color response indicates that probe 1 could be used as a visual tool for detection of H 2 S. H 2 S can be detected quantitatively in the concentration range 0 to 25 μM and the detection limit was 30 nM. Moreover, probe 1 was successfully used as a sensor to determine H 2 S levels in red wine and beer. Fluorescent probe 1 could be employed as a visible sensor for H 2 S. Probe 1 could be used to detect H 2 S quantitatively in food simple. © 2017 Institute of Food Technologists®.

Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore.

PubMed

Chib, Rahul; Mummert, Mark; Bora, Ilkay; Laursen, Bo W; Shah, Sunil; Pendry, Robert; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal

2016-05-01

In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.

The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

PubMed

Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

2014-09-01

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

Probing thermal stability of the β-lactoglobulin-oleic acid complex by fluorescence spectroscopy and molecular modeling

NASA Astrophysics Data System (ADS)

Simion (Ciuciu), Ana-Maria; Aprodu, Iuliana; Dumitrașcu, Loredana; Bahrim, Gabriela Elena; Alexe, Petru; Stănciuc, Nicoleta

2015-09-01

Bovine β-lactoglobulin is able to interact with different bioactive compounds, thus being an important candidate in the development of delivery systems with improved functionality. The heat induced changes in the β-lactoglobulin-oleic acid complex were examined by means of fluorescence spectroscopy and molecular modeling techniques. Fluorescence spectroscopy results indicated a rigid protein structure in the temperature range 25-70 °C, whereas at temperatures over 75 °C, the rearrangements of the polypeptide chains led to higher exposure of hydrophobic residues. The most significant increase of the accessible surface area with temperature increase was identified in case of Tyr99 and Tyr102. The phase diagram method indicated an all or none transition between two conformations. Due to conformational changes, no contact between Ile56 or Lys60 and the fatty acid could be identified at 85 °C, but new non-bonding interaction were established with Ile12 and Val15. The results obtained in this study provide important details about thermal induced changes in the conformation of β-lactoglobulin-oleic acid complex. Significant conformational changes were registered above 75 °C, suggesting the possibility of obtaining highly functional complexes between whey proteins and natural unsaturated fatty acids.

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

PubMed

Liu, Xianjun; Xiang, Meihao; Tong, Zongxuan; Luo, Fengyan; Chen, Wen; Liu, Feng; Wang, Fenglin; Yu, Ru-Qin; Jiang, Jian-Hui

2018-05-01

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 μm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

A novel reaction-based fluorescent probe for the detection of cysteine in milk and water samples.

PubMed

Wang, Jialin; Wang, Hao; Hao, Yanfeng; Yang, Shaoxiang; Tian, Hongyu; Sun, Baoguo; Liu, Yongguo

2018-10-01

A novel fluorescent probe 3'-hydroxy-3-oxo-3H-spiro [isobenzofuran-1,9'-xanthene]-6'-yl-2,4-dinitrobenzenesulfonate (probe 1) was designed and synthesized as a visual sensor for the detection of cysteine levels in milk and water samples. The addition of cysteine to the solution of probe 1 resulted in an increase in fluorescence intensity and color change, from light yellow to yellow-green. The distinct color response indicated that probe 1 could be used as a visual sensor for cysteine. Cysteine can be detected quantitatively at concentrations between 0 and 400 μM and the detection limit of the fluorescence response to the probe was 6.5 μM. This suggests that probe 1 could be used as a signaling tool to determine the cysteine levels in samples, such as milk and water. Copyright © 2018 Elsevier Ltd. All rights reserved.

A G-quadruplex based fluorescent oligonucleotide turn-on probe towards iodides detection in real samples.

PubMed

Li, Qian; Li, Shuaihua; Chen, Xiu; Bian, Liujiao

2017-09-01

A basket-type G-quadruplex (GQ) fluorescent oligonucleotide (OND) probe is designed to detect iodides dependent on thymine-Hg(II)-thymine (T-Hg(II)-T) base pairs and the intrinsic fluorescence quenching capacity of GQ. In the presence of Hg(II) ions (Hg 2+ ), the two hexachloro-fluorescein-labeled ONDs form a hairpin structure and the fluorophores are dragged close to the GQ, leading to fluorescence quenching of the probe due to photoinduced electron transfer. Upon addition of iodide anions, Hg 2+ are extracted from T-Hg(II)-T complexes which attributes to the stronger binding with iodide anions, resulting in the fluorescence recovery. Through performing the fluorescence quenching and recovery processes, this probe developed a fluorescence turn-on sensor for iodide anions determination over a linear range of 20-200nmol/L with a limit of detection of 5nmol/L. The practical use of the turn-on technology was demonstrated by its application in determination of iodides in water, food, pharmaceutical products and biological samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

PubMed

Huang, S K; Cheng, M; Hui, S W

1990-11-01

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

Target-cancer cell specific activatable fluorescence imaging Probes: Rational Design and in vivo Applications

PubMed Central

Kobayashi, Hisataka; Choyke, Peter L.

2010-01-01

CONSPECTUS Conventional imaging methods, such as angiography, computed tomography, magnetic resonance imaging and radionuclide imaging, rely on contrast agents (iodine, gadolinium, radioisotopes) that are “always on”. While these agents have proven clinically useful, they are not sufficiently sensitive because of the inadequate target to background ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, i.e. only “turned on” under certain conditions. These probes can be designed to emit signal only after binding a target tissue, greatly increasing sensitivity and specificity in the detection of disease. There are two basic types of activatable fluorescence probes; 1) conventional enzymatically activatable probes, which exist in the quenched state until activated by enzymatic cleavage mostly outside of the cells, and 2) newly designed target-cell specific activatable probes, which are quenched until activated in targeted cells by endolysosomal processing that results when the probe binds specific cell-surface receptors and is subsequently internalized. Herein, we present a review of the rational design and in vivo applications of target-cell specific activatable probes. Designing these probes based on their photo-chemical (e.g. activation strategy), pharmacological (e.g. biodistribution), and biological (e.g. target specificity) properties has recently allowed the rational design and synthesis of target-cell specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photo-chemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include: self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal

Investigation of Drug-Induced Hepatotoxicity and Its Remediation Pathway with Reaction-Based Fluorescent Probes.

PubMed

Cheng, Dan; Xu, Wang; Yuan, Lin; Zhang, Xiaobing

2017-07-18

Drug-induced liver injury (DILI) is considered a serious problem related to public health, due to its unpredictability and acute response. The level of peroxynitrite (ONOO - ) generated in liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we present two reaction-based fluorescent probes (Naph-ONOO - and Rhod-ONOO - ) for ONOO - through a novel and universally applicable mechanism: ONOO - -mediated deprotection of α-keto caged fluorophores. Among them, Rhod-ONOO - can selectively accumulate and react in mitochondria, one of the main sources of ONOO - , with a substantial lower nanomolar sensitivity of 43 nM. The superior selectivity and sensitivity of two probes enable real-time imaging of peroxynitrite generation in lipopolysaccharide-stimulated live cells, with a remarkable difference from cells doped with other interfering reactive oxygen species, in either one- or two-photon imaging modes. More importantly, we elucidated the drug-induced hepatotoxicity pathway with Rhod-ONOO - and revealed that CYP450/CYP2E1-mediated enzymatic metabolism of acetaminophen leads to ONOO - generation in liver cells. This is the first time to showcase the drug-induced hepatotoxicity pathways by use of a small-molecule fluorescent probe. We hence conclude that fluorescent probes can engender a deeper understanding of reactive species and their pathological revelations. The reaction-based fluorescent probes will be a potentially useful chemical tool to assay drug-induced hepatotoxicity.

Intrinsic Fluorescence as a Spectral Probe for Protein Denaturation Studies in the Presence of Honey

NASA Astrophysics Data System (ADS)

Wong, Y. H.; Kadir, H. A.; Tayyab, S.

2015-11-01

Honey was found to quench the intrinsic fluorescence of bovine serum albumin (BSA) in a concentration dependent manner, showing complete quenching in the presence of 5% (w/v) honey. Increasing the protein concentration up to 5.0 μM did not lead to the recovery of the protein fluorescence. Urea denaturation of BSA, which otherwise shows a two-step, three-state transition, using intrinsic fluorescence of the protein as the probe failed to produce any result in the presence of 5% (w/v) honey. Thus, intrinsic fluorescence cannot be used as a spectral probe for protein denaturation studies in the presence of honey.

Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

PubMed

Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

2014-06-11

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

Dual lanthanide-doped complexes: the development of a time-resolved ratiometric fluorescent probe for anthrax biomarker and a paper-based visual sensor.

PubMed

Wang, Qi-Xian; Xue, Shi-Fan; Chen, Zi-Han; Ma, Shi-Hui; Zhang, Shengqiang; Shi, Guoyue; Zhang, Min

2017-08-15

In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO 2 -Eu/GMP) has been designed for detecting anthrax biomarker (dipicolinic acid, DPA), a unique and major component of anthrax spores. In such complexes-based probe, Tb/DPA@SiO 2 can serve as a stable reference signal with green fluorescence and Eu/GMP act as a sensitive response signal with red fluorescence for ratiometric fluorescent sensing DPA. Additionally, the probe exhibits long fluorescence lifetime, which can significantly reduce the autofluorescence interferences from biological samples by using time-resolved fluorescence measurement. More significantly, a paper-based visual sensor for DPA has been devised by using filter paper embedded with Tb/DPA@SiO 2 -Eu/GMP, and we have proved its utility for fluorescent detection of DPA, in which only a handheld UV lamp is used. In the presence of DPA, the paper-based visual sensor, illuminated by a handheld UV lamp, would result in an obvious fluorescence color change from green to red, which can be easily observed with naked eyes. The paper-based visual sensor is stable, portable, disposable, cost-effective and easy-to-use. The feasibility of using a smartphone with easy-to-access color-scanning APP as the detection platform for quantitative scanometric assays has been also demonstrated by coupled with our proposed paper-based visual sensor. This work unveils an effective method for accurate, sensitive and selective monitoring anthrax biomarker with backgroud-free and self-calibrating properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Challenges and advances in quantum dot fluorescent probes to detect reactive oxygen and nitrogen species: a review.

PubMed

Adegoke, Oluwasesan; Forbes, Patricia B C

2015-03-03

The pathological and physiological effects of reactive oxygen and nitrogen species (ROS/RNS) have instigated increasing awareness in the scientific field with respect to the development of suitable probes for their detection. Among the various probes developed to date, semiconductor quantum dots (QDs) fluorescent probes have attracted significant attention. The unfavourable properties of ROS/RNS with respect to their detection, such as their short lifetimes and the competitive presence of various endogenous reactive species, capable of interfering with the probe in biological matrices, have hindered the effective performance of most probes as well as complicating the design of suitable probes. The development of novel QD fluorescent probes capable of circumventing these problems is thus, of scientific interest. In this review, we highlight the challenges faced, pros and cons and published developments to date, with respect to QD fluorescent probes for ROS/RNS such as H2O2, O2(·-), ·OH, HOCl, NO and ONOO(-). Copyright © 2014 Elsevier B.V. All rights reserved.

Reaction-based small-molecule fluorescent probes for chemoselective bioimaging

PubMed Central

Chan, Jefferson; Dodani, Sheel C.; Chang, Christopher J.

2014-01-01

The dynamic chemical diversity of elements, ions and molecules that form the basis of life offers both a challenge and an opportunity for study. Small-molecule fluorescent probes can make use of selective, bioorthogonal chemistries to report on specific analytes in cells and in more complex biological specimens. These probes offer powerful reagents to interrogate the physiology and pathology of reactive chemical species in their native environments with minimal perturbation to living systems. This Review presents a survey of tools and tactics for using such probes to detect biologically important chemical analytes. We highlight design criteria for effective chemical tools for use in biological applications as well as gaps for future exploration. PMID:23174976

Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives.

PubMed

Restrepo-Ortiz, C X; Merbt, S N; Barrero-Canossa, J; Fuchs, B M; Casamayor, E O

2018-04-28

The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient, and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments. Copyright © 2018 Elsevier GmbH. All rights reserved.

Silver ions-mediated conformational switch: facile design of structure-controllable nucleic acid probes.

PubMed

Wang, Yongxiang; Li, Jishan; Wang, Hao; Jin, Jianyu; Liu, Jinhua; Wang, Kemin; Tan, Weihong; Yang, Ronghua

2010-08-01

Conformationally constraint nucleic acid probes were usually designed by forming an intramolecular duplex based on Watson-Crick hydrogen bonds. The disadvantages of these approaches are the inflexibility and instability in complex environment of the Watson-Crick-based duplex. We report that this hydrogen bonding pattern can be replaced by metal-ligation between specific metal ions and the natural bases. To demonstrate the feasibility of this principle, two linear oligonucleotides and silver ions were examined as models for DNA hybridization assay and adenosine triphosphate detection. The both nucleic acids contain target binding sequences in the middle and cytosine (C)-rich sequences at the lateral portions. The strong interaction between Ag(+) ions and cytosines forms stable C-Ag(+)-C structures, which promises the oligonucleotides to form conformationally constraint formations. In the presence of its target, interaction between the loop sequences and the target unfolds the C-Ag(+)-C structures, and the corresponding probes unfolding can be detected by a change in their fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using Ag(+) ion complexes instead of traditional Watson-Crick-based duplex. In particular, the intrinsic feature of the metal-ligation motif facilitates the design of functional nucleic acids probes by independently varying the concentration of Ag(+) ions in the medium.

Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.

PubMed

Cabrera, Mynthia; Natarajan, Jayakumar; Paguio, Michelle F; Wolf, Christian; Urbach, Jeffrey S; Roepe, Paul D

2009-10-13

Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.

[Fluorescence property of a chemical probe for naked-eye and detection of Fe3+].

PubMed

Song, Yu-Min; Ma, Xin-Xian; Yang, Wu

2012-12-01

A higher selective and sensitive probe for the detection of Fe(III) in aqueous media was made using 2,4-Diisocyanatotoluene (TDI) as a bridge to couple Fe3 O4 nanoparticles(NPs) and Rhodamine-6G hydrazide. The characterization of composite materials with Infrared spectra(IR), Thermal Gravimetric analysis(TGA) and Transmission Emission Microscopy(TEM) points to the graft of Rhodamine-6G hydrazide onto the surface of the Fe3O4. The obvious color change of the probe solution from light grey to pink upon the addition of Fe3+ demonstrated the probe could be used for "naked-eye" detection of Fe3+ in water at pH 7. The presence of 1 equivalent (10 micromol x L(-1) microm) of each of these metal ions, including Mn2+, Ni2+, Y2+, Eu3+, Ce3+, La3+, Pr3+, Cd2+, Cr3+, Sm3+, Fe2+, Cu2+ and Zn2+ ions, did not demonstrate any obvious fluorescence change of the probe water solution, which confirmed the probe was a probe with remarkable selectivity for Fe3+. And the fluorescence images of HeLa cells in physiological solutions after incubation with Fe3+ and then further incubated with the probe leading to a strong intracellular fluorescence, which suggested the probe could penetrate the HeLa cell membrane and could respond to Fe3+ in intracellular within living cells.

Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells.

PubMed

Liu, Yuan-Yuan; Wu, Ming; Zhu, Li-Na; Feng, Xi-Zeng; Kong, De-Ming

2015-06-01

pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water-soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH-dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH-sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1-8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing phospholipase a(2) with fluorescent phospholipid substrates.

PubMed

Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

2007-09-03

The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.

Dopamine-functionalized InP/ZnS quantum dots as fluorescence probes for the detection of adenosine in microfluidic chip.

PubMed

Ankireddy, Seshadri Reddy; Kim, Jongsung

2015-01-01

Microbeads are frequently used as solid supports for biomolecules such as proteins and nucleic acids in heterogeneous microfluidic assays. Chip-based, quantum dot (QD)-bead-biomolecule probes have been used for the detection of various types of DNA. In this study, we developed dopamine (DA)-functionalized InP/ZnS QDs (QDs-DA) as fluorescence probes for the detection of adenosine in microfluidic chips. The photoluminescence (PL) intensity of the QDs-DA is quenched by Zn(2+) because of the strong coordination interactions. In the presence of adenosine, Zn(2+) cations preferentially bind to adenosine, and the PL intensity of the QDs-DA is recovered. A polydimethylsiloxane-based microfluidic chip was fabricated, and adenosine detection was confirmed using QDs-DA probes.

Dopamine-functionalized InP/ZnS quantum dots as fluorescence probes for the detection of adenosine in microfluidic chip

PubMed Central

Ankireddy, Seshadri Reddy; Kim, Jongsung

2015-01-01

Microbeads are frequently used as solid supports for biomolecules such as proteins and nucleic acids in heterogeneous microfluidic assays. Chip-based, quantum dot (QD)-bead-biomolecule probes have been used for the detection of various types of DNA. In this study, we developed dopamine (DA)-functionalized InP/ZnS QDs (QDs-DA) as fluorescence probes for the detection of adenosine in microfluidic chips. The photoluminescence (PL) intensity of the QDs-DA is quenched by Zn2+ because of the strong coordination interactions. In the presence of adenosine, Zn2+ cations preferentially bind to adenosine, and the PL intensity of the QDs-DA is recovered. A polydimethylsiloxane-based microfluidic chip was fabricated, and adenosine detection was confirmed using QDs-DA probes. PMID:26347351

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

PubMed

Nakagawa, Kazuhiro; Yamamoto, Takuro; Yasuda, Akio

2010-09-15

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms. 2010 Elsevier Inc. All rights reserved.

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Nakashima, K.; Xing, Shaoyong; Gong, Yongkuan; Miyajima, Toru

2008-07-01

We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda's rule.

Two colorimetric and ratiometric fluorescence probes for hydrogen sulfide based on AIE strategy of α-cyanostilbenes

NASA Astrophysics Data System (ADS)

Zhao, Baoying; Yang, Binsheng; Hu, Xiangquan; Liu, Bin

2018-06-01

Aggregation-induced emission (AIE) active fluorescent probes have attracted great potential in biological sensors. In this paper two cyanostilbene based fluorescence chemoprobe Cya-NO2 (1) and Cya-N3 (2) were developed and evaluated for the selective and sensitive detection of hydrogen sulfide (H2S). Both of these probes behave aggression-induced emission (AIE) activity which fluoresces in the red region with a large Stokes shift. They exhibit rapid response to H2S with enormous colorimetric and ratiometric fluorescent changes. They are readily employed for assessing intracellular H2S levels.

A novel and simple fluorescence probe for detecting main group magnesium ion in HeLa cells and Arabidopsis.

PubMed

Yu, Tingting; Sun, Ping; Hu, Yijie; Ji, Yinggang; Zhou, Hongping; Zhang, Baowei; Tian, Yupeng; Wu, Jieying

2016-12-15

A simple-molecule fluorescence probe L has been designed, synthesized and characterized, which shows high selectivity and sensitivity for the main group magnesium ion through fluorescence "turn-on" response in ethanol solution, and no interference from calcium ion in particular. Detection limit of probe L is 1.47×10(-6) M and the rapid response could reach about 15-20s. The recognition mechanism has been established by fluorescence spectra, (1)H NMR study. Moreover, probe L presents a great photostability, low toxicity and cellular permeability, then we have carried out fluorescent bio-imaging of the probe L for magnesium ions in HeLa cells, which showed that probe L could be utilized to detect the intracellular magnesium ion. Furthermore, it is successfully used as a magnesium ion developer in plant tissues, which shows that it not only can be well tracking the transport of magnesium ion but also make a corresponding fluorescence response to different concentrations magnesium ion. These results would make this probe a great potential application for detecting Mg(2+) in biological system. Copyright © 2016 Elsevier B.V. All rights reserved.

NIR fluorescent dyes: versatile vehicles for marker and probe applications

NASA Astrophysics Data System (ADS)

Patonay, Gabor; Chapman, Gala; Beckford, Garfield; Henary, Maged

2013-02-01

The use of the NIR spectral region (650-900 nm) is advantageous due to the inherently lower background interference and the high molar absorptivities of NIR chromophores. Near-Infrared (NIR) dyes are increasingly used in the biological and medical field. The binding characteristics of NIR dyes to biomolecules are possibly controlled by several factors, including hydrophobicity, size and charge just to mention a few parameters. Binding characteristics of symmetric carbocyanines and found that the hydrophobic nature of the NIR dye is only partially responsible for the binding strength. Upon binding to biomolecules significant fluorescence enhancement can be observed for symmetrical carbocyanines. This fluorescence amplification facilitates the detection of the NIR dye and enhances its utility as NIR reporter. This manuscript discusses some probe and marker applications of such NIR fluorescent dyes. One application discussed here is the use of NIR dyes as markers. For labeling applications the fluorescence intensity of the NIR fluorescent label can significantly be increased by enclosing several dye molecules in nanoparticles. To decrease self quenching dyes that have relatively large Stokes' shift needs to be used. This is achieved by substituting meso position halogens with amino moiety. This substitution can also serve as a linker to covalently attach the dye molecule to the nanoparticle backbone. We report here on the preparation of NIR fluorescent silica nanoparticles. Silica nanoparticles that are modified with aminoreactive moieties can be used as bright fluorescent labels in bioanalytical applications. A new bioanalytical technique to detect and monitor the catalytic activity of the sulfur assimilating enzyme using NIR dyes is reported as well. In this spectroscopic bioanalytical assay a family of Fischer based n-butyl sulfonate substituted dyes that exhibit distinct variation in absorbance and fluorescence properties and strong binding to serum albumin as its

A novel fluorescence probe based on triphenylamine Schiff base for bioimaging and responding to pH and Fe3.

PubMed

Wang, Lei; Yang, Xiaodong; Chen, Xiuli; Zhou, Yuping; Lu, Xiaodan; Yan, Chenggong; Xu, Yikai; Liu, Ruiyuan; Qu, Jinqing

2017-03-01

A novel fluorescence probe 1 based on triphenylamine was synthesized and characterized by NMR, IR, high resolution mass spectrometry and elemental analysis. Its fluorescence was quenched when pH below 2. There was a linear relationship between the fluorescence intensity and pH value ranged from 2 to 7. And its fluorescence emission was reversibility in acidic and alkaline solution. Furthermore, it exhibited remarkable selectivity and high sensitivity to Fe 3+ and was able to detect Fe 3+ in aqueous solution with low detection limit of 0.511μM. Job plot showed that the binding stoichiometry of 1 with Fe 3+ was 1:1. Further observations of 1 H NMR titration suggested that coordination interaction between Fe 3+ and nitrogen atom on CN bond promoted the intramolecular charge transfer (ICT) or energy transfer process causing fluorescence quenching. Additionally, 1 was also able to be applied for detecting Fe 3+ in living cell and bioimaging. Copyright © 2016. Published by Elsevier B.V.

Novel Benzothiazole Derivatives as Fluorescent Probes for Detection of β-Amyloid and α-Synuclein Aggregates.

PubMed

Watanabe, Hiroyuki; Ono, Masahiro; Ariyoshi, Taisuke; Katayanagi, Rikako; Saji, Hideo

2017-08-16

Deposits of β-amyloid (Aβ) and α-synuclein (α-syn) are the hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The detection of these protein aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In this study, we newly designed and synthesized three push-pull benzothiazole (PP-BTA) derivatives as fluorescent probes for detection of Aβ and α-syn aggregates. Fluorescence intensity of all PP-BTA derivatives significantly increased upon binding to Aβ(1-42) and α-syn aggregates in solution. In in vitro saturation binding assays, PP-BTA derivatives demonstrated affinity for both Aβ(1-42) (K d = 40-148 nM) and α-syn (K d = 48-353 nM) aggregates. In particular, PP-BTA-4 clearly stained senile plaques composed of Aβ aggregates in the AD brain section. Moreover, it also labeled Lewy bodies composed of α-syn aggregates in the PD brain section. These results suggest that PP-BTA-4 may serve as a promising fluorescent probe for the detection of Aβ and α-syn aggregates.

Hydroethidine: a fluorescent redox probe for locating hypoxic cells in spheroids and murine tumours.

PubMed

Olive, P L

1989-09-01

The fluorescent redox probe hydroethidine was accumulated and metabolised about five times faster in aerobic than in hypoxic mammalian cells. Patterns of fluorescence in Chinese hamster V79 spheroids also indicated that internal hypoxic cells were less able to metabolise the drug; toxicity was observed in cells only when cell fluorescence exceeded about 500 times background. In medium equilibrated with air or nitrogen, cell accumulation of the stain was rapid, and began to plateau after 30 min; loss of ethidium was initially rapid, with a slower component after 30 min, and transfer of the metabolite ethidium between stained and unstained cells was observed after 2 h co-incubation. Sorting cells from irradiated spheroids on the basis of ethidium fluorescence provided good separation of aerobic radiosensitive and hypoxic radioresistant cells, although separation using the perfusion probe, Hoechst 33342, was superior. Similar experiments with the murine SCCVII squamous cell carcinoma suggested that hydroethidine might be a useful indirect stain for locating hypoxic cells in experimental tumours when used in combination with a perfusion probe such as Hoechst 33342.

Two sugar-rhodamine "turn-on" fluorescent probes for the selective detection of Fe3 +

NASA Astrophysics Data System (ADS)

Chen, Qing; Fang, Zhijie

2018-03-01

Two new sugar-rhodamine fluorescent probes (RDG1 and RDG2) have been synthesized and characterized by 1H NMR, 13C NMR and HRMS. Their UV-Vis, fluorescence spectra and fluorescence-response to Fe3 + are investigated and discussed. RDG1 had a very nice linear relationship between UV absorbance and Fe3 + concentration with the correlation coefficient as high as 0.997 and the detection limit is 3.46 × 10- 6 M. Upon the addition of Fe3 +, the spirolactam ring of RDG1 was opened and a 1:1 metal ligand complex was formed from Job's plot. The results showed that RDG1 can be used as an effective fluorescent probe for selective detection of Fe3 + in water. RDG2 was incorporated the well-known rhodamine group and a water-soluble D-glucose group within one molecule and can be used for detecting Fe3 + in natural water as a selective fluorescent sensor. The addition of Fe3 + into RDG2 resulted in a strongly enhanced fluorescence as well as color change of solution from colorless to pink. Job's plot of RDG2 indicated 1:1 stoichiometry of RDG2-Fe3 +. RDG2 can serve as a probe for Fe3 + between pH = 4.0 to 7.0 and it's detection limit is 2.09 × 10- 6 M. The OFF-ON fluorescent mechanisms of RDG1-Fe3 + and RDG2-Fe3 + are proposed.

RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vascular imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Belfield, Kevin D.; Yue, Xiling; Morales, Alma R.; Githaiga, Grace W.; Woodward, Adam W.; Tang, Simon; Sawada, Junko; Komatsu, Masanobu; Liu, Xuan

2016-03-01

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 μm in tumor tissue.

Synthesis and Antibacterial Activities of Antibacterial Peptides with a Spiropyran Fluorescence Probe

PubMed Central

Chen, Lei; Zhu, Yu; Yang, Danling; Zou, Rongfeng; Wu, Junchen; Tian, He

2014-01-01

In this report, antibacterial peptides1-3 were prepared with a spiropyran fluorescence probe. The probe exhibits a change in fluorescence when isomerized from a colorless spiro-form (spiropyran, Sp) to a colored open-form (merocyanine, Mc) under different chemical environments, which can be used to study the mechanism of antimicrobial activity. Peptides 1-3 exhibit a marked decrease in antimicrobial activity with increasing alkyl chain length. This is likely due to the Sp-Mc isomers in different polar environments forming different aggregate sizes in TBS, as demonstrated by time-dependent dynamic light scattering (DLS). Moreover, peptides 1-3 exhibited low cytotoxicity and hemolytic activity. These probe-modified peptides may provide a novel approach to study the effect of structural changes on antibacterial activity, thus facilitating the design of new antimicrobial agents to combat bacterial infection. PMID:25358905

Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

PubMed

Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

2017-03-01

This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

PubMed

Fu, Hualong; Tu, Peiyu; Zhao, Liu; Dai, Jiapei; Liu, Boli; Cui, Mengchao

2016-02-02

The formation of extracellular amyloid-β (Aβ) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aβ plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aβ plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aβ aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aβ plaques in the brain.

Sensitive and Selective Ratiometric Fluorescence Probes for Detection of Intracellular Endogenous Monoamine Oxidase A.

PubMed

Wu, Xiaofeng; Li, Lihong; Shi, Wen; Gong, Qiuyu; Li, Xiaohua; Ma, Huimin

2016-01-19

Monoamine oxidase A (MAO-A) is known to widely exist in most cell lines in the body, and its dysfunction (unusually high or low levels of MAO-A) is thought to be responsible for several psychiatric and neurological disorders. Thus, a sensitive and selective method for evaluating the relative MAO-A levels in different live cells is urgently needed to better understand the function of MAO-A, but to our knowledge such a method is still lacking. Herein, we rationally design two new ratiometric fluorescence probes (1 and 2) that can sensitively and selectively detect MAO-A. The probes are constructed by incorporating a recognition group of propylamine into the fluorescent skeleton of 1,8-naphthalimide, and the detection mechanism is based on amine oxidation and β-elimination to release the fluorophore (4-hydroxy-N-butyl-1,8-naphthalimide), which is verified by HPLC analysis. Reaction of the probes with MAO-A produces a remarkable fluorescence change from blue to green, and the ratio of fluorescence intensity at 550 and 454 nm is directly proportional to the concentration of MAO-A in the ranges of 0.5-1.5 and 0.5-2.5 μg/mL with detection limits of 1.1 and 10 ng/mL (k = 3) for probes 1 and 2, respectively. Surprisingly, these probes show strong fluorescence responses to MAO-A but almost none to MAO-B (one of two isoforms of MAO), indicating superior ability to distinguish MAO-A from MAO-B. The high specificity of the probes for MAO-A over MAO-B is further supported by different inhibitor experiments. Moreover, probe 1 displays higher sensitivity than probe 2 and is thus investigated to image the relative MAO-A levels in different live cells, such as HeLa and NIH-3T3 cells. It is found that the concentration of endogenous MAO-A in HeLa cells is approximately 1.8 times higher than that in NIH-3T3 cells, which is validated by the result from an ELISA kit. Additionally, the proposed probes may find more uses in the specific detection of MAO-A between the two isoforms of MAO

Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

PubMed

Liras, M; Simoncelli, S; Rivas-Aravena, A; García, O; Scaiano, J C; Alarcon, E I; Aspée, A

2016-04-26

A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered.

Synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group

NASA Astrophysics Data System (ADS)

He, Guangjie; Liu, Xiangli; Xu, Jinhe; Ji, Liguo; Yang, Linlin; Fan, Aiying; Wang, Songjun; Wang, Qingzhi

2018-02-01

A highly selective copper ions fluorescent probe based on the coumarin-type Schiff base derivative 1 (probe) was produced by condensation reaction between coumarin carbohydrazide and 1H-indazole-3-carbaldehyde. The UV-vis spectroscopy showed that the maximum absorption peak of compound 1 appeared at 439 nm. In the presence of Cu2 + ions, the maximum peak decreased remarkably compared with other physiological important metal ions and a new absorption peak at 500 nm appeared. The job's plot experiments showed that complexes of 1:2 binding mode were formed in CH3CN:HEPES (3:2, v/v) solution. Compound 1 exhibited a strong blue fluorescence. Upon addition of copper ions, the fluorescence gradually decreased and reached a plateau with the fluorescence quenching rate up to 98.73%. The detection limit for Cu2 + ions was estimated to 0.384 ppm. Fluorescent microscopy experiments demonstrated that probe 1 had potential to be used to investigate biological processes involving Cu2 + ions within living cells.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

PubMed

Zhang, Suge; Sun, Hongxia; Chen, Hongbo; Li, Qian; Guan, Aijiao; Wang, Lixia; Shi, Yunhua; Xu, Shujuan; Liu, Meirong; Tang, Yalin

2018-05-01

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells. Copyright © 2018. Published by Elsevier B.V.

A new class of fast-response and highly selective fluorescent probes for visualizing peroxynitrite in live cells, subcellular organelles, and kidney tissue of diabetic rats.

PubMed

Miao, Junfeng; Huo, Yingying; Liu, Qian; Li, Zhe; Shi, Heping; Shi, Yawei; Guo, Wei

2016-11-01

Peroxynitrite (ONOO(-)) is an extremely powerful oxidant in biological systems, and can react with a wide variety of molecular targets including proteins, lipids, and nucleic acids, eventually resulting in a series of disease states such as diabetes, Alzheimer's disease, cancer, arthritis, autoimmune, and other disorders. In this work, we present a new class of ONOO(-) fluorescent probes by exploiting the ONOO(-)-triggered N-oxidation and N-nitrosation reactions of aromatic tertiary amine for the first time. The as-obtained fluorescent probe A2 could detect ONOO(-) with quite fast fluorescence off-on response (within seconds), ultrasensitivity (detection limit: <2 nM), and excellent selectivity over a series of biologically relevant reactive oxygen species as well as metal cations. With the probe, the endogenous ONOO(-) in activated RAW264.7 murine macrophage, EA.hy926 endothelial cells after oxygen glucose deprivation and reoxygenation (OGD/RO), and kidney tissue of diabetic rats has been successfully visualized. Based on the molecular platform of A2, we further develop its mitochondria- and lysosome-targetable fluorescent probes Mito-A2 and Lyso-A2 by installing the corresponding targeting groups to alkoxy unit of A2, and confirm their abilities to image ONOO(-) in mitochondria and lysosomes, respectively, by co-localization assays. It is greatly expected that these probes can serve as useful imaging tools for clarifying the distribution and pathophysiological functions of ONOO(-) in cells, subcellular organelles, and animal tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

Diffuse fluorescence fiber probe for in vivo detection of circulating cells

NASA Astrophysics Data System (ADS)

Pera, Vivian; Tan, Xuefei; Runnels, Judith; Sardesai, Neha; Lin, Charles P.; Niedre, Mark

2017-03-01

There has been significant recent interest in the development of technologies for enumeration of rare circulating cells directly in the bloodstream in many areas of research, for example, in small animal models of circulating tumor cell dissemination during cancer metastasis. We describe a fiber-based optical probe that allows fluorescence detection of labeled circulating cells in vivo in a diffuse reflectance configuration. We validated this probe in a tissue-mimicking flow phantom model in vitro and in nude mice injected with fluorescently labeled multiple myeloma cells in vivo. Compared to our previous work, this design yields an improvement in detection signal-to-noise ratio of 10 dB, virtually eliminates problematic motion artifacts due to mouse breathing, and potentially allows operation in larger animals and limbs.

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

Complexation of β-cyclodextrin with dual molecular probes bearing fluorescent and paramagnetic moieties linked by short polyether chains.

PubMed

Mocanu, S; Matei, I; Ionescu, S; Tecuceanu, V; Marinescu, G; Ionita, P; Culita, D; Leonties, A; Ionita, Gabriela

2017-10-18

Electron paramagnetic resonance (EPR) and fluorescence spectroscopies provide molecular-level insights on the interaction of paramagnetic and fluorescent species with the microenvironment. A series of dual molecular probes bearing fluorescent and paramagnetic moieties linked by flexible short polyether chains have been synthesized. These new molecular probes open the possibility to investigate various multi-component systems such as host-guest systems, polymeric micelles, gels and protein solutions by using EPR and fluorescence spectroscopies concertedly. The EPR and fluorescence spectra of these compounds show that the dependence of the rotational correlation time and fluorescence quantum yield on the chain length of the linker is not linear, due to the flexibility of the polyether linker. The quenching effect of the nitroxide moiety on the fluorescence intensity of the pyrene group varies with the linker length and flexibility. The interaction of these dual molecular probes with β-cyclodextrin, in solution and in polymeric gels, was evaluated and demonstrated by analysis of EPR and fluorescence spectra.

Discovery of Novel Inhibitors and Fluorescent Probe Targeting NAMPT.

PubMed

Wang, Xia; Xu, Tian-Ying; Liu, Xin-Zhu; Zhang, Sai-Long; Wang, Pei; Li, Zhi-Yong; Guan, Yun-Feng; Wang, Shu-Na; Dong, Guo-Qiang; Zhuo, Shu; Le, Ying-Ying; Sheng, Chun-Quan; Miao, Chao-Yu

2015-07-31

Nicotinamide phosphoribosyltransferase (NAMPT) is a promising antitumor target. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for development of antitumor agents. Using high throughput screening system targeting NAMPT on a chemical library of 30000 small-molecules, we found a non-fluorescent compound F671-0003 and a fluorescent compound M049-0244 with excellent in vitro activity (IC50: 85 nM and 170 nM respectively) and anti-proliferative activity against HepG2 cells. These two compounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and π-π interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 (3 μM) significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Our findings provide novel antitumor lead compounds and a "first-in-class" fluorescent probe for imaging NAMPT.

Near-infrared fluorescence probes for enzymes based on binding affinity modulation of squarylium dye scaffold.

PubMed

Oushiki, Daihi; Kojima, Hirotatsu; Takahashi, Yuki; Komatsu, Toru; Terai, Takuya; Hanaoka, Kenjiro; Nishikawa, Makiya; Takakura, Yoshinobu; Nagano, Tetsuo

2012-05-15

We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye-protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye's protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye's protein affinity is restored, protein binding occurs, and the dye's fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes.

Development of Functional Fluorescent Molecular Probes for the Detection of Biological Substances

PubMed Central

Suzuki, Yoshio; Yokoyama, Kenji

2015-01-01

This review is confined to sensors that use fluorescence to transmit biochemical information. Fluorescence is, by far, the most frequently exploited phenomenon for chemical sensors and biosensors. Parameters that define the application of such sensors include intensity, decay time, anisotropy, quenching efficiency, and luminescence energy transfer. To achieve selective (bio)molecular recognition based on these fluorescence phenomena, various fluorescent elements such as small organic molecules, enzymes, antibodies, and oligonucleotides have been designed and synthesized over the past decades. This review describes the immense variety of fluorescent probes that have been designed for the recognitions of ions, small and large molecules, and their biological applications in terms of intracellular fluorescent imaging techniques. PMID:26095660

NIR fluorescence lifetime sensing through a multimode fiber for intravascular molecular probing

NASA Astrophysics Data System (ADS)

Ingelberts, H.; Hernot, S.; Debie, P.; Lahoutte, T.; Kuijk, M.

2016-04-01

Coronary artery disease (CAD) contributes to millions of deaths each year. The identification of vulnerable plaques is essential to the diagnosis of CAD but is challenging. Molecular probes can improve the detection of these plaques using intravascular imaging methods. Fluorescence lifetime sensing is a safe and robust method to image these molecular probes. We present two variations of an optical system for intravascular near-infrared (NIR) fluorescence lifetime sensing through a multimode fiber. Both systems are built around a recently developed fast and efficient CMOS detector, the current-assisted photonic sampler (CAPS) that is optimized for sub-nanosecond NIR fluorescence lifetime sensing. One system mimics the optical setup of an epifluorescence microscope while the other uses a practical fiber optic coupler to separate fluorescence excitation and emission. We test both systems by measuring the lifetime of several NIR dyes in DMSO solutions and we show that these systems are capable of detecting lifetimes of solutions with concentrations down to 370 nM and this with short acquisition times. These results are compared with time-correlated single photon counting (TCSPC) measurements for reference.

Fluorescent porous silicon biological probes with high quantum efficiency and stability.

PubMed

Tu, Chang-Ching; Chou, Ying-Nien; Hung, Hsiang-Chieh; Wu, Jingda; Jiang, Shaoyi; Lin, Lih Y

2014-12-01

We demonstrate porous silicon biological probes as a stable and non-toxic alternative to organic dyes or cadmium-containing quantum dots for imaging and sensing applications. The fluorescent silicon quantum dots which are embedded on the porous silicon surface are passivated with carboxyl-terminated ligands through stable Si-C covalent bonds. The porous silicon bio-probes have shown photoluminescence quantum yield around 50% under near-UV excitation, with high photochemical and thermal stability. The bio-probes can be efficiently conjugated with antibodies, which is confirmed by a standard enzyme-linked immunosorbent assay (ELISA) method.

Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe.

PubMed

Zhou, Jin; Shi, Wen; Li, Lihong; Gong, Qiuyu; Wu, Xiaofeng; Li, Xiaohua; Ma, Huimin

2016-04-19

Tyrosinase is regarded as an important biomarker of melanoma cancer, and its metabolism is closely related to some severe skin diseases such as vitiligo. Since tyrosinase is mainly located in the melanosomes of melanocytes, a probe that can specifically detect and image tysosinase in melanosomes would be in urgent demand to study the behavior of the enzyme in cells, but unfortunately, no melanosome-targeting tyrosinase fluorescent probe has been reported so far to the best of our knowledge. In this work, we have developed such a new probe, Mela-TYR, which bears morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group. The probe exhibits not only a highly sensitive and selective off-on response to tyrosinase via oxidization cleavage, but also an accurate targeting ability toward the acidic organelles of melanosomes and lyososomes, which is validated by colocalization experiments with mCherry-tagged melanosomes as well as DND-99 (a commercial dye). The probe has been used to image the relative contents of tyrosinase in different cells. Notably, because of the tyrosinase deficiency in normal lysosomes, the probe only fluoresces in melanosomes in principle although it can accumulate in other acidic organelles like lysosomes. By virtue of this property, the misdistribution of tyrosinase from melanosomes to lysosomes in murine melanoma B16 cells under the stimulation of inulavosin is imaged in real time for the first time. Moreover, the upregulation of melanosomal tyrosinase in live B16 cells under the stimulation of psoralen/ultraviolet A is detected with our probe, and this upregulation is further verified by standard colorimetric assay. The probe provides a simple, visual method to study the metabolism of tyrosinase in cells and shows great potential in clinical diagnosis and treatments of tyrosinase-associated diseases.

A new simple phthalimide-based fluorescent probe for highly selective cysteine and bioimaging for living cells

NASA Astrophysics Data System (ADS)

Shen, Youming; Zhang, Xiangyang; Zhang, Youyu; Zhang, Chunxiang; Jin, Junling; Li, Haitao

2017-10-01

A new turn-on phthalimide fluorescent probe has designed and synthesized for sensing cysteine (Cys) based on excited state intramolecular proton transfer (ESIPT) process. It is consisted of a 3-hydroxyphthalimide derivative moiety as the fluorophore and an acrylic ester group as a recognition receptor. The acrylic ester acts as an ESIPT blocking agent. Upon addition of cystein, intermolecular nucleophilic attack of cysteine on acrylic ester releases the fluorescent 3-hydroxyphthalimide derivative, thereby enabling the ESIPT process and leading to enhancement of fluorescence. The probe displays high sensitivity, excellent selectivity and with large Stokes shift toward cysteine. The linear interval range of the fluorescence titration ranged from 0 to 1.0 × 10- 5 M and detection limit is low (6 × 10- 8 M). In addition, the probe could be used for bio-imaging in living cells.

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

PubMed

Anumala, Upendra Rao; Gu, Jiamin; Lo Monte, Fabio; Kramer, Thomas; Heyny-von Haußen, Roland; Hölzer, Jana; Goetschy-Meyer, Valerie; Schön, Christian; Mall, Gerhard; Hilger, Ingrid; Czech, Christian; Herms, Jochen; Schmidt, Boris

2013-09-01

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Colorimetric and Fluorescent Probe for the Detection of Cu2+ in a Complete Aqueous Solution.

PubMed

Xu, Jing; Wang, Zuokai; Liu, Caiyun; Xu, Zhenghe; Zhu, Baocun; Wang, Ning; Wang, Kun; Wang, Jiangting

2018-01-01

The fluorescent probe has become an important method for the detection of heavy metal ions. In the present work, a new and simple fluorescent probe, Cu-P, for detecting copper ion (Cu 2+ ) was designed and synthesized. The probe has shown high sensitivity and selectivity toward Cu 2+ . The detection limit was 13 nM (based on the 3σ/slope). A significant color change from yellow to pink was observed; thus, the probe Cu-P could serve as a "naked-eye" indicator for Cu 2+ . Furthermore, the proposed probe was used to detect Cu 2+ in real water and soil extract samples, with the result being satisfactory. Therefore, our proposed probe would provide a promising method for the detection of Cu 2+ in the environment.

A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging.

PubMed

Pan, Deng; Hu, Zhe; Qiu, Fengwu; Huang, Zhen-Li; Ma, Yilong; Wang, Yina; Qin, Lingsong; Zhang, Zhihong; Zeng, Shaoqun; Zhang, Yu-Hui

2014-11-20

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

An intramolecular crossed-benzoin reaction based KCN fluorescent probe in aqueous and biological environments.

PubMed

Lee, Jae Hong; Jang, Joo Hee; Velusamy, Nithya; Jung, Hyo Sung; Bhuniya, Sankarprasad; Kim, Jong Seung

2015-05-04

A turn-on fluorescent probe was designed for selective cyanide anion sensing in aqueous and biological environments. The probe underwent an intramolecular crossed-benzoin reaction in the presence of KCN to expel the fluorophore resorufin. This probe was sensitive to KCN concentrations as low as 4 nM in aqueous media.

Theoretical Studies on Two-Photon Fluorescent Hg2+ Probes Based on the Coumarin-Rhodamine System.

PubMed

Zhang, Yujin; Leng, Jiancai

2017-07-20

The development of fluorescent sensors for Hg 2+ has attracted much attention due to the well-known adverse effects of mercury on biological health. In the present work, the optical properties of two newly-synthesized Hg 2+ chemosensors based on the coumarin-rhodamine system (named Pro1 and Pro2) were systematically investigated using time-dependent density functional theory. It is shown that Pro1 and Pro2 are effective ratiometric fluorescent Hg 2+ probes, which recognize Hg 2+ by Förster resonance energy transfer and through bond energy transfer mechanisms, respectively. To further understand the mechanisms of the two probes, we have developed an approach to predict the energy transfer rate between the donor and acceptor. Using this approach, it can be inferred that Pro1 has a six times higher energy transfer rate than Pro2. Thus the influence of spacer group between the donor and acceptor on the sensing performance of the probe is demonstrated. Specifically, two-photon absorption properties of these two probes are calculated. We have found that both probes show significant two-photon responses in the near-infrared light region. However, only the maximum two-photon absorption cross section of Pro1 is greatly enhanced with the presence of Hg 2+ , indicating that Pro1 can act as a potential two-photon excited fluorescent probe for Hg 2+ . The theoretical investigations would be helpful to build a relationship between the structure and the optical properties of the probes, providing information on the design of efficient two-photon fluorescent sensors that can be used for biological imaging of Hg 2+ in vivo.

A novel fluorescent probe for rapid and sensitive detection of hydrogen sulfide in living cells

NASA Astrophysics Data System (ADS)

Pan, Jian; Xu, Junchao; Zhang, Youlai; Wang, Liang; Qin, Caiqin; Zeng, Lintao; Zhang, Yue

2016-11-01

A novel fluorescent probe for H2S was developed based on a far-red emitting indole-BODIPY, which was decorated with morpholine and 2,4-dinitrobenzenesulfonyl (DNBS) group. This probe showed rapid response (t1/2 = 3 min), high selectivity and sensitivity for H2S with significant colorimetric and fluorescence OFF-ON signals, which was triggered by cleavage of 2,4-dinitrobenzenesulfonyl group. This probe could quantitatively detect the concentrations of H2S ranging from 0 to 60 μM, and the detection of limit was found to be as low as 26 nM. Cell imaging results indicated that the probe could detect and visualize H2S in the living cells.

Layer-by-layer assembled fluorescent probes in the second near-infrared window for systemic delivery and detection of ovarian cancer

PubMed Central

Dang, Xiangnan; Gu, Li; Qi, Jifa; Correa, Santiago; Zhang, Geran; Belcher, Angela M.; Hammond, Paula T.

2016-01-01

Fluorescence imaging in the second near-infrared window (NIR-II, 1,000–1,700 nm) features deep tissue penetration, reduced tissue scattering, and diminishing tissue autofluorescence. Here, NIR-II fluorescent probes, including down-conversion nanoparticles, quantum dots, single-walled carbon nanotubes, and organic dyes, are constructed into biocompatible nanoparticles using the layer-by-layer (LbL) platform due to its modular and versatile nature. The LbL platform has previously been demonstrated to enable incorporation of diagnostic agents, drugs, and nucleic acids such as siRNA while providing enhanced blood plasma half-life and tumor targeting. This work carries out head-to-head comparisons of currently available NIR-II probes with identical LbL coatings with regard to their biodistribution, pharmacokinetics, and toxicities. Overall, rare-earth-based down-conversion nanoparticles demonstrate optimal biological and optical performance and are evaluated as a diagnostic probe for high-grade serous ovarian cancer, typically diagnosed at late stage. Successful detection of orthotopic ovarian tumors is achieved by in vivo NIR-II imaging and confirmed by ex vivo microscopic imaging. Collectively, these results indicate that LbL-based NIR-II probes can serve as a promising theranostic platform to effectively and noninvasively monitor the progression and treatment of serous ovarian cancer. PMID:27114520

Design and Investigation of Optical Properties of N-(Rhodamine-B)-Lactam-Ethylenediamine (RhB-EDA) Fluorescent Probe.

PubMed

Soršak, Eva; Volmajer Valh, Julija; Korent Urek, Špela; Lobnik, Aleksandra

2018-04-14

This study presents chemical modification of a Rhodamine B (RhB) sensor probe by ethylenediamine (EDA), and investigation of its spectral as well as sensor properties to the various metals. The synthesised N -(Rhodamine-B)-lactam-ethylenediamine (RhB-EDA) fluorescent probe shows interesting optical sensor properties, and high sensitivity and selectivity to Ag⁺ ions among all the tested metal ions (K⁺, Mg 2+ , Cu 2+ , Ni 2+ , Fe 2+ , Pb 2+ , Na⁺, Mn 2+ , Li⁺, Al 3+ , Co 2+ , Hg 2+ , Sr 2+ , Ca 2+ , Ag⁺, Cd 2+ and Zn 2+ ), while the well-known Rhodamine B (RhB) fluorescent probe shows much less sensitivity to Ag⁺ ions, but high sensitivity to Fe 2+ ions. The novel fluorescent sensor probe RhB-EDA has the capabilities to sense Ag⁺ ions up to µM ranges by using the fluorescence quenching approach. The probe displayed a dynamic response to Ag⁺ in the range of 0.43 × 10 -3 -10 -6 M with a detection limit of 0.1 μM. The sensing system of an RhB-EDA novel fluorescent probe was optimised according to the spectral properties, effect of pH and buffer, photostability, incubation time, sensitivity, and selectivity. Since all the spectral and sensing properties were tested in green aqueous media, although many other similar sensor systems rely on organic solvent solutions, the RhB-EDA sensing probe may be a good candidate for measuring Ag⁺ ions in real-life applications.

Fast-Response Turn-on Fluorescent Probes Based on Thiolysis of NBD Amine for H2 S Bioimaging.

PubMed

Wang, Runyu; Li, Zhifei; Zhang, Changyu; Li, Yanyan; Xu, Guoce; Zhang, Qiang-Zhe; Li, Lu-Yuan; Yi, Long; Xi, Zhen

2016-05-17

Hydrogen sulfide (H2 S) is an important endogenous signaling molecule with multiple biological functions. New selective fluorescent turn-on probes based on fast thiolyling of NBD (7-nitro-1,2,3-benzoxadiazole) amine were explored for sensing H2 S in aqueous buffer and in living cells. The syntheses of both probes are simple and quite straightforward. The probes are highly sensitive and selective toward H2 S over other biologically relevant species. The fluorescein-NBD-based probe showed 65-fold green fluorescent increase upon H2 S activation. The rhodamine-NBD-based probe reacted rapidly with H2 S (t1/2 ≈1 min) to give a 4.5-fold increase in red fluorescence. Moreover, both probes were successfully used for monitoring H2 S in living cells and in mice. Based on such probe-based tools, we could observe H2 O2 -induced H2 S biogenesis in a concentration-dependent and time-dependent fashion in living cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An excited-state intramolecular photon transfer fluorescence probe for localizable live cell imaging of cysteine

NASA Astrophysics Data System (ADS)

Liu, Wei; Chen, Wen; Liu, Si-Jia; Jiang, Jian-Hui

2017-03-01

Small molecule probes suitable for selective and specific fluorescence imaging of some important but low-concentration intracellular reactive sulfur species such as cysteine (Cys) pose a challenge in chemical biology. We present a readily available, fast-response fluorescence probe CHCQ-Ac, with 2-(5‧-chloro-2-hydroxyl-phenyl)-6-chloro-4(3 H)-quinazolinone (CHCQ) as the fluorophore and acrylate group as the functional moiety, that enables high-selectivity and high-sensitivity for detecting Cys in both solution and biological system. After specifically reacted with Cys, the probe undergoes a seven-membered intramolecular cyclization and released the fluorophore CHCQ with excited-state intramolecular photon transfer effect. A highly fluorescent, insoluble aggregate was then formed to facilitate high-sensitivity and high-resolution imaging. The results showed that probe CHCQ-Ac affords a remarkably large Stokes shift and can detect Cys under physiological pH condition with no interference from other analytes. Moreover, this probe was proved to have excellent chemical stability, low cytotoxicity and good cell permeability. Our design of this probe provides a novel potential tool to visualize and localize cysteine in bioimaging of live cells that would greatly help to explore various Cys-related physiological and pathological cellular processes in cell biology and diagnostics.

Theoretical Design of a Two-Photon Fluorescent Probe for Nitric Oxide with Enhanced Emission Induced by Photoninduced Electron Transfer.

PubMed

Zhang, Yujin; Leng, Jiancai; Hu, Wei

2018-04-25

In the present work, we systematically investigate the sensing abilities of two recently literature-reported two-photon fluorescent NO probes, i.e., the o-phenylenediamine derivative of Nile Red and the p-phenylenediamine derivative of coumarin. The recognition mechanisms of these probes are studied by using the molecular orbital classifying method, which demonstrates the photoinduced electron transfer process. In addition, we have designed two new probes by swapping receptor units present on fluorophores, i.e., the p-phenylenediamine derivative of Nile Red and the o-phenylenediamine derivative of coumarin. However, it illustrates that only the latter has ability to function as off-on typed fluorescent probe for NO. More importantly, calculations on the two-photon absorption properties of the probes demonstrate that both receptor derivatives of coumarin possess larger TPA cross-sections than Nile Red derivatives, which makes a better two photon fluorescent probe. Our theoretical investigations reveal that the underlying mechanism satisfactorily explain the experimental results, providing a theoretical basis on the structure-property relationships which is beneficial to developing new two-photon fluorescent probes for NO.

Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

PubMed

Xue, Yuyuan; Liang, Wanshan; Li, Yuan; Wu, Ying; Peng, Xinwen; Qiu, Xueqing; Liu, Jinbin; Sun, Runcang

2016-12-28

A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pK a of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

Energy Donor Effect on the Sensing Performance for a Series of FRET-Based Two-Photon Fluorescent Hg2+ Probes.

PubMed

Zhang, Yujin; Hu, Wei

2017-01-25

Nonlinear optical properties of a series of newly-synthesized molecular fluorescent probes for Hg 2+ containing the same acceptor (rhodamine group) are analyzed by using time-dependent density functional theory in combination with analytical response theory. Special emphasis is placed on evolution of the probes' optical properties in the absence and presence of Hg 2+ . These compounds show drastic changes in their photoabsorption and photoemission properties when they react with Hg 2+ , indicating that they are excellent candidates for ratiometric and colorimetric fluorescent chemosensors. Most importantly, the energy donor moiety is found to play a dominant role in sensing performance of these probes. Two-photon absorption cross sections of the compounds are increased with the presence of Hg 2+ , which theoretically suggests the possibility of the probes to be two-photon fluorescent Hg 2+ sensors. Moreover, analysis of molecular orbitals is presented to explore responsive mechanism of the probes, where the fluorescence resonant energy transfer process is theoretically demonstrated. Our results elucidate the available experimental measurements. This work provides guidance for designing efficient two-photon fluorescent probes that are geared towards biological and chemical applications.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

PubMed

Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

2017-05-01

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy.

PubMed Central

Buehler, C; Dong, C Y; So, P T; French, T; Gratton, E

2000-01-01

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-microm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from approximately 30 to approximately 150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell. PMID:10866979

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

Quenching of photoexcited states of the proteins chromophores and introduced into the protein macromolecules fluorescent probes by heavy metal ions

NASA Astrophysics Data System (ADS)

Melnikov, A. G.; Dyachuk, O. A.; Melnikov, G. V.

2015-03-01

We have studied the processes of quenching of photoexcited states of fluorescent probes and quenching of the fluorescence of the chromophores of human serum albumin (HSA) by heavy metal ions (HM): cations Tl+, Pb2+, Cu2+, Cd2+, and the anion of iodine (I-). We used the dye from xanthene series - eosin as a fluorescent probe. By quenching of the fluorescence of protein chromophores we found an influence of HM on the structure of proteins, resulting in a shift of the peak of the fluorescence of HSA tryptophanyl. This can be explained by proteins denaturation under the influence of heavy metals and penetration of water into the inner environment of HSA tryptophan. It was established that the constant of the quenching of the probe phosphorescence is much higher than the fluorescence, which is explained by significantly longer lifetime of the photoexcited states of fluorescent probes in the triplet state than in the singlet.

Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis

NASA Astrophysics Data System (ADS)

Goswami, Linee; Pratihar, Sanjay; Dasgupta, Suman; Bhattacharyya, Pradip; Mudoi, Pronab; Bora, Jayanta; Bhattacharya, Satya Sundar; Kim, Ki Hyun

2016-07-01

Metal contamination from coal ashes (CAs) is widely recognized as a significant environmental concern. To learn more about metal detoxification and accumulation potential of earthworm species, metal-rich tea factory coal ashes (TFCA) were fed to Eisenia fetida and Lampito mauritii by employing a fluorescent tag detection method. Fascinatingly, on feeding fluorescence probed Zn and Cd along with cow dung to Eisenia fetida, the detection of the gut-proteins with a molecular mass higher than 100 kDa was a distinct evidence of metal binding. Significant increases were observed in the content of humified organic C [humic acid (HAC) and fulvic acid C (FAC)] and degree of humification during vermicomposting. Concurrently, considerably large amount of toxic metals (Cr, Cd, Pb, and Zn) was transformed from exchangeable to recalcitrant (organic matter and mineral bound) fractions. Moreover, total metal concentrations were reduced with high removal efficiency upon vermicomposting.