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Sample records for acid fluorescent probes

  1. Fluorescent hybridization probes for nucleic acid detection.

    PubMed

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  2. Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes

    NASA Astrophysics Data System (ADS)

    Zhou, Yunyou; Bian, Guirong; Wang, Leyu; Dong, Ling; Wang, Lun; Kan, Jian

    2005-06-01

    This paper describes the preparation of organic nanoparticles by reprecipitation method under sonication and vigorous stirring. Transmission electron microscopy (TEM) was used to characterize the size and size distribution of the luminescent nanoparticles. Their average diameter was about 25 nm with a size variation of ±18%. The fluorescence decay lifetime of the nanoparticles also was determined on a self-equipped fluorospectrometer with laser light source. The lifetime (˜0.09 μs) of nanoparticles is about three times long as that of the monomer. The nanoparticles were in abundant of hydrophilic groups, which increased their miscibility in aqueous solution. These organic nanoparticles have high photochemical stability, excellent resistance to chemical degradation and photodegradation, and a good fluorescence quantum yield (25%). The fluorescence can be efficiently quenched by nucleic acids. Based on the fluorescence quenching of nanoparticles, a fluorescence quenching method was developed for determination of microamounts of nucleic acids by using the nanoparticles as a new fluorescent probe. Under optimal conditions, maximum fluorescence quenching is produced, with maximum excitation and emission wavelengths of 345 and 402 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.4-19.0 μg ml -1 for calf thymus DNA (ct-DNA) and 0.3-19.0 μg ml -1 for fish sperm DNA (fs-DNA). The corresponding detection limits are 0.25 μg ml -1 for ct-DNA and 0.17 μg ml -1 for fs-DNA. The relative standard deviation of six replicate measurements is 1.3-2.1%. The method is simple, rapid and sensitive with wide linear range. The recovery and relative standard deviation are very satisfactory.

  3. Fluorescence probe for the convenient and sensitive detection of ascorbic acid

    PubMed Central

    Matsuoka, Yuta; Yamato, Mayumi; Yamada, Ken-ichi

    2016-01-01

    Ascorbic acid is an important antioxidant that plays an essential role in the biosynthesis of numerous bioactive substances. The detection of ascorbic acid has traditionally been achieved using high-performance liquid chromatography and absorption spectrophotometry assays. However, the development of fluorescence probes for this purpose is highly desired because they provide a much more convenient and highly sensitive technique for the detection of this material. OFF-ON-type fluorescent probes have been developed for the detection of non-fluorescent compounds. Photo-induced electron transfer and fluorescence resonance energy transfer are the two main fluorescence quenching mechanisms for the detection of ascorbic acid, and several fluorescence probes have been reported based on redox-responsive metals and quantum dots. Profluorescent nitroxide compounds have also been developed as non-metal organic fluorescence probes for ascorbic acid. These nitroxide systems have a stable unpaired electron and can therefore react with ascorbic acid and a strong fluorescence quencher. Furthermore, recent synthetic advances have allowed for the synthesis of α-substituted nitroxides with varying levels of reactivity towards ascorbic acid. In this review, we have discussed the design strategies used for the preparation of fluorescent probes for ascorbic acid, with particular emphasis on profluorescent nitroxides, which are unique radical-based redox-active fluorescent probes. PMID:26798193

  4. Fluorescent macrocyclic probes with pendant functional groups as markers of acidic organelles within live cells.

    PubMed

    Wadhavane, Prashant D; Izquierdo, M Ángeles; Lutters, Dennis; Burguete, M Isabel; Marín, María J; Russell, David A; Galindo, Francisco; Luis, Santiago V

    2014-02-07

    A new family of acidity sensitive fluorescent macrocycles has been synthesized and fully characterized. Their photophysical properties including emission quantum yield and fluorescence lifetime have been determined. The acid-base properties of the new molecules can be tuned by the incorporation of pendant functional groups. The nature of such functional groups (carboxylic acid or ester) influences dramatically the pKa of the probes, two compounds of which exhibit low values. Preliminary intracellular studies using confocal microscopy together with emission spectra of the probes from the cellular environment have shown that the synthesized fluorescent macrocycles mark the acidic organelles of RAW 264.7 macrophage cells.

  5. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  6. A novel acidic pH fluorescent probe based on a benzothiazole derivative.

    PubMed

    Ma, Qiujuan; Li, Xian; Feng, Suxiang; Liang, Beibei; Zhou, Tiqiang; Xu, Min; Ma, Zhuoyi

    2017-04-15

    A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H(+) in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

  7. A novel acidic pH fluorescent probe based on a benzothiazole derivative

    NASA Astrophysics Data System (ADS)

    Ma, Qiujuan; Li, Xian; Feng, Suxiang; Liang, Beibei; Zhou, Tiqiang; Xu, Min; Ma, Zhuoyi

    2017-04-01

    A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H+ in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1 min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

  8. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

  9. Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

    PubMed Central

    Mergny, J L; Boutorine, A S; Garestier, T; Belloc, F; Rougée, M; Bulychev, N V; Koshkin, A A; Bourson, J; Lebedev, A V; Valeur, B

    1994-01-01

    The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes. PMID:8152922

  10. Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria.

    PubMed

    Blasco, Lucía; Ferrer, Sergi; Pardo, Isabel

    2003-08-08

    A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).

  11. Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids.

    PubMed

    El-Yazbi, Amira F; Loppnow, Glen R

    2013-07-05

    Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb(3+)). Single-stranded oligonucleotides greatly enhance the Tb(3+) emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb(3+)/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb(3+), producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb(3+)/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb(3+)/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36±1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.

  12. 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

  13. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.

    PubMed

    Macnaughton, S J; O'Donnell, A G; Embley, T M

    1994-10-01

    The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

  14. 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.

  15. Boronic Acid: A Bio-Inspired Strategy To Increase the Sensitivity and Selectivity of Fluorescent NADH Probe.

    PubMed

    Wang, Lu; Zhang, Jingye; Kim, Beomsue; Peng, Juanjuan; Berry, Stuart N; Ni, Yong; Su, Dongdong; Lee, Jungyeol; Yuan, Lin; Chang, Young-Tae

    2016-08-24

    Fluorescent probes have emerged as an essential tool in the molecular recognition events in biological systems; however, due to the complex structures of certain biomolecules, it remains a challenge to design small-molecule fluorescent probes with high sensitivity and selectivity. Inspired by the enzyme-catalyzed reaction between biomolecule and probe, we present a novel combination-reaction two-step sensing strategy to improve sensitivity and selectivity. Based on this strategy, we successfully prepared a turn-on fluorescent reduced nicotinamide adenine dinucleotide (NADH) probe, in which boronic acid was introduced to bind with NADH and subsequently accelerate the sensing process. This probe shows remarkably improved sensitivity (detection limit: 0.084 μM) and selectivity to NADH in the absence of any enzymes. In order to improve the practicality, the boronic acid was further modified to change the measurement conditions from alkalescent (pH 9.5) to physiological environment (pH 7.4). Utilizing these probes, we not only accurately quantified the NADH weight in a health care product but also evaluated intracellular NADH levels in live cell imaging. Thus, these bio-inspired fluorescent probes offer excellent tools for elucidating the roles of NADH in biological systems as well as a practical strategy to develop future sensitive and selective probes for complicated biomolecules.

  16. New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

    PubMed Central

    Mateo, C R; Souto, A A; Amat-Guerri, F; Acuña, A U

    1996-01-01

    The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A. Images FIGURE 10 PMID:8889194

  17. 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

  18. A carbon dot-based "off-on" fluorescent probe for highly selective and sensitive detection of phytic acid.

    PubMed

    Gao, Zhao; Wang, Libing; Su, Rongxin; Huang, Renliang; Qi, Wei; He, Zhimin

    2015-08-15

    We herein report a facile, one-step pyrolysis synthesis of photoluminescent carbon dots (CDs) using citric acid as the carbon source and lysine as the surface passivation reagent. The as-prepared CDs show narrow size distribution, excellent blue fluorescence and good photo-stability and water dispersivity. The fluorescence of the CDs was found to be effectively quenched by ferric (Fe(III)) ions with high selectivity via a photo-induced electron transfer (PET) process. Upon addition of phytic acid (PA) to the CDs/Fe(III) complex dispersion, the fluorescence of the CDs was significantly recovered, arising from the release of Fe(III) ions from the CDs/Fe(III) complex because PA has a higher affinity for Fe(III) ions compared to CDs. Furthermore, we developed an "off-on" fluorescence assay method for the detection of phytic acid using CDs/Fe(III) as a fluorescent probe. This probe enables the selective detection of PA with a linear range of 0.68-18.69 μM and a limit of detection (signal-to-noise ratio is 3) of 0.36 μM. The assay method demonstrates high selectivity, repeatability, stability and recovery ratio in the detection of the standard and real PA samples. We believe that the facile operation, low-cost, high sensitivity and selectivity render this CD-based "off-on" fluorescent probe an ideal sensing platform for the detection of PA.

  19. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  20. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

    PubMed

    Sholokh, Marianna; Zamotaiev, Oleksandr M; Das, Ranjan; Postupalenko, Viktoriia Y; Richert, Ludovic; Dujardin, Denis; Zaporozhets, Olga A; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2015-02-12

    Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

  1. 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.

  2. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH.

  3. 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 (H2O2), 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 H2O2 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 H2O2 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.

  4. Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

    PubMed Central

    Oliveira, Kenneth; Haase, Gerhard; Kurtzman, Cletus; Hyldig-Nielsen, Jens Jo/rgen; Stender, Henrik

    2001-01-01

    The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis. PMID:11682542

  5. A high-resolution mitochondria-targeting ratiometric fluorescent probe for detection of the endogenous hypochlorous acid

    NASA Astrophysics Data System (ADS)

    Zhou, Liyi; Lu, Dan-Qing; Wang, Qianqian; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (Cdbnd C) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210 nm) and the two well-resolved emission peaks separated by 140 nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50 s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution.

  6. Preparation and application of cysteine-capped ZnS nanoparticles as fluorescence probe in the determination of nucleic acids

    NASA Astrophysics Data System (ADS)

    Li, Yongxin; Chen, Jinlong; Zhu, Changqin; Wang, Lun; Zhao, Danhua; Zhuo, Shujuan; Wu, Yuqin

    2004-07-01

    Cysteine-capped ZnS nanometer-sized fluorescent particles were produced by a colloidal aqueous synthesis. The functionalized nanoparticles are water-soluble and suitable for biological application. A synchronous fluorescence method has been developed for the rapid determination of DNA with functionalized nano-ZnS as a fluorescence probe, based on the synchronous fluorescence enhancement of cysteine-capped nano-ZnS in the presence of DNA. When Δ λ=190 nm, maximum synchronous fluorescence is produced at 267 nm at pH 5.12. Under optimum conditions, the synchronous fluorescence intensity is proportional to the concentration of nucleic acids in the range 0.1-1.2 μg ml -1 for calf thymus DNA, 0.1-0.6 μg ml -1 for fish sperm DNA. The corresponding detection limit is 32.9 ng ml -1 for calf thymus DNA and 24.6 ng ml -1 for fish sperm DNA. This method is simple, inexpensive, rapid and sensitive. The recovery and relative standard deviation are satisfactory.

  7. 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

  8. A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.

    PubMed

    Moreno, Alfredo; SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Alvarez, Javier

    2010-12-01

    Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques.

  9. Novel emissive bio-inspired non-proteinogenic coumarin-alanine amino acid: fluorescent probe for polyfunctional systems.

    PubMed

    Oliveira, Elisabete; Capelo, José Luis; Lima, João Carlos; Lodeiro, Carlos

    2012-10-01

    Two new bio-inspired non-proteinogenic compounds L1 and L2, containing coumarin and/or acridine chromophores and bearing as spacer an alanine amino acid were successfully synthesized and fully characterized by elemental analysis, (1)H and (13)C NMR, infrared spectroscopy (KBr discs), melting point, ESI-TOF (electrospray ionization-time of flight-mass), UV-vis absorption and emission spectroscopy, fluorescence quantum yields and lifetime measurements. A relative fluorescence quantum yield of 0.02 was determined for both compounds. In L2 the presence of an intramolecular energy transfer from the coumarin to the acridine unit was observed. L1 and L2 are quite sensitive to the basicity of the environment. At alkaline values both compounds show a strong quenching in the fluorescence emission, attributed to the photoinduced electron transfer (PET). However, both deprotonated forms recover the emission with the addition of Zn(2+), Cd(2+) and Al(3+) metal ions. As multifunctional emissive probes, the titration of L1 and L2 with lanthanides (III), Eu(3+) and Tb(3+) was also explored as new visible bio-probes in the absence and in the presence of liposomes. In a liposomal environment a lower energy transfer was observed.

  10. Hydrothermal synthetic mercaptopropionic acid stabled CdTe quantum dots as fluorescent probes for detection of Ag⁺.

    PubMed

    Gan, Ting-Ting; Zhang, Yu-Jun; Zhao, Nan-Jing; Xiao, Xue; Yin, Gao-Fang; Yu, Shao-Hui; Wang, Huan-Bo; Duan, Jing-Bo; Shi, Chao-Yi; Liu, Wen-Qing

    2012-12-01

    Mercaptopropionic acid (MPA) capped CdTe quantum dots (QDs) with particle size 3 nm have been successfully synthesized in aqueous medium by hydrothermal synthesis method. And the effects of different metal ions on MPA capped CdTe QDs fluorescence were studied using fluorescence spectrometry. The results demonstrated that at the same concentration level, Ag(+) could strongly quench CdTe QDs fluorescence, and the other metal ions had little effect on CdTe QDs fluorescence except Cu(2+). On the basis of this fact, a rapid, simple, highly sensitive and selective method based on fluorescence quenching principle for Ag(+) detection in aqueous solution was proposed. Under optimal conditions, the quenched fluorescence intensity (F(0)-F) increased linearly with the concentration of Ag(+) ranging from 4 × 10(-7) to 32 × 10(-7)mol L(-1). The limit of detection for Ag(+) was 4.106 × 10(-8)mol L(-1). The obtained plot of F(0)/F versus [Ag(+)] was an upward curvature, concave towards the y-axis, rather than a straight line. The modified form of the Stern-Volmer equation was third order in Ag(+) concentration. According to the modified Stern-Volmer equation, it can be inferred that dynamic quenching and static quenching simultaneously occurred when Ag(+) interacted with MPA capped CdTe QDs. At the same time other factors might also influence the quenching process. Based on this study, hydrothermal synthesized MPA capped CdTe QDs with particle size 3 nm may be used as a novel fluorescence probe to quantificationally and selectively detect Ag(+).

  11. 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.

  12. Mercaptopropionic acid-capped CdTe quantum dots as fluorescence probe for the determination of salicylic acid in pharmaceutical products.

    PubMed

    Bunkoed, Opas; Kanatharana, Proespichaya

    2015-11-01

    Mercaptopropionic acid (MPA)-capped cadmium telluride (CdTe) quantum dot (QDs) fluorescent probes were synthesized in aqueous solution and used for the determination of salicylic acid. The interaction between the MPA-capped CdTe QDs and salicylic acid was studied using fluorescence spectroscopy and some parameters that could modify the fluorescence were investigated to optimize the measurements. Under optimum conditions, the quenched fluorescence intensity of MPA-capped CdTe QDs was linearly proportional to the concentration of salicylic acid in the range of 0.5-40 µg mL(-1) with a coefficient of determination of 0.998, and the limit of detection was 0.15 µg mL(-1). The method was successfully applied to the determination of salicylic acid in pharmaceutical products, and satisfactory results were obtained that were in agreement with both the high pressure liquid chromatography (HPLC) method and the claimed values. The recovery of the method was in the range 99 ± 3% to 105 ± 9%. The proposed method is simple, rapid, cost effective, highly sensitivity and eminently suitable for the quality control of pharmaceutical preparation. The possible mechanisms for the observed quenching reaction was also discussed.

  13. 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.

  14. DAPI: a DNA-specific fluorescent probe.

    PubMed

    Kapuscinski, J

    1995-09-01

    DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

  15. Synthesis of fluorescent D-amino acids (FDAAs) and their use for probing peptidoglycan synthesis and bacterial growth in situ

    PubMed Central

    Kuru, Erkin; Tekkam, Srinivas; Hall, Edward

    2015-01-01

    Fluorescent D-amino acids (FDAAs) are efficiently incorporated into the peptidoglycan of diverse bacterial species at the sites of active peptidoglycan biosynthesis, allowing specific and covalent probing of bacterial growth with minimal perturbation. Here, we provide a protocol for the synthesis of four FDAAs emitting light in blue, green or red and for their use in peptidoglycan labeling of live bacteria. Our modular synthesis protocol gives easy access to a library of different FDAAs made with commercially available fluorophores. FDAAs can be synthesized in a typical chemistry laboratory in 2–3 days. The simple labeling procedure involves addition of the FDAAs to the bacterial sample for the desired labeling duration and stopping further label incorporation by fixation or by washing away excess dye. We discuss several scenarios for the use of these labels including short or long labeling durations, and the combination of different labels in pure culture or complex environmental samples. Depending on the experiment, FDAA labeling can take as little as 30 s for a rapidly growing species such as Escherichia coli. PMID:25474031

  16. A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes.

    PubMed

    Lee, Kyungmyung; Park, Hyun-Chul; An, Sanghyun; Ahn, Eu-Ree; Lee, Yang-Han; Kim, Mi-Jung; Lee, Eun-Jung; Park, Jae Sin; Jung, Jin Wook; Lim, Sikeun

    2015-09-01

    ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n=5], A/O01 or A/O02 [n=23], B/B [n=3], B/O01 or B/O02 [n=18], A/B [n=9], O01/O01 or O01/O02 or O02/O02 [n=20], cis-AB01/A [n=1], and cis-AB01/O01 or cis-AB01/O02 [n=1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.

  17. 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.

  18. Preparation of Au Nanoclusters-Modified Polylactic Acid Fiber with Bright Red Fluorescence and its Use as Sensing Probe.

    PubMed

    Zhu, Wenli; Li, Huili; Wan, Ajun; Liu, Lanbo

    2017-01-01

    In present work, the Au nanoclusters-modified polylactic acid fiber (PLA-Au NCs) with bright red fluorescence were fabricated by the encapsulation of Au nanoclusters (Au NCs) in the PLA fiber treated with H2O2. The Au25 nanoclusters stabilized by bovine serum albumin (BSA-Au NCs) were prepared via an improved "green" synthetic routine. With pretreatment of the PLA fiber in H2O2 concentration of 12 and 18 %, the as-prepared PLA-Au NCs exhibited brighter red emission with a strong peak centered at ~640 nm than BSA-Au NCs. The fluorescence can be quenched by nitric oxide (NO). A good linear relationship between the relative fluorescence quenching intensity of the as-prepared PLA-Au NCs and the concentration of NO can be obtained in the range of 0.0732 to 0.7320 mM, and the detection limit was 0.0070 mM.

  19. ANTS-anchored Zn-Al-CO3-LDH particles as fluorescent probe for sensing of folic acid

    NASA Astrophysics Data System (ADS)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan; Li, Lei

    2016-09-01

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO3-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn2+ ions of Zn-Al-CO3-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO3-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO3-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO3 groups in ANTS-anchored on the surface of Zn-Al-CO3-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO3-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution.

  20. Preparation and application of L-cysteine-modified CdSe/CdS core/shell nanocrystals as a novel fluorescence probe for detection of nucleic acid

    NASA Astrophysics Data System (ADS)

    Huang, Fenghua; Chen, Guonan

    2008-07-01

    The water-soluble L-cysteine-modified CdSe/CdS core/shell nanocrystals (expressed as CdSe/CdS/Cys nanocrystals) have been synthesized in aqueous by using L-cysteine as stabilizer. The size, shape, component and spectral property of CdSe/CdS/Cys nanocrystals were characterized by high-resolution transmission electron microscope (HRTEM), energy dispersive X-ray fluorescence (EDX), infrared spectrum (IR) and photoluminescence (PL). The results showed that the spherical CdSe/CdS/Cys nanocrystals with an average diameter of 2.3 nm have favorable fluorescent property, theirs photostability and fluorescence intensity are enhanced greatly after overcoating with CdS. The cysteine modified on the surface of core/shell CdSe/CdS nanocrystals renders the nanocrystals water-soluble and biocompatible. Based on the fluorescence quenching of the nanocrystals in the presence of calf thymus deoxyribonucleic acid (ct-DNA), a fluorescence quenching method has been developed for the determination of ct-DNA by using the nanocrystals as a novel fluorescence probe. The pH value of the system was selected at pH 7.4, with excitation and emission wavelength at 380 and 522 nm, respectively. Under the optimal conditions, the fluorescence quenching intensity of the system is linear with the concentration of ct-DNA in the range of 0.1-3.5 μg/mL ( r = 0.9987). The detection limit is 0.06 μg/mL. And two synthetic samples were analyzed satisfactorily.

  1. Dendrimer Probes for Enhanced Photostability and Localization in Fluorescence Imaging

    PubMed Central

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

    2013-01-01

    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. PMID:23561533

  2. Carbon Nanoparticle-based Fluorescent Bioimaging Probes

    PubMed Central

    Bhunia, Susanta Kumar; Saha, Arindam; Maity, Amit Ranjan; Ray, Sekhar C.; Jana, Nikhil R.

    2013-01-01

    Fluorescent nanoparticle-based imaging probes have advanced current labelling technology and are expected to generate new medical diagnostic tools based on their superior brightness and photostability compared with conventional molecular probes. Although significant progress has been made in fluorescent semiconductor nanocrystal-based biological labelling and imaging, the presence of heavy metals and the toxicity issues associated with heavy metals have severely limited the application potential of these nanocrystals. Here, we report a fluorescent carbon nanoparticle-based, alternative, nontoxic imaging probe that is suitable for biological staining and diagnostics. We have developed a chemical method to synthesise highly fluorescent carbon nanoparticles 1–10 nm in size; these particles exhibit size-dependent, tunable visible emission. These carbon nanoparticles have been transformed into various functionalised nanoprobes with hydrodynamic diameters of 5–15 nm and have been used as cell imaging probes. PMID:23502324

  3. Fluorescence sensing of phosdrin pesticide by the luminescent Eu(III)- and Tb(III)-bis(coumarin-3-carboxylic acid) probes

    NASA Astrophysics Data System (ADS)

    Hussein, Belal H. M.; Khairy, Gasser M.; Kamel, Rasha M.

    2016-04-01

    Luminescence quenching of the Eu(III)- and Tb(III)-bis (coumarin-3-carboxylic acid) (Ln(III)-(CCA)2) probes has been studied in the presence of organophosphorus or organochlorine pesticides; Phosdrin (P1), Malathion (P2), Profenofos (P3), Formothion (P4), Heptachlor (P5), and Endosulfan (P6). The luminescence intensity of lanthanide complex probes Ln(III)-(CCA)2 decreases as the concentration of the Phosdrin pesticide increases, while the other investigated pesticides have no significant influence on the lanthanide fluorescent intensities. It is observed that the quenching of Eu(III) and Tb(III)-coumarin-3-carboxylic acid by Phosdrin proceeds via static quenching processes according to Stern-Volmer plot. The binding constants (K) and the thermodynamic parameters of the interaction of Ln(III)-(CCA)2 with Phosdrin have been determined. A direct method for the determination of the Phosdrin in ethanol has been developed based on the luminescence changes of the Ln(III)-(CCA)2-phosdrin ternary complexes. The detection limits of P1 were 6.28 and 1.07 μM in case of Eu(III) and Tb(III)-complex, respectively. The influence of various interfering species on the detection of P1 has been investigated to assess the analytical applicability of the method. The new method was applied to determine the Phosdrin pesticide in different types of water samples.

  4. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    PubMed

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  5. Naphthoxazole-based singlet oxygen fluorescent probes.

    PubMed

    Ruiz-González, Rubén; Zanocco, Renzo; Gidi, Yasser; Zanocco, Antonio L; Nonell, Santi; Lemp, Else

    2013-01-01

    In this study, we report the synthesis and photochemical behavior of a new family of photoactive compounds to assess its potential as singlet oxygen ((1)O2) probes. The candidate dyads are composed by a (1)O2 trap plus a naphthoxazole moiety linked directly or through an unsaturated bond to the oxazole ring. In the native state, the inherent great fluorescence of the naphthoxazole moiety is quenched; but in the presence of (1)O2, generated by the addition and appropriate irradiation of an external photosensitizer, a photooxidation reaction occurs leading to the formation of a new chemical entity whose fluorescence is two orders of magnitude higher than that of the initial compound, at the optimal selected wavelength. The presented dyads outperform the commonly used indirect fluorescent (1)O2 probes in terms of fluorescence enhancement maintaining the required specificity for (1)O2 detection in solution.

  6. Introduction to fluorescence probing of biological membranes.

    PubMed

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

    2015-01-01

    Fluorescence is one of the most powerful and commonly used tools in biophysical studies of biomembrane structure and dynamics that can be applied on different levels, from lipid monolayers and bilayers to living cells, tissues, and whole animals. Successful application of this method relies on proper design of fluorescence probes with optimized photophysical properties. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Being smaller than the membrane width they can sense the gradients of these parameters across the membrane. We present examples of novel dyes that achieve increased spatial resolution and information content of the probe responses. In this respect, multiparametric environment-sensitive probes feature considerable promise.

  7. Fluorescent probe based on heteroatom containing styrylcyanine: pH-sensitive properties and bioimaging in vivo.

    PubMed

    Yang, Xiaodong; Gao, Ya; Huang, Zhibing; Chen, Xiaohui; Ke, Zhiyong; Zhao, Peiliang; Yan, Yichen; Liu, Ruiyuan; Qu, Jinqing

    2015-01-01

    A novel fluorescent probe based on heteroatom containing styrylcyanine is synthesized. The fluorescence of probe is bright green in basic and neutral media but dark orange in strong acidic environments, which could be reversibly switched. Such behavior enables it to work as a fluorescent pH sensor in the solution state and a chemosensor for detecting acidic and basic volatile organic compounds. Analyses by NMR spectroscopy confirm that the protonation or deprotonation of pyridinyl moiety is responsible for the sensing process. In addition, the fluorescent microscopic images of probe in live cells and zebrafish are achieved successfully, suggesting that the probe has good cell membrane permeability and low cytotoxicity.

  8. Molecular modulated cysteine-selective fluorescent probe.

    PubMed

    Jung, Hyo Sung; Pradhan, Tuhin; Han, Ji Hye; Heo, Kyung Jun; Lee, Joung Hae; Kang, Chulhun; Kim, Jong Seung

    2012-11-01

    We have synthesized a series of coumarins (1-3) that can emit fluorescence in a turn-on manner through a Michael-type reaction with thiol-containing compounds. The only difference among the coumarins is the position of a carboxyl group on its benzene ring moiety near the double-bond conjugated coumarin. Their selectivity for Cys, GSH, and Hcy as well as the associated fluorogenic mechanism were illustrated by fluorescence spectroscopy, DFT calculations, and kinetic studies. All isomers prefer Cys over GSH in the reaction from 48.6 (probe 3) to 111-fold (probe 1) as demonstrated in a second order kinetics. The high selectivity of probe 1 to Cys might be achieved since the ortho carboxyl group on its benzene ring prefers a less negatively charged nucleophile. During intracellular Cys detection using 1, a possible interference by a large amount of GSH in the HepG2 cells was evaluated. The cells were treated with l-buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, providing an experimental condition where the cells could not synthesize GSH from Cys or other species. Then, the fluorescence intensity of 1 in HepG2 cells under BSO-H(2)O(2) treatment was strongly enhanced by N-acetylcysteine (NAC), a precursor of Cys, implicating that the fluorescence signal from the cells is mainly associated with changes in intracellular [Cys] rather than that in intracellular [GSH].

  9. Gold nanoclusters as switch-off fluorescent probe for detection of uric acid based on the inner filter effect of hydrogen peroxide-mediated enlargement of gold nanoparticles.

    PubMed

    Liu, Yanyan; Li, Hongchang; Guo, Bin; Wei, Lijuan; Chen, Bo; Zhang, Youyu

    2017-05-15

    Herein we report a novel switch-off fluorescent probe for highly selective determination of uric acid (UA) based on the inner filter effect (IFE), by using poly-(vinylpyrrolidone)-protected gold nanoparticles (PVP-AuNPs) and chondroitin sulfate-stabilized gold nanoclusters (CS-AuNCs) as the IFE absorber/fluorophore pair. In this IFE-based fluorometric assay, the newly designed CS-AuNCs were explored as an original fluorophore and the hydrogen peroxide (H2O2) -driven formed PVP-AuNPs can be a powerful absorber to influence the excitation of the fluorophore, due to the complementary overlap between the absorption band of PVP-AuNPs and the emission band of CS-AuNCs. Under the optimized conditions, the extent of the signal quenching depends linearly on the H2O2 concentration in the range of 1-100μM (R(2) =0.995) with a detection limit down to 0.3μM. Based on the H2O2-dependent fluorescence IFE principle, we further developed a new assay strategy to enable selective sensing of UA by using a specific uricase-catalyzed UA oxidation as the in situ H2O2 generator. The proposed uricase-linked IFE-based assay exhibited excellent analytical performance for measuring UA over the concentration ranging from 5 to 100μM (R(2)=0.991), and can be successfully applied to detection of UA as low as 1.7μM (3σ) in diluted human serum samples.

  10. Boronic acid functionalized N-doped carbon quantum dots as fluorescent probe for selective and sensitive glucose determination

    NASA Astrophysics Data System (ADS)

    Jiang, Guohua; Jiang, Tengteng; Li, Xia; Wei, Zheng; Du, Xiangxiang; Wang, Xiaohong

    2014-04-01

    Nitrogen doped carbon quantum dots (NCQDs) of about 10 nm in diameter have been obtained by hydrothermal reaction from collagen. Because of the superiority of water dispersion, low toxicity and ease of functionlization, the NCQDs were designed as a glucose sensor after covalent grafting by 3-aminophenylboronic (APBA) (APBA-NCQDs). The as-prepared APBA-NCQDs were imparted with glucose sensitivity and selectivity from other saccharides via fluorescence (FL) quenching effect at physiological pH and at room temperature, which show high sensitivity and specificity for glucose determination with a wide range from 1 mM to 14 mM. FL quenching mechanism of APBA-NCQDs was also investigated by adding an external quencher. The APBA-NCQDs-based platform is an environmentally friendly way to substitute inorganic quantum dots containing heavy metals which offer a facile and low cost detection method.

  11. 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.

  12. Nitrogen-doped carbon dots as multifunctional fluorescent probes

    NASA Astrophysics Data System (ADS)

    Du, Fengyi; Jin, Xin; Chen, Junhui; Hua, Ye; Cao, Mulan; Zhang, Lirong; Li, Jianan; Zhang, Li; Jin, Jie; Wu, Chaoyang; Gong, Aihua; Xu, Wenrong; Shao, Qixiang; Zhang, Miaomiao

    2014-11-01

    Highly fluorescent nitrogen-doped carbon dots (NCDs) were prepared through the hydrothermal carbonization of citric acid and ammonium acetate. The resulting NCDs were quasi-spherical particles with an average diameter of approximately 2.1 nm. They exhibited excellent photoluminescent properties and had favorable solubility in water. Furthermore, the NCDs had low cytotoxicity and were readily integrated with cytoplasm. This makes them particularly suitable for multicolor bioimaging. Most importantly, NCDs internalized by cancer cells can be detected at four channels simultaneously with flow cytometry, which further demonstrates that the NCDs can be used as multifunctional fluorescent probes for biomedical applications.

  13. 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.

  14. A genetically encoded fluorescent probe in mammalian cells.

    PubMed

    Chatterjee, Abhishek; Guo, Jiantao; Lee, Hyun Soo; Schultz, Peter G

    2013-08-28

    Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

  15. Fluorescent polyene ceramide analogues as membrane probes.

    PubMed

    Nieves, Ingrid; Artetxe, Ibai; Abad, José Luis; Alonso, Alicia; Busto, Jon V; Fajarí, Lluís; Montes, L Ruth; Sot, Jesús; Delgado, Antonio; Goñi, Félix M

    2015-03-03

    Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively, at C5 (Penta5dox) and at C16 (Penta16dox) positions of the N-acyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 and 478 nm, respectively. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid-water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel-fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.

  16. 3-Aminophenylboronic acid-functionalized CuInS2 quantum dots as a near-infrared fluorescence probe for the detection of dicyandiamide.

    PubMed

    Liu, Siyu; Pang, Shu; Huang, Hui; Su, Xingguang

    2014-11-21

    In this paper, a simple and highly selective method for the determination of dicyandiamide (DCD) was developed based on the fluorescence quenching of functionalized CuInS2 quantum dots (QDs). Water-soluble CuInS2 QDs, capped by mercaptopropionic acid, were directly synthesized in aqueous solution and then covalently linked to 3-aminophenylboronic acid molecules to form the 3-aminophenylboronic acid-functionalized CuInS2 QDs (F-CuInS2 QDs) that had a fairly symmetric fluorescence emission centered at 736 nm. Based on the cyclization of the guanidine group of DCD with 2,3-butanedione and 3-aminophenylboronic acid, the fluorescence of the F-CuInS2 QDs is quenched by DCD in the presence of 2,3-butanedione. This method effectively distinguishes DCD from other amino acids and nitrogen pollutants, such as melamine, in real milk samples. Under optimum conditions, there was a good linear relationship between the fluorescence intensity of F-CuInS2 QDs and the concentration of DCD in the range of 2.0 × 10(-6) to 2.0 × 10(-3) mol L(-1), with a detection limit of 0.6 μmol L(-1).

  17. A ratiometric fluorescent probe for oxalate based on alkyne-conjugated carboxamidoquinolines in aqueous solution and imaging in living cells.

    PubMed

    He, Chunsheng; Qian, Xuhong; Xu, Yufang; Yang, Chunmei; Yin, Liyan; Zhu, Weiping

    2011-02-07

    A novel ratiometric fluorescent probe for oxalic acid was designed and synthesized, based on the zinc-containing [DAQZ@2Zn(2+)] complex. It shows highly selective "on-off" fluorescence changes with a more than 20 nm blue shift in wavelength for oxalic acids in aqueous solution. Moreover, it can fluorescently respond to oxalic acid in living cells.

  18. Focused fluorescent probe library for metal cations and biological anions.

    PubMed

    Rhee, Hyun-Woo; Lee, Sang Wook; Lee, Jun-Seok; Chang, Young-Tae; Hong, Jong-In

    2013-09-09

    A focused fluorescent probe library for metal cations was developed by combining metal chelators and picolinium/quinolinium moieties as combinatorial blocks connected through a styryl group. Furthermore, metal complexes derived from metal chelators having high binding affinities for metal cations were used to construct a focused probe library for phosphorylated biomolecules. More than 250 fluorescent probes were screened for identifying an ultraselective probe for dTTP.

  19. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    PubMed

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  20. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

    PubMed Central

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  1. A facile carbon dots based fluorescent probe for ultrasensitive detection of ascorbic acid in biological fluids via non-oxidation reduction strategy.

    PubMed

    Kong, Weiheng; Wu, Di; Li, Guoliang; Chen, Xuefeng; Gong, Peiwei; Sun, Zhiwei; Chen, Guang; Xia, Lian; You, Jinmao; Wu, Yongning

    2017-04-01

    A rapid, facile and ultrasensitive fluorescence sensing system based on nitrogen-doped carbon dots (N-doped CDs) for the detection of ascorbic acid (AA) was developed. The highly photoluminescent N-doped CDs with excellent solubility in water and good biocompatibility were prepared by a one-step hydrothermal synthesis and gave the fluorescence quantum yield of 47%. The addition of AA can intensively suppress the fluorescence of the N-doped CDs through the synergistic effect of the inner filter effect (IFE) and the static quenching effect (SQE). Benefited from the remarkable synergistic effect of IFE and SQE, a facile and ultrasensitive sensor was constructed successfully for AA sensing. The detection procedure was achieved within 2min. The linear response range of AA was obtained from 10(-3) to 10(-8) M with a detection limit of 5nM. This developed method enjoyed many merits including more simplicity, lost cost, high sensitivity, good selectivity, rapid response and excellent biocompatibility. Notably, the proposed fluorescent sensor exhibits excellent performance and applicability for AA determination in human serum and rat brain microdialysate, and may provide a fast yet facile route for AA detection in physiological and pathological fields.

  2. Aurones: Small Molecule Visible Range Fluorescent Probes Suitable for Biomacromolecules

    PubMed Central

    Shanker, Natasha; Dilek, Ozlem; Mukherjee, Kamalika; McGee, Dennis W.; Bane, Susan L.

    2017-01-01

    Aurones, derivatives of 2-benylidenebenzofuran-3 (2H)-one, are natural products that serve as plant pigments. There have been reports that some of these substances fluoresce, but little information about their optical properties is in the literature. In this report, series of aurone derivatives were synthesized as possible fluorescent probes that can be excited by visible light. We found that an amine substituent shifted the lowest energy absorption band from the near-UV to the visible region of the electromagnetic spectrum. Four amine-substituted aurone derivatives were synthesized to explore the effect of this substituent on the absorption and emission properties of the aurone chromophore. The emission maxima and intensities of the molecules are strongly dependent on the nature of the substituent and the solvent polarity. Overall, the emission intensity increases and the maximum wavelength decreases in less polar solvents; thus, the aurones may be useful probes for hydrophobic sites on biological molecules. A limited investigation with model protein, nucleic acid and fixed cells supports this idea. It is known that the sulfur analog of aurone can undergo photoinduced E/Z isomerization. This possibility was investigated for one of the aminoaurones, which was observed to reversible photoisomerize. The two isomers have similar absorption spectra, but the emission properties are distinct. We conclude that appropriately substituted aurones are potentially useful as biological probes and photoswitches. PMID:21748237

  3. Novel pyrazoline-based fluorescent probe for detecting thiols and its application in cells.

    PubMed

    Zhang, Rong-Rong; Zhang, Jin-Feng; Wang, Sheng-Qing; Cheng, Yan-Long; Miao, Jun-Ying; Zhao, Bao-Xiang

    2015-02-25

    A new compound, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-acrylamide (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting thiol from other amino acids. On being mixed with thiol in buffered DMSO:HEPES=1:1 solution at pH 7.4, the probe exhibited the blue emission at 474 nm. This probe is very sensitive and displayed a linear fluorescence off-on response to thiol. The fluorescence emission of the probe is pH independent in the physiological pH range. Living cell imaging of HeLa cells confirmed its cell permeability and its ability to selectively detect thiol in cells. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy analysis.

  4. Novel pyrazoline-based fluorescent probe for detecting thiols and its application in cells

    NASA Astrophysics Data System (ADS)

    Zhang, Rong-Rong; Zhang, Jin-Feng; Wang, Sheng-Qing; Cheng, Yan-Long; Miao, Jun-Ying; Zhao, Bao-Xiang

    2015-02-01

    A new compound, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-acrylamide (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting thiol from other amino acids. On being mixed with thiol in buffered DMSO:HEPES = 1:1 solution at pH 7.4, the probe exhibited the blue emission at 474 nm. This probe is very sensitive and displayed a linear fluorescence off-on response to thiol. The fluorescence emission of the probe is pH independent in the physiological pH range. Living cell imaging of HeLa cells confirmed its cell permeability and its ability to selectively detect thiol in cells. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy analysis.

  5. 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

  6. DBD dyes as fluorescent probes for sensing lipophilic environments.

    PubMed

    Wawrzinek, Robert; Wessig, Pablo; Möllnitz, Kristian; Nikolaus, Jörg; Schwarzer, Roland; Müller, Peter; Herrmann, Andreas

    2012-09-01

    Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).

  7. A general sensing strategy for detection of Fe3+ by using amino acid-modified graphene quantum dots as fluorescent probe

    NASA Astrophysics Data System (ADS)

    Ma, Qi; Song, Jinping; Wang, Shangzhi; Yang, Jie; Guo, Yong; Dong, Chuan

    2016-12-01

    Amino acid-modified graphene quantum dots (GQDs) were synthesized through acylation and amination reactions. The as-synthesized GQDs were characterized by high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and UV-vis absorption and fluorescence spectroscopy. A general sensing strategy for detection of Fe3+ was successfully developed. The detection limit can reach as low as 50 nM. Moreover, the proposed sensing system was successfully employed to detect Fe3+ in real water sample, and satisfactory results were obtained. This work will open up new avenues to develop potential applications of GQDs materials in environmental monitoring.

  8. 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.

  9. 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.

  10. Fluorescent probes for hydrogen sulfide detection and bioimaging.

    PubMed

    Yu, Fabiao; Han, Xiaoyue; Chen, Lingxin

    2014-10-21

    In comparison with other biological detection technologies, fluorescence bioimaging technology has become a powerful supporting tool for intracellular detection, and can provide attractive facilities for investigating physiological and pathological processes of interest with high spatial and temporal resolution, less invasiveness, and a rapid response. Due to the versatile roles of hydrogen sulfide (H2S) in cellular signal transduction and intracellular redox status regulation, fluorescent probes for the detection of this third signalling gasotransmitter have rapidly increased in number in recent years. These probes can offer powerful means to investigate the physiological actions of H2S in its native environments without disturbing its endogenous distribution. In this feature article, we address the synthesis and design strategies for the development of fluorescent probes for H2S based on the reaction type between H2S and the probes. Moreover, we also highlight fluorescent probes for other reactive sulfur species, such as sulfane sulfurs and SO2 derivatives.

  11. 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.

  12. A simple fluorescent probe for sensing cysteine over homocysteine and glutathione based on PET

    NASA Astrophysics Data System (ADS)

    Fan, Wenlong; Huang, Ximing; Shi, Xiaomin; Wang, Zhuo; Lu, Zhengliang; Fan, Chunhua; Bo, Qibing

    2017-02-01

    A big challenge is the discrimination of sulfhydryl-containing amino acids due to their structural similarity. We designed and synthesized a simple fluorescent probe 3 for specific detection of cysteine based on photo-induced electron transfer (PET). The acrylate and BODIPY moieties in probe 3 act as a reaction site and reporter group, respectively. So the synergistic effect of the substituent groups endows probe 3 very strong green fluorescence at 525 nm (λex = 500 nm). The cleavage reaction induced by cysteine leads to acrylate hydrolysis, and thereby triggers PET on, which effectively quench the fluorescence of 3. Probe 3 exhibited a rapid response towards cysteine over homocysteine and glutathione. Probe 3 is successfully applied for sensing and imaging cysteine in vitro or in vivo cells with low cytotoxicity.

  13. Folic acid-conjugated core/shell ZnS:Mn/ZnS quantum dots as targeted probes for two photon fluorescence imaging of cancer cells.

    PubMed

    Geszke, Malgorzata; Murias, Marek; Balan, Lavinia; Medjahdi, Ghouti; Korczynski, Jaroslaw; Moritz, Michal; Lulek, Janina; Schneider, Raphaël

    2011-03-01

    This work presents a novel approach to producing water soluble manganese-doped core/shell ZnS/ZnS quantum dots (ZnS:Mn/ZnS). The Mn-doped ZnS core was prepared through a nucleation doping strategy and a ZnS shell was grown on ZnS:Mn d-dots by decomposition of Zn(2+)-3-mercaptopropionic acid (MPA) complexes at 100 °C. It was found that the Mn2+(4)T1→6A1 fluorescence emission at ∼590 nm significantly increased after growth of the shell when the Mn2+ doping content was 4.0 at.%. A photoluminescence quantum yield of ∼22% was obtained for core/shell nanocrystals. The nanoparticles were structurally and compositionally characterized by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and dynamic light scattering. The surface MPA molecules favor the dispersion of ZnS:Mn/ZnS QDs in aqueous media and make possible conjugation with targeting folic acid molecules. The folate receptor-mediated delivery of folic acid-conjugated ZnS:Mn/ZnS QDs was demonstrated using confocal microscopy with biphotonic excitation. Bare and folate-conjugated QDs exhibit only weak cytotoxicity towards folate receptor-positive T47D cancer cells and MCF-7 cells, used as a reference, at high concentrations (mmolar range) after 72h incubation.

  14. Combined fiber probe for fluorescence lifetime and Raman spectroscopy

    PubMed Central

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-01-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. PMID:26093843

  15. Visual and fluorescent detection of tyrosinase activity by using a dual-emission ratiometric fluorescence probe.

    PubMed

    Yan, Xu; Li, Hongxia; Zheng, Weishi; Su, Xingguang

    2015-09-01

    In this work, we designed a dual-emission ratiometric fluorescence probe by hybridizing two differently colored quantum dots (QDs), which possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. Red emissive QDs were embedded in the silica nanoparticle as reference while the green emissive QDs were covalently linked to the silica nanoparticle surface to form ratiometric fluorescence probes (RF-QDs). Dopamine (DA) was then conjugated to the surface of RF-QDs via covalent bonding. The ratiometric fluorescence probe functionalized with dopamine (DA) was highly reactive toward tyrosinase (TYR), which can catalyze the oxidization of DA to dopamine quinine and therefore quenched the fluorescence of the green QDs on the surface of ratiometric fluorescence probe. With the addition of different amounts of TYR, the ratiometric fluorescence intensity of the probe continually varied, leading to color changes from yellow-green to red. So the ratiometric fluorescence probe could be utilized for sensitive and selective detection of TYR activity. There was a good linear relationship between the ratiometric fluorescence intensity and TYR concentration in the range of 0.05-5.0 μg mL(-1), with the detection limit of 0.02 μg mL(-1). Significantly, the ratiometric fluorescence probe has been used to fabricate paper-based test strips for visual detection of TYR activity, which validates the potential on-site application.

  16. Fluorescent probes for biomedical applications (2009-2014).

    PubMed

    Warrier, Sona; Kharkar, Prashant S

    2014-09-01

    The discovery and subsequent development of fluorescent probes was one of the most exciting innovations in life sciences, which marked the beginning of interpretation of numerous biological phenomena. Today, fluorescent probes are used for a wide range of biomedical applications, such as pharmaceutical research, clinical diagnostics and high-throughput screening, to name a few. Despite the availability of a large number of these probes, efforts to invent newer versions utilizing novel chemistry to address limitations of the current approaches continue. This review article gives a rundown on 'small-molecule fluorescent probes' patents/patent applications from January 2009 to March 2014. The patent literature was classified based on 'preparation' and 'biomedical applications' of these 'fluorescing wonders'.

  17. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  18. Functional nucleic acid probes and uses thereof

    DOEpatents

    Nilsen-Hamilton, Marit

    2006-10-03

    The present invention provides functional nucleic acid probes, and methods of using functional nucleic acid probes, for binding a target to carry out a desired function. The probes have at least one functional nucleic acid, at least one regulating nucleic acid, and at least one attenuator. The functional nucleic acid is maintained in an inactive state by the attenuator and activated by the regulating nucleic acid only in the presence of a regulating nucleic acid target. In its activated state the functional nucleic acid can bind to its target to carry out a desired function, such as generating a signal, cleaving a nucleic acid, or catalyzing a reaction.

  19. Nucleic acid based fluorescent nanothermometers.

    PubMed

    Ebrahimi, Sara; Akhlaghi, Yousef; Kompany-Zareh, Mohsen; Rinnan, Asmund

    2014-10-28

    Accurate thermometry at micro- and nanoscales is essential in many nanobiotechnological applications. The nanothermometers introduced in this paper are composed of labeled molecular beacons (MBs) comprising gold nanoparticles (AuNPs) on which, depending on application, many MBs of one or more types are immobilized. In this design, three differently labeled MBs with different thermostabilities function as the sensing elements, and AuNPs act as carriers of the MBs and also quenchers of their fluorophores. This flexible design results in a number of nanothermometers with various temperature-sensing ranges. At the lowest temperature, the MBs are in the closed form, where they are quenched. By increasing the temperature, the MBs start to open with respect to their melting points (Tm), and as a result, the fluorescence emission will increase. The temperature resolution of the nanoprobes over a range of 15-60 °C is less than 0.50 °C, which indicates their high sensitivity. Such a good temperature resolution is a result of the specific design of the unusual less stable MBs and also presence of many MBs on AuNPs. The reproducibility and precision of the probes are also satisfactory. The multiplex MB nanoprobe is suitable for thermal imaging by fluorescence microscopy.

  20. 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.

  1. Doped semiconductor nanocrystal based fluorescent cellular imaging probes.

    PubMed

    Maity, Amit Ranjan; Palmal, Sharbari; Basiruddin, S K; Karan, Niladri Sekhar; Sarkar, Suresh; Pradhan, Narayan; Jana, Nikhil R

    2013-06-21

    Doped semiconductor nanocrystals such as Mn doped ZnS, Mn doped ZnSe and Cu doped InZnS, are considered as new classes of fluorescent biological probes with low toxicity. Although the synthesis in high quality of such nanomaterials is now well established, transforming them into functional fluorescent probes remains a challenge. Here we report a fluorescent cellular imaging probe made of high quality doped semiconductor nanocrystals. We have identified two different coating approaches suitable for transforming the as synthesized hydrophobic doped semiconductor nanocrystals into water-soluble functional nanoparticles. Following these approaches we have synthesized TAT-peptide- and folate-functionalized nanoparticles of 10-80 nm hydrodynamic diameter and used them as a fluorescent cell label. The results shows that doped semiconductor nanocrystals can be an attractive alternative for conventional cadmium based quantum dots with low toxicity.

  2. 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.

  3. Doped semiconductor nanocrystal based fluorescent cellular imaging probes

    NASA Astrophysics Data System (ADS)

    Maity, Amit Ranjan; Palmal, Sharbari; Basiruddin, Sk; Karan, Niladri Sekhar; Sarkar, Suresh; Pradhan, Narayan; Jana, Nikhil R.

    2013-05-01

    Doped semiconductor nanocrystals such as Mn doped ZnS, Mn doped ZnSe and Cu doped InZnS, are considered as new classes of fluorescent biological probes with low toxicity. Although the synthesis in high quality of such nanomaterials is now well established, transforming them into functional fluorescent probes remains a challenge. Here we report a fluorescent cellular imaging probe made of high quality doped semiconductor nanocrystals. We have identified two different coating approaches suitable for transforming the as synthesized hydrophobic doped semiconductor nanocrystals into water-soluble functional nanoparticles. Following these approaches we have synthesized TAT-peptide- and folate-functionalized nanoparticles of 10-80 nm hydrodynamic diameter and used them as a fluorescent cell label. The results shows that doped semiconductor nanocrystals can be an attractive alternative for conventional cadmium based quantum dots with low toxicity.Doped semiconductor nanocrystals such as Mn doped ZnS, Mn doped ZnSe and Cu doped InZnS, are considered as new classes of fluorescent biological probes with low toxicity. Although the synthesis in high quality of such nanomaterials is now well established, transforming them into functional fluorescent probes remains a challenge. Here we report a fluorescent cellular imaging probe made of high quality doped semiconductor nanocrystals. We have identified two different coating approaches suitable for transforming the as synthesized hydrophobic doped semiconductor nanocrystals into water-soluble functional nanoparticles. Following these approaches we have synthesized TAT-peptide- and folate-functionalized nanoparticles of 10-80 nm hydrodynamic diameter and used them as a fluorescent cell label. The results shows that doped semiconductor nanocrystals can be an attractive alternative for conventional cadmium based quantum dots with low toxicity. Electronic supplementary information available: Characterization details of coating and

  4. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA.

    PubMed

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  5. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  6. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao

    2012-03-01

    This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the

  7. Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

    PubMed

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2015-11-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

  8. A simple and effective strategy to increase the sensitivity of fluorescence probes in living cells.

    PubMed

    Izumi, Saki; Urano, Yasuteru; Hanaoka, Kenjiro; Terai, Takuya; Nagano, Tetsuo

    2009-07-29

    Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists, and fluorescence probes are powerful tools for this purpose. Because many target molecules are present in only trace amounts, high sensitivity is very important, and it is common to improve the sensitivity of fluorescence probes by focusing on high reaction velocity, K(d). (Gee, K. R.; Archer, E. A.; Lapham, L. A.; Leonard, M. E.; Zhou, Z.; Bingham, J.; Diwu, Z. Bioorg. Med. Chem. Lett. 2000, 10, 1515-1518.) So far, we have designed and synthesized various highly sensitive fluorescence probes based on the above concepts. (Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357-3367. Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447-13454.) Nevertheless, they were sometimes insufficiently sensitive to detect biomolecules in living cells, despite high chemical sensitivity in cuvette. In this report, we suggest a new approach to increase the sensitivity of fluorescence probes, focusing on their intracellular retention. Since calcein is well-retained, we investigated its structural, chemical, and optical characteristics and found that the iminodiacetic acid group (IAG) is a key structure for the intracellular retention. We next designed and synthesized novel fluorescence probes containing IAGs. They showed superior intracellular retention, making it possible to visualize low concentrations of target molecules that would be difficult to observe with conventional probes and permitting long-term observation in living cells. Improvement of intracellular retention of fluorescence probes holds great promise as a strategy for developing a wide range of highly sensitive probes for studies on various biological phenomena.

  9. Fluorescent probe ABM for screening gastrointestinal patient's immune state.

    PubMed

    Kalnina, Inta; Klimkane, Laura; Kirilova, Elena; Toma, Maija Malda; Kizane, Gunta; Meirovics, Imants

    2007-11-01

    The fluorescent probe-aminoderivative of benzanthrone, ABM (developed at Riga Technical University, Riga, Latvia) was used to characterize the membranes of lymphocytes of cancer patients: 46 patients with gastrointestinal diseases, 13 patients having different primary localizations with massive metastases and intoxication. Patients were divided into three groups: (1) with decreased fluorescence intensity, (2) normal fluorescence intensity, (3) increased fluorescence intensity. The lymphocytes distribution among subsets differed between groups, in correspondence to the level of fluorescence intensity. Surgical treatment affected the main immunological parameters and elevated the functional activity of lymphocytes. In the advanced tumors group, fluorescence intensity correlates with the survival rate. Results suggest that determination of lymphocytes functional activity by ABM can aid evaluation of the immune status in cancer patients.

  10. Zinc(II)-selective ratiometric fluorescent probe based on perylene bisimide derivative.

    PubMed

    Zhao, Yingjie; Sun, Juanjuan; Shi, Zhiqiang; Pan, Cuicui; Xu, Maoyou

    2011-01-01

    A fluorescent probe of N,N'-biscyclohexyl-1,7-di(3-pyridoxy)-perylene-3,4:9,10-tetracarboxylic acid diimide has been synthesized, and exhibited excellent selectivity and sensitivity for Zn(2+) over other competing biological cations. The Zn(2+) -selective fluorescence blue-shift and enhancing property in conjunction with a visible colorimetric change from orange to green could be observed. With favorable photophysical properties in the visible region, the perylene bisimide derivatives remarkably improved the performance of the probe.

  11. 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.

  12. Sensitivity of activatable reactive oxygen species probes by fluorescence spectroelectrochemistry

    PubMed Central

    Wang, Steven T.; Zhegalova, Natalia G.; Gustafson, Tiffany P.; Zhou, Andy; Sher, Joel; Achilefu, Samuel; Berezinand, Oleg Y.

    2013-01-01

    We have developed a new analytical method of evaluating activatable fluorescent probes for ROS detection using integrated fluorescence spectroelectrochemistry. Tafel formalism was applied to describe the process of the probes’ oxidation under electrochemical conditions and identify a novel parameter defined as the threshold oxidation potential. This potential can serve as an approximation to the equilibrium potential and can be utilized for determining the sensitivity of a probe to oxidation. Based upon the measured values of threshold potentials, the order of sensitivity towards oxidation among several mostly used probes was determined to be following (from highest to lowest): 2,7-dichlorodihydrofluorescein > dihydroethidium > dihydrorhodamine 123 > dihydrorhodamine 6G. The presented approach opens up a new direction in synthesizing and screening novel ROS probes with a well-defined sensitivity for in vitro and in vivo applications. PMID:23736882

  13. 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.

  14. Superior fluorescent probe for detection of cardiolipin.

    PubMed

    Leung, Chris W T; Hong, Yuning; Hanske, Jonas; Zhao, Engui; Chen, Sijie; Pletneva, Ekaterina V; Tang, Ben Zhong

    2014-01-21

    Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated.

  15. A fluorescence enhancement probe based on BODIPY for the discrimination of cysteine from homocysteine and glutathione.

    PubMed

    Gong, Deyan; Tian, Yuejun; Yang, Chengduan; Iqbal, Anam; Wang, Zhiping; Liu, Weisheng; Qin, Wenwu; Zhu, Xiangtao; Guo, Huichen

    2016-11-15

    Herein, a fluorescent probe BODIPY-based glyoxal hydrazone (BODIPY-GH) (1) for cysteine based on inhibiting of intramolecular charge transfer (ICT) quenching process upon reaction with the unsaturated aldehyde has been synthesized, which exhibits longer excitation wavelength, selective and sensitive colorimetric and fluorimetric response toward cysteine in natural media. The probe shows highly selectivity towards cysteine over homocysteine and glutathione as well as other amino acids with a significant fluorescence enhancement response within 15min In the presence of 50 equiv. of homocysteine, the emission increased slightly within 15min and completed in 2.5h to reach its maximum intensity. Therefore, the discrimination of cysteine from homocysteine and glutathione can be achieved through detection of probe 1. It shows low cytotoxicity and excellent membrane permeability toward living cells, which was successfully applied to detect and image intracellular cysteine effectively by confocal fluorescence imaging.

  16. A Microfluidic Microbeads Fluorescence Assay with Quantum Dots-Bead-DNA Probe.

    PubMed

    Ankireddy, S R; Kim, Jongsung

    2016-03-01

    A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA.

  17. Fluorescence-based Broad Dynamic Range Viscosity Probes.

    PubMed

    Dragan, Anatoliy; Graham, August E; Geddes, Chris D

    2014-03-01

    We introduce two new fluorescent viscosity probes, SYBR Green (SG) and PicoGreen (PG), that we have studied over a broad range of viscosity and in collagen solutions. In water, both dyes have low quantum yields and excited state lifetimes, while in viscous solvents or in complex with DNA both parameters dramatically (300-1000-fold) increase. We show that in log-log scale the dependence of the dyes' quantum yield vs. viscosity is linear, the slope of which is sensitive to temperature. Application of SG and PG, as a fluorescence-based broad dynamic range viscosity probes, to the life sciences is discussed.

  18. Nucleic acid probes in diagnostic medicine

    NASA Technical Reports Server (NTRS)

    Oberry, Phillip A.

    1991-01-01

    The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed.

  19. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo

    PubMed Central

    Sawa, Masaaki; Hsu, Tsui-Ling; Itoh, Takeshi; Sugiyama, Masakazu; Hanson, Sarah R.; Vogt, Peter K.; Wong, Chi-Huey

    2006-01-01

    Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycan-labeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates. To address this issue, we developed a fluorescent labeling technique based on the Cu(I)-catalyzed [3 + 2] cycloaddition, or click chemistry, which allows rapid, versatile, and specific covalent labeling of cellular glycans bearing azide groups. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by clicking the fluorescent trigger, the alkyne at the 4 position, with an azido-modified sugar. Using this click-activated fluorescent probe, we demonstrate incorporation of an azido-containing fucose analog into glycoproteins via the fucose salvage pathway. Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or biotinylated alkyne. The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy. This technique will allow dynamic imaging of cellular fucosylation and facilitate studies of fucosylated glycoproteins and glycolipids. PMID:16895981

  20. Synthesis, metal coordination, and cellular internalization of a siderophore-bearing NIR fluorescent carbocyanine probe

    NASA Astrophysics Data System (ADS)

    Ye, Yunpeng; Xu, Baogang; Bloch, Sharon; Achilefu, Samuel

    2006-02-01

    In order to explore novel NIR fluorescent probes for optical imaging in biomedicines, one desferrioxamine (DFO)-bearing NIR fluorescent probe was designed and synthesized based on a dicarboxylic acid-containing carbocyanine (Cypate). Similar to the free DFO, the resulting conjugate Cypate-DFO showed high binding affinity with Fe(III) and Ga(III) as identified by ES-MS. Nevertheless, the iron binding was found to quench its fluorescent emission significantly, suggesting that the siderophore moiety might perturb the spectroscopic properties of the attached carbocyanine fluorophore through metal binding. As observed by fluorescence microscopy, Cypate-DFO showed significant cellular internalization in A549 cells in vitro. Further studies on novel Cypate-DFO derivatives of this type may reveal some exciting properties and biological activities.

  1. 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.

  2. Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.

    PubMed

    Han, Zhou; Jin, Lei; Chen, Fuyi; Loturco, Joseph J; Cohen, Lawrence B; Bondar, Alexey; Lazar, Josef; Pieribone, Vincent A

    2014-01-01

    ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

  3. 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.

  4. 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.

  5. Fluorescence Studies Of A Cholesterol-Analogue Probe

    NASA Astrophysics Data System (ADS)

    Drew, Jacinta; Szabo, Arthur G.; Morand, Peter

    1988-06-01

    A novel cholesterol-analogue probe1,2 with a diene-(2-naphthyl) fluorophore in the sidechain (Figure 1), hereafter referred to as DN-Chol, has had its steady-state and time-resolved fluorescence properties characterized in solvents and in various viscosity mineral oils.

  6. Two-photon fluorescent probe for cadmium imaging in cells.

    PubMed

    Liu, Yongyou; Dong, Xiaohu; Sun, Jian; Zhong, Cheng; Li, Boheng; You, Ximeng; Liu, Bifeng; Liu, Zhihong

    2012-04-21

    A novel two-photon excited fluorescent probe for cadmium (named as TPCd) was designed and synthesized utilizing a prodan (6-acetyl-2-methoxynaphthalene) derivative as the two-photon fluorophore and an o-phenylenediamine derivative as the Cd(2+) chelator, which possessed favorable photophysical properties and good water-solubility. The probe was designed with a photoinduced electron transfer (PET) mechanism and thus was weakly fluorescent itself. After binding with Cd(2+) which blocked the PET process, the fluorescence intensity of the probe was enhanced by up to 15-fold under one-photon excitation (OPE) and 27-fold under two-photon excitation (TPE), respectively. The two-photon action cross-section (Φδ) of the TPCd-Cd complex at 740 nm reached 109 GM compared to 3.6 GM for free TPCd, indicating the promising prospect of the probe in two-photon application. TPCd chelated Cd(2+) with 1 : 1 stoichiometry, and the apparent dissociation constant (K(d)) was 6.1 × 10(-5) M for the one-photon mode and 7.2 × 10(-5) M for the two-photon mode. The probe responded to Cd(2+) over a wide linear range from 0.1 to 30 μM with a detection limit of 0.04 μM. High selectivity of the probe towards Cd(2+) was acquired in Tris-HCl/sodium phosphate buffer. The probe was pH-independent in the biologically relevant pH range and non-toxic to living cells at reasonable concentration levels, warranting its in vivo applications. Through two-photon microscopy imaging, the probe was successfully applied to detect Cd(2+) uptake in living HepG2 cells.

  7. A hyperspectral fluorescence lifetime probe for skin cancer diagnosis

    NASA Astrophysics Data System (ADS)

    De Beule, P. A. A.; Dunsby, C.; Galletly, N. P.; Stamp, G. W.; Chu, A. C.; Anand, U.; Anand, P.; Benham, C. D.; Naylor, A.; French, P. M. W.

    2007-12-01

    The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.

  8. New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging.

    PubMed

    Resa, Sandra; Orte, Angel; Miguel, Delia; Paredes, Jose M; Puente-Muñoz, Virginia; Salto, Rafael; Giron, Maria D; Ruedas-Rama, Maria J; Cuerva, Juan M; Alvarez-Pez, Jose M; Crovetto, Luis

    2015-10-12

    The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

  9. Probe for intracellular concentrations of drugs: delayed fluorescence from acridine orange

    SciTech Connect

    Wardman, P.; Dennis, M.F.; White, J.

    1989-04-01

    The aim of this work is to develop fluorescent probes that will indicate effective concentrations of therapeutic agents, or endogenous protectors, at important cellular sites. Acridine orange associates with nucleic acids and emits a 'delayed' fluorescence signal. This signal is quenched by oxidants such as oxygen, nitroaryl radiosensitizers, adriamycin and mitomycin-c, and reductants such as thiols, ascorbate and other radioprotectors. The quenching of the acridine orange delayed fluorescence reflects the effective concentration of these therapeutically-important oxidants and reductants near DNA. The relative concentration of basic radiosensitizers such as pimonidazole (Ro 03-8799) near the DNA is greater than that of misonidazole. Thiols quench the delayed fluorescence signal according to the degree of ionization of the thiol function; this may model the reactivity of thiols with guanine radical sites in DNA. Ascorbate and aminopyrine do not quench the delayed fluorescence from cells stained with acridine orange as these compounds are taken up by cells very inefficiently.

  10. Fluorescence In Situ Hybridization Probe Preparation.

    PubMed

    Tolomeo, Doron; Stanyon, Roscoe R; Rocchi, Mariano

    2017-01-01

    The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A "golden path" of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800-1000 bp) at each end allowed them to be very precisely mapped on the human genome.The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56-64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.

  11. 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.

  12. An enzymatically activated fluorescence probe for targeted tumor imaging

    PubMed Central

    Kamiya, Mako; Kobayashi, Hisataka; Hama, Yukihiro; Koyama, Yoshinori; Bernardo, Marcelino; Nagano, Tetsuo; Choyke, Peter L.; Urano, Yasuteru

    2008-01-01

    β-Galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-βGal (J. Am. Chem. Soc., 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-βGal, provides a dramatic fluorescence enhancement upon reaction with β-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-βGal, after confirming that tumors in the model could be labeled with avidin-β-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-βGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 μm. This strategy has potential clinical application, for example in video-assisted laparoscopic tumor resection. PMID:17352471

  13. Fluorescent Probes for H2S Detection and Quantification.

    PubMed

    Feng, Wei; Dymock, Brian W

    2015-01-01

    Many diverse, sensitive and structurally novel fluorescent probes have recently been reported for H2S detection. Quantification of H2S requires a selective chemosensor which will react only with H2S against a background of high concentrations of other thiols or reducing agents. Most published probes are able to quantify H2S selectively in a simple in vitro system with the most sensitive probes able to detect H2S at below 100 nM concentrations. A subset of probes also have utility in sensing H2S in living cells, and there are now several with specific sub-cellular localization and a few cases of in vivo applications. Biologists studying H2S now have a wide range of tools to assist them to aid further understanding of the role of H2S in biology.

  14. Fluorescent cyanine probe for DNA detection and cellular imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Yong-Chao; Zheng, Mei-Ling; Zhao, Zhen-Sheng; Duan, Xuan-Ming

    2014-03-01

    In our study, two carbazole-based cyanines, 3,6-bis[2-(1-methylpyridinium)vinyl]-9-methyl carbazole diiodide (A) and 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3yl)methane diiodide (B) were synthesized and employed as light-up probes for DNA and cell imaging. Both of the cyanine probes possess a symmetric structure and bis-cationic center. The obvious induced circular dichroism signals in circular dichroism spectra reveal that the molecules can specifically interact with DNA. Strong fluorescence enhancement is observed when these two cyanines are bound to DNA. These cyanine probes show high binding affinity to oligonucleotides but different binding preferences to various secondary structures. Confocal microscopy images of fixed cell stained by the probes exhibit strong brightness and high contrast in nucleus with a very low cytoplasmic background.

  15. 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.

  16. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed

    Colarusso, Pina; Spring, Kenneth R

    2002-02-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane.

  17. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed Central

    Colarusso, Pina; Spring, Kenneth R

    2002-01-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane. PMID:11806917

  18. New fluorescent probes for detection and characterization of amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Gorbenko, Galyna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Kalnina, Inta; Vasilev, Aleksey; Kaloyanova, Stefka; Deligeorgiev, Todor

    2010-08-01

    The applicability of the novel fluorescent probes, aminoderivative of benzanthrone ABM, squaraine dye SQ-1 and polymethine dye V2 to identification and structural analysis of amyloid fibrils has been evaluated using the lysozyme model system in which fibrillar aggregates have been formed in concentrated ethanol solution. The association constant, binding stoichiometry and molar fluorescence of the bound dye have been determined. ABM was found to surpass classical amyloid marker ThT in the sensitivity to the presence of fibrillar aggregates. Resonance energy transfer measurements involving ABM-SQ-1 and SQ-1-V2 donor-acceptor pairs yielded the limits for fractal-like dimension of lysozyme fibrils.

  19. Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers.

    PubMed

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

    2004-05-01

    A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.

  20. Dual-emissive fluorescence measurements of hydroxyl radicals using a coumarin-activated silica nanohybrid probe.

    PubMed

    Liu, Saisai; Zhao, Jun; Zhang, Kui; Yang, Lei; Sun, Mingtai; Yu, Huan; Yan, Yehan; Zhang, Yajun; Wu, Lijun; Wang, Suhua

    2016-04-07

    This work reports a novel dual-emissive fluorescent probe based on dye hybrid silica nanoparticles for ratiometric measurement of the hydroxyl radical (˙OH). In the probe sensing system, the blue emission of coumarin dye (coumarin-3-carboxylic acid, CCA) immobilized on the nanoparticle surface is selectively enhanced by ˙OH due to the formation of a coumarin hydroxylation product with strong fluorescence, whereas the emission of red fluorescent dye encapsulated in the silica nanoparticle is insensitive to ˙OH as a self-referencing signal, and so the probe provides a good quantitative analysis based on ratiometric fluorescence measurement with a detection limit of 1.65 μM. Moreover, the probe also shows high selectivity for ˙OH determination against metal ions, other reactive oxygen species and biological species. More importantly, it exhibits low cytotoxicity and high biocompatibility in living cells, and has been successfully used for cellular imaging of ˙OH, showing its promising application for monitoring of intracellular ˙OH signaling events.

  1. Reaction Fields in the Environment of Fluorescent Probes: Polarity Profiles in Membranes

    PubMed Central

    Marsh, Derek

    2009-01-01

    Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains. PMID:19348740

  2. Reaction fields in the environment of fluorescent probes: polarity profiles in membranes.

    PubMed

    Marsh, Derek

    2009-04-08

    Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains.

  3. Off-On-Off fluorescence behavior of an intramolecular charge transfer probe toward anions and CO2

    NASA Astrophysics Data System (ADS)

    Ali, Rashid; Razi, Syed S.; Shahid, Mohammad; Srivastava, Priyanka; Misra, Arvind

    2016-11-01

    The photophysical behavior of a newly developed fluorescent probe, tricyanoethylphenyl phenanthroimidazole (TCPPI) has been studied. Upon interaction of different class of anions TCPPI displayed naked-eye sensitive fluorescence "turn-on" response to detect selectively F- (0.98 μM, 18.62 ppb) and CN- (1.12 μM, 29.12 ppb) anions in acetonitrile (MeCN). Job's plot analysis revealed a 1:1 binding stoichiometry between probe and anions. The spectral data analysis and 1H NMR titration studies suggested about the affinity of F- and CN- anions with moderately acidic - NH fragment of imidazolyl unit of probe through deprotonation and H-bonding interaction. Moreover, the anion activated probe upon interaction with CO2 revived photophysical properties of probe, "On-Off-On" type fluorescence and enabled anion-induced CO2 sensing in the medium.

  4. Two-photon fluorescent probes for biological Mg(2+) detection based on 7-substituted coumarin.

    PubMed

    Yin, Haijing; Zhang, Buchang; Yu, Haizhu; Zhu, Lin; Feng, Yan; Zhu, Manzhou; Guo, Qingxiang; Meng, Xiangming

    2015-05-01

    Two novel water-soluble coumarin-based compounds (OC7, NC7) were designed and synthesized as two-photon fluorescent probes for biological Mg(2+) detection. The compounds feature a β-keto acid as a high selective binding site for Mg(2+) and the coumarin framework as the two-photon fluorophore. OC7 and NC7 show significant "off-on" detecting signals (9.05-fold and 23.8-fold fluorescence enhancement) and lower detection limits compared with previous reported two-photon fluorescent probes for Mg(2+). Moreover, OC7-Mg(2+) and NC7-Mg(2+) exhibit large two-photon absorption cross sections (340 and 615 GM) at the near-infrared wavelengths (740 and 860 nm), which indicates that the probes are very suitable for detection of Mg(2+) in vivo. Both OC7 and NC7 are pH-insensitive and of low cytotoxicity and can be applied to image intracellular Mg(2+) under two-photon microscopy (TPM). Our results provide a strategy to modify the coumarin fluorophore to get better two-photon fluorescent properties. And the results also suggest that electronic density of β-keto acid plays a very important role in the recognition of Mg(2+).

  5. A novel fluorescent probe: europium complex hybridized T7 phage.

    PubMed

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  6. Multiparametric probing of microenvironment with solvatochromic fluorescent dyes.

    PubMed

    Klymchenko, Andrey S; Demchenko, Alexander P

    2008-01-01

    We describe new methodology for multiparametric probing of weak non-covalent interactions in the medium based on response of environment-sensitive fluorescent dyes. The commonly used approach is based on correlation of one spectroscopic parameter (e.g. wavelength shift) with environment polarity, which describes a superposition of universal and specific (such as hydrogen bonding) interactions. In our approach, by using several independent spectroscopic parameters of a dye, we monitor simultaneously each individual type of the interactions. For deriving these extra parameters the selected dye should exist in several excited and/or ground states. In the present work, we applied 4'-(diethylamino)-3-hydroxyflavone, which undergoes the excited-state intramolecular proton transfer (ESIPT) and thus exhibits an additional emission band belonging to an ESIPT product (tautomer) form of the dye. The spectroscopic characteristics of the excited normal and the tautomer states as well as of the ESIPT reaction of the dye are differently sensitive to the different types of interactions with microenvironment and therefore can be used for its multiparametric description. The new methodology allowed us to monitor simultaneously three fundamental physicochemical parameters of probe microenvironment: polarity, electronic polarizability and H-bond donor ability. The applications of this approach to binary solvent mixtures, reverse micelles, lipid bilayers and binding sites of proteins are presented and the limitations of this approach are discussed. We believe that the methodology of multiparametric probing will extend the capabilities of fluorescent probes as the tools in biomolecular and cellular research.

  7. Heterogeneous rotational diffusion of a fluorescent probe in lipid monolayers

    PubMed Central

    Dadashvand, Neda; Williams, LaNell A.

    2014-01-01

    The rotational correlation time of the lipid probe 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) is measured using fluorescence anisotropy for two lipid species. We measure the rotational diffusion in a monolayer of 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DPPC) which displays a phase transition at room temperature from the liquid-expanded to the liquid-condensed phase. The constant rotational diffusion of the probe throughout the phase transition reflects the measurement of dynamics in only the liquid-expanded phase. We contrast the dynamic changes during this phase coexistence to the continuous density increase observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at room temperature. We observe a non-exponential decay of the probe diffusion consistent with heterogeneity of the orientational dynamics. PMID:26798782

  8. Heterogeneous rotational diffusion of a fluorescent probe in lipid monolayers.

    PubMed

    Dadashvand, Neda; Williams, LaNell A; Othon, Christina M

    2014-09-01

    The rotational correlation time of the lipid probe 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) is measured using fluorescence anisotropy for two lipid species. We measure the rotational diffusion in a monolayer of 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DPPC) which displays a phase transition at room temperature from the liquid-expanded to the liquid-condensed phase. The constant rotational diffusion of the probe throughout the phase transition reflects the measurement of dynamics in only the liquid-expanded phase. We contrast the dynamic changes during this phase coexistence to the continuous density increase observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at room temperature. We observe a non-exponential decay of the probe diffusion consistent with heterogeneity of the orientational dynamics.

  9. 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).

  10. Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity.

    PubMed

    Zeng, Yan-Syun; Gao, Ruo-Cing; Wu, Ting-Wei; Cho, Chien; Tan, Kui-Thong

    2016-08-17

    Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes to overcome these two problems. The probes were constructed by following the current probe design for enzymes and reactive small molecules but with an additional benzylguanine moiety for selective SNAP-tag conjugation. The SNAP-tag conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma, whereas analyte concentrations were overestimated up to 700-fold when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically, as our probes displayed 390-fold fluorescence enhancement upon SNAP-tag conjugation, in stark contrast to the weak fluorescence of the free probes in aqueous solutions. Compared with the conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcame many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

  11. Heterogeneity in maize starch granule internal architecture deduced from diffusion of fluorescent dextran probes.

    PubMed

    Dhital, Sushil; Shelat, Kinnari J; Shrestha, Ashok K; Gidley, Michael J

    2013-04-02

    Heterogeneity in maize starch granules was investigated by studying the diffusion of fluorescent dextran probes (20, 70 and 150kDa) inside granules using fluorescence recovery after photobleaching combined with confocal microscopy. Access of probes to the interior of granules was greatly enhanced by limited (2.4%) amylolysis. The diffusion of probes within granules was found to be either 'fast' with diffusion coefficients in the order of 10(-6)cm(2)s(-1) or 'slow' with diffusion coefficients in the order of 10(-7)cm(2)s(-1), independent of the size of dextran probes or prior treatment of the granules by α-amylase. Results were compared with observations of pores and channels in granules by electron microscopy and by confocal microscopy after labelling with 8-amino-1,3,6-pyrenetrisulfonic acid. It is proposed that there is an inherent heterogeneity of internal architecture in maize starch granules due to the presence or absence in individual granules of (a) pores leading to a central cavity, resulting in 'fast' diffusion of dextran probes and (b) accessibility of the starch polymer matrix to dextran probes, leading to 'slow' diffusion behaviour. The observed heterogeneity of maize starch granule porosity has implications for chemical modification reactions and the kinetics of digestion with amylases.

  12. Sensitive fluorescence response of ZnSe(S) quantum dots: an efficient fluorescence probe

    NASA Astrophysics Data System (ADS)

    Saikia, K.; Deb, P.; Kalita, E.

    2013-06-01

    An efficient fluorescence probe based on ZnSe(S) alloyed quantum dots (QDs) has been reported here. The alloyed QDs were prepared through an aqueous route, where 3-mercaptopropionic acid (MPA) was employed as the effective precursor for both the sulfur source and stabilizer in the development of the alloyed system. Five-fold quantum yield (QY) enhancement was obtained for the ZnSe(S) QDs compared to the ZnSe QDs, formed in the initial stage of the refluxing process. The ultimate alloyed systems retained their high biocompatibility characteristics similar to the conventional ZnSe QDs. The photoluminescence of the ZnSe(S) QDs showed pH dependence, which was also evidenced in mammalian lymphocyte cells suspended in biological buffer over a wide pH range of 4.00-12.00. These characteristics make our prepared ZnSe(S) an efficient system for development of cell tracking, monitoring and sensing intracellular nanoprobes and devices.

  13. Fluorescence probes for studying the mechanisms of transcription activation

    NASA Astrophysics Data System (ADS)

    Heyduk, Tomasz; Callaci, Sandhya

    1994-08-01

    Regulation of transcription involves a complex interplay between protein-ligand, protein-DNA, and protein-protein interactions. Fluorescence probes seem to be very well suited to study such complex systems since the selectivity and sensitivity of fluorescence makes possible to select only a part of the system for observation leaving the rest of it transparent to the technique. We have used fluorescence spectroscopy to study the activation of E.coli RNA polymerase by cAMP receptor protein (CRP). The cAMP interactions with CRP, domain flexibility in CRP molecule, the structure of CRP-DNA complex, and interaction of CRP with RNA-polymerase have been studied. Here we report the preparation and properties of 5-OH-Trp derivative of the sigma subunit of E.coli RNA polymerase. This subunit is responsible for specific promoter recognition. The obtained results show that the biological activities of the derivative are identical as observed for the native protein. Comparison of fluorescence properties of the 5-OH-Trp sigma derivative free and bound to the core RNA polymerase suggests a conformational change in the sigma protein induced by this interaction. These data show that replacement of Trp residues with 5-OH-Trp can be a very useful approach to prepare specific fluorescence derivatives of multimeric proteins.

  14. 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

  15. 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

  16. Probing Lipid Membrane Rafts (Microdomains) with Fluorescent Phospholipids

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Mitchel, Drake

    2011-10-01

    Membrane rafts are enriched in sphingolipids and cholesterol, they exist in a more ordered state (the liquid-ordered phase; lo) than the bulk membrane (the liquid-disordered phase; ld). Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form membrane rafts over a wide range of molar ratios. We are examining the ability of two fluorescent probes, NBD linked to di-16:0 PE which partitions into the lo phase, and NBD linked to di-18:1 PE which partitions into the ld phase, to detect these two phases. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the size and stability of POPC/SPM/Chol membrane rafts. We report on the fluorescence lifetime and anisotropy decay dynamics of two fluorescent probes. Data were acquired via frequency-domain measurements from 5 to 250 MHz.

  17. Fluorescence probes to detect lipid-derived radicals.

    PubMed

    Yamada, Ken-Ichi; Mito, Fumiya; Matsuoka, Yuta; Ide, Satsuki; Shikimachi, Kazushige; Fujiki, Ayano; Kusakabe, Daiki; Ishida, Yuma; Enoki, Masataka; Tada, Arisa; Ariyoshi, Miyuki; Yamasaki, Toshihide; Yamato, Mayumi

    2016-08-01

    Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

  18. 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.

  19. 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

  20. 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.

  1. Two photon fluorescence imaging of lipid membrane domains and potentials using advanced fluorescent probes

    NASA Astrophysics Data System (ADS)

    Kilin, Vasyl; Darwich, Zeinab; Richert, Ludovic; Didier, Pascal; Klymchenko, Andrey; Mély, Yves

    2013-02-01

    Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.

  2. Boronic acids for fluorescence imaging of carbohydrates.

    PubMed

    Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

    2016-02-28

    "Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

  3. Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds.

    PubMed

    Neto, Brenno A D; Carvalho, Pedro H P R; Correa, Jose R

    2015-06-16

    This Account describes the origins, features, importance, and trends of the use of fluorescent small-molecule 2,1,3-benzothiadiazole (BTD) derivatives as a new class of bioprobes applied to bioimaging analyses of several (live and fixed) cell types. BTDs have been successfully used as probes for a plethora of biological analyses for only a few years, and the impressive responses obtained by using this important class of heterocycle are fostering the development of new fluorescent BTDs and expanding the biological applications of such derivatives. The first use of a fluorescent small-molecule BTD derivative as a selective cellular probe dates back to 2010, and since then impressive advances have been described by us and others. The well-known limitations of classical scaffolds urged the development of new classes of bioprobes. Although great developments have been achieved by using classical scaffolds such as coumarins, BODIPYs, fluoresceins, rhodamines, cyanines, and phenoxazines, there is still much to be done, and BTDs aim to succeed where these dyes have shown their limitations. Important organelles and cell components such as nuclear DNA, mitochondria, lipid droplets, and others have already been successfully labeled by fluorescent small-molecule BTD derivatives. New technological systems that use BTDs as the fluorophores for bioimaging experiments have been described in recent scientific literature. The successful application of BTDs as selective bioprobes has led some groups to explore their potential for use in studying membrane pores or tumor cells under hypoxic conditions. Finally, BTDs have also been used as fluorescent tags to investigate the action mechanism of some antitumor compounds. The attractive photophysical data typically observed for π-extended BTD derivatives is fostering interest in the use of this new class of bioprobes. Large Stokes shifts, large molar extinction coefficients, high quantum yields, high stability when stored in solution or

  4. Fluorescent probes for shock compression spectroscopy of microstructured materials

    NASA Astrophysics Data System (ADS)

    Christensen, James M.; Banishev, Alexandr A.; Dlott, Dana D.

    2017-01-01

    We are developing fluorescent probes to obtain dynamic two-dimensional pressure maps of shocked microstructured materials. We have fabricated silica nano-or micro-spheres doped with rhodamine 6G dye (R6G) which fluoresce strongly, and which may be dispersed throughout a microstructured sample. Alternatively we can grow thin skin layers of dye-doped silica on the surface of particles. The emissive microspheres were embedded in poly-methyl methacrylate (PMMA) and were excited by a quasi-continuous laser. When the samples were shocked to 3-8.4 GPa using laser-driven flyer plates, the emission redshifted and lost intensity. When encapsulating the dye in silica, the emission became brighter and the intensity-loss response became fast enough to monitor nanosecond shock effects. Preliminary data are reported showing the intensity loss in a shocked microstructured medium, an artificial sand, consisting of dye-coated silica microspheres.

  5. Fluorescent probes for super-resolution imaging in living cells.

    PubMed

    Fernández-Suárez, Marta; Ting, Alice Y

    2008-12-01

    In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

  6. Determination of amantadine and rimantadine using a sensitive fluorescent probe

    NASA Astrophysics Data System (ADS)

    Wang, Guang-Quan; Qin, Yan-Fang; Du, Li-Ming; Li, Jun-Fei; Jing, Xu; Chang, Yin-Xia; Wu, Hao

    2012-12-01

    Amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) are non-fluorescent in aqueous solutions. This property makes their determination through direct fluorescent method difficult. The competing reactions and the supramolecular interaction mechanisms between the two drugs and coptisine (COP) as they fight for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AMA or RIM in their pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.0040 to 1.0 μg mL-1 with a detection limit ranging from 0.0012 to 0.0013 μg mL-1. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

  7. ESIPT-Based Photoactivatable Fluorescent Probe for Ratiometric Spatiotemporal Bioimaging

    PubMed Central

    Zhou, Xiaohong; Jiang, Yuren; Zhao, Xiongjie; Guo, Dong

    2016-01-01

    Photoactivatable fluorophores have become an important technique for the high spatiotemporal resolution of biological imaging. Here, we developed a novel photoactivatable probe (PHBT), which is based on 2-(2-hydroxyphenyl)benzothiazole (HBT), a small organic fluorophore known for its classic luminescence mechanism through excited-state intramolecular proton transfer (ESIPT) with the keto form and the enol form. After photocleavage, PHBT released a ratiometric fluorophore HBT, which showed dual emission bands with more than 73-fold fluorescence enhancement at 512 nm in buffer and more than 69-fold enhancement at 452 nm in bovine serum. The probe displayed a high ratiometric imaging resolution and is believed to have a wide application in biological imaging. PMID:27754338

  8. Neutral red as a specific light-up fluorescent probe for i-motif DNA.

    PubMed

    Xu, Lijun; Wang, Jine; Sun, Na; Liu, Min; Cao, Yi; Wang, Zhili; Pei, Renjun

    2016-12-06

    We report a specific light-up fluorescent probe for i-motif DNA for the first time. Compared with the previously reported probes, neutral red could selectively interact with an i-motif and show a significant increase in its fluorescence. This feature makes it advantageous for designing label-free fluorescent sensing systems.

  9. 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.

  10. 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

  11. Recent advances in the synthesis and application of fluorescent α-amino acids.

    PubMed

    Harkiss, Alexander H; Sutherland, Andrew

    2016-09-26

    Fluorescence spectroscopy has become a powerful technique for probing a range of complex biological processes including enzyme mechanisms and protein-protein interactions. While the application of this technique uses a number of strategies, many of these rely on the use of fluorescent α-amino acids. This review highlights the recent synthetic methods developed for the incorporation of highly conjugated chromophores into the side-chain of α-amino acids and the application of these compounds as probes for imaging in medicine and biology. In particular, the design and synthesis of α-amino acids bearing coumarin, flavone and polyaromatic derived chromophores is described.

  12. Dimethylaminostyrylmethylpyridiniumiodine (daspmi) as a fluorescent probe for mitochondria in situ.

    PubMed

    Bereiter-Hahn, J

    1976-01-15

    An investigation has been made on the properties of dimethylaminostyrylmethylpyridiniumiodine (DASPMI), its reaction with isolated pigeon heart mitochondria and its suitability as a vital strain for mitochondria in situ. DASPMI is a low toxicity specific vital stain for mitochondria in living cells. In vitro dye concentrations over 6 nmol/mg protein inhibit fast (state 3) respiration after a preincubation time of more than 5 min in the presence of substrate. No uncoupling was observed. Energization of pigeon heart mitochondria by addition of ATP or various substrated yields an average 8.5-fold increase in fluorescence intensity in relation to DASPMI-stained mitochondria that are under anoxia, substrate deficiency, or under the influence of respiratory inhibitors, or uncouplers. The alterations in fluorescence intensity are not primarily due to ion movements or pH changes. The amount of dye (2.96+/-0.8 nmol) yielding maximal fluorescence response with 1 mg mitochondrial protein remains constant during energization of mitochondria. As indicated by electron microscopic studies the observed changes in emission intensity may be related to changes in the fine structural organisation of cristae. A remarkable difference exists between isolated mitochondria and mitochondria in situ with respect to the reaction to cyanide. According to the reported results DASPMI will be a useful probe for the investigation of mitochondrial activities in living cells.

  13. Characterization of photodynamic and sonodynamic cytotoxicity by fluorescent probes

    NASA Astrophysics Data System (ADS)

    Kessel, David

    1993-06-01

    A variety of porphyrins and related structures can sensitize cells to light; many of these agents can also promote ultrasound-induced cytotoxicity. Subcellular sites of localization sensitizers with a sufficient fluorescence yield can be assessed by fluorescence microscopy, but this becomes difficult when (Phi) F is low. We have explored several indirect procedures for assessing examining loci of photodamage and sonodamage. Damage to lysosomal structures was probed with acridine orange, mitochondria with Rhodamine 123 and the plasma membrane with several diphenylhexatriene (DPH) derivatives. Additional information on alterations in heterogeneity of binding of diphenylhexatriene derivatives to photodamaged cells was provided by a distributed fluorescent lifetime study. Using a sulfonated benzochlorin, which photosensitizes cell-surface loci, we evaluated four DPH derivatives for their sensitivity to membrane damage. Anionic or cationic DPH derivatives were the most sensitive in this regard. Enhanced cytotoxicity associated with ultrasound + porphyrins yielded no detectable effects on mitochondrial or lysosomal structures, and barely detectable changes in membrane interactions with DPH derivatives, suggesting an 'all or none' effect.

  14. Rapid and sensitive determination of clenbuterol in porcine muscle and swine urine using a fluorescent probe

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Bai, Bing; Zhang, Chenxuan; Wu, Wenying; Du, Liming; Liu, Hailong; Yao, Guojun

    2015-02-01

    The feed additive Clenbuterol hydrochloric acid (CLB) is non-fluorescent, thus it is difficult to quantify through direct fluorescent method. Palmatine (PAL) can react with cucurbit[7]uril (CB[7]) to form stable complexes as a fluorescent probe. Significant quenching of the fluorescence intensity of the CB[7]-PAL complex was observed with the addition of CLB. Based on the significant quenching of the supramolecular complex fluorescence intensity, a novel spectrofluorimetric method with high convenience, selectivity and sensitivity was developed for the determination of CLB. The fluorescence quenching values (ΔF) showed good linear relationship with CLB concentrations from 0.011 μg mL-1 to 4.2 μg mL-1 with a detection limit 0.004 μg mL-1. In this research, an ultrasound treatment replaced the former time-consuming shake method to form stable complexes. The proposed spectrofluorimetric method had been successfully applied to the determination of CLB in porcine muscle and swine urine with good precision and accuracy. The competing reaction and the supramolecular interaction mechanisms between the CLB and PAL as they fight for occupancy of the CB[7] cavity were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Interestingly, results indicate that two stable CB[7]-CLB complexes were formed.

  15. Rapid and sensitive determination of clenbuterol in porcine muscle and swine urine using a fluorescent probe.

    PubMed

    Jing, Xu; Bai, Bing; Zhang, Chenxuan; Wu, Wenying; Du, Liming; Liu, Hailong; Yao, Guojun

    2015-02-05

    The feed additive Clenbuterol hydrochloric acid (CLB) is non-fluorescent, thus it is difficult to quantify through direct fluorescent method. Palmatine (PAL) can react with cucurbit[7]uril (CB[7]) to form stable complexes as a fluorescent probe. Significant quenching of the fluorescence intensity of the CB[7]-PAL complex was observed with the addition of CLB. Based on the significant quenching of the supramolecular complex fluorescence intensity, a novel spectrofluorimetric method with high convenience, selectivity and sensitivity was developed for the determination of CLB. The fluorescence quenching values (ΔF) showed good linear relationship with CLB concentrations from 0.011 μg mL(-1) to 4.2 μg mL(-1) with a detection limit 0.004 μg mL(-1). In this research, an ultrasound treatment replaced the former time-consuming shake method to form stable complexes. The proposed spectrofluorimetric method had been successfully applied to the determination of CLB in porcine muscle and swine urine with good precision and accuracy. The competing reaction and the supramolecular interaction mechanisms between the CLB and PAL as they fight for occupancy of the CB[7] cavity were studied using spectrofluorimetry, (1)H NMR, and molecular modeling calculations. Interestingly, results indicate that two stable CB[7]-CLB complexes were formed.

  16. A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited-state intramolecular proton transfer.

    PubMed

    Fan, Chunlei; Luo, Shengxu; Qi, Haiping

    2016-03-01

    A ratiometric fluorescent probe 2-(benzimidazol-2-yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(2'-hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl3 , HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris-HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I430/I360) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP.

  17. Fluorescence encoded super resolution imaging based on a location estimation algorithm for high-density fluorescence probes

    NASA Astrophysics Data System (ADS)

    Nishimura, Takahiro; Kimura, Hitoshi; Ogura, Yusuke; Tanida, Jun

    2016-11-01

    In this paper, we propose a fluorescence encoded super resolution technique based on an estimation algorithm to determine locations of high-density fluorescence emitters. In our method, several types of fluorescence coded probes are employed to reduce densities of target molecules labeled with individual codes. By applying an estimation algorithm to each coded image, the locations of the high density probes can be determined. Due to multiplexed fluorescence imaging, this approach will provide fast super resolution microscopy. In experiments, we evaluated the performance of the method using probes with different fluorescence wavelengths. Numerical simulation results show that the locations of probes with the density of 200 μ m^{-2} , which is a typical membrane-receptor expression level, are determined with acquisition of 16 different coded images.

  18. Fluorescent Probe Encapsulated in Avidin Protein to Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity in Blood Serum.

    PubMed

    Wu, Ting-Wei; Lee, Fang-Hong; Gao, Ruo-Cing; Chew, Chee Ying; Tan, Kui-Thong

    2016-08-16

    Quantitative detection of trace amounts of a biomarker in protein rich human blood plasma using fluorescent probes is a great challenge as the real signal is usually obscured by nonspecific fluorescence. This problem occurs because most of the fluorescent dyes bind very tightly with blood proteins to produce a large fluorescence increase, resulting in overestimation of the biomarker concentrations and false positive diagnosis. In this paper, we report that biotinylated fluorescent probes encapsulated in avidin protein can generate very specific fluorescence in blood serum by blocking out nonspecific dye-protein interactions. We applied our novel probe design to detect two different types of biomolecules, hydrogen sulfide and nitroreductase. Our Avidin conjugated probes achieved quantitative analyte detection in blood serum; whereas concentrations were overestimated up to 320-fold when bare fluorescent probes were employed. As compared to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple approach successfully overcomes many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

  19. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  20. Fluorescent probe environment and the structural and charge changes in energy coupling of mitochondrial membranes.

    PubMed

    Chance, B

    1970-10-01

    The use of fluorescent probes to give continuous readouts of the structural states of mitochondrial membranes during energy coupling seems a logical extension of their use in the study of protein structural changes. A clear correlation of the probes' fluorescence characteristics with the acquisition of energy coupling can be demonstrated in fragmented and natural membrane using 1-anilinonaphthalene-8-sulfonate (ANS) and ethidium bromide respectively. The present contribution attempts to bring together contemporary viewpoints of this and other laboratories and the recent experimental data and give some detailed information on probe environment and on the structural or charge changes occurring upon energization. The energy-dependent region of the membrane is located at an aqueous interface between an outer layer of proteins (presumably cytochromes) and the membrane permeability barrier; the aromatic portion of ANS appears to be located in the lipid phase and the sulfonic acid group in the aqueous phase. The aqueous phase is probably a structured water region near paramagnetic membrane components such as cytochrome. Membrane energization arising from altered redox potential changes of cytochromes (b(T)) is communicated to the water structure through altered structural states of the hemoproteins, causing a decreased volume of the structured water region and increased interaction with the paramagnetic components in the energized state. Attendant alterations of protonic equilibria of membrane components induce both local and transmembrane changes in charge distribution, with consequent movements of ions, including the probe molecules themselves.

  1. A photochromic-acidochromic HCl fluorescent probe. An unexpected chloride-directed recognition.

    PubMed

    Jiménez-Sánchez, Arturo; Santillan, Rosa

    2016-06-20

    Non-classical protomerism of Schiff bases offers several advantages; for example, specific interactions in the -C[double bond, length as m-dash]N- linkage can be controlled and differentiated because the interactions are not governed by keto-enol tautomerism. Herein, the pH sensing properties of a new protomeric Schiff base probe () are reported. In particular, among several acids, the probe displays significant optical responses upon interaction with hydrochloric acid (HCl). X-ray structural analysis confirmed the existence of an intermolecular interaction with HCl through a -C[double bond, length as m-dash]NH-ClO- linkage. Moreover, an optical response via a second channel is manifested as photochromic fluorescence behavior. The properties of were investigated by UV-vis and fluorescence spectroscopy in a solution and the solid state. Its strong acidofluorochromic behavior was analyzed and its pKa and values were determined, which revealed a photobasic character. Positive solvatochromism that resulted from specific interactions taking place in was studied using four different solvent scales, namely, Lippert-Mataga, Kamlet-Taft, Catalán and the recently proposed scale of Laurence et al., which yielded consistent results. Finally, theoretical calculations were conducted to analyze the mechanism of the probe in terms of natural transition orbitals (NTOs) and the spatial extent of charge transfer excitations.

  2. A resorufin-based colorimetric and fluorescent probe for live-cell monitoring of hydrazine.

    PubMed

    Qian, Yong; Lin, Jie; Han, Lujing; Lin, Lin; Zhu, Hailiang

    2014-08-15

    We report a novel colorimetric and red-emitting fluorescent probe for hydrazine detection based on resorufin platform. This OFF-ON fluorescent probe shows a large (117nm) red-shifted absorption spectrum and the color changes from colorless to red upon addition of hydrazine in the aqueous solution, which can serve as a "naked-eye" probe for hydrazine. Moreover, this probe also shows a significant fluorescence increase (~16 folds) and excellent linear relationship at physiological pH. Utilizing this sensitive and selective probe, we have successfully detected hydrazine in living cells.

  3. DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

    PubMed Central

    Beierlein, Frank R.; Paradas Palomo, Miguel; Sharapa, Dmitry I.; Zozulia, Oleksii; Mokhir, Andriy; Clark, Timothy

    2016-01-01

    Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries. PMID:27467071

  4. Near-Infrared Fluorescent Probe with High Quantum Yield and Its Application in the Selective Detection of Glutathione in Living Cells and Tissues.

    PubMed

    Xie, Jun-Ying; Li, Chun-Yan; Li, Yong-Fei; Fei, Junjie; Xu, Fen; Ou-Yang, Juan; Liu, Juan

    2016-10-04

    Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small-molecular biothiols that play key roles in various biological systems. Among these biothiols, GSH is the most abundant intracellular thiol. Until now, a small number of the near-infrared (NIR) fluorescent probes have been designed for the detection of GSH. Unfortunately, most of these NIR probes are based on cyanine dyes, which generally suffer low fluorescence quantum yield (Φ < 0.25), which are not suitable for bioimaging. In addition, some probes are difficult to effectively distinguish GSH from Cys and Hcy. In this work, an NIR fluorescent probe with high fluorescence quantum yield is developed by introducing a rigid coplanar structure such as rhodamine dyes, and the NIR probe (CyR) with spirolactam structure is first synthesized and used to recognize GSH. The characteristics of this NIR probe are as follows: (1) probe CyR exhibits high fluorescence quantum yield (Φ = 0.43) after the addition of GSH and high sensitivity toward GSH with 75-fold fluorescence enhancement. (2) The probe is highly selective, which will not interfere with the other biological thiols (Cys, Hcy) and amino acids. (3) A possible reaction mechanism of the NIR probe CyR and GSH (Cys, Hcy) can be proposed and proved by (1)H NMR, (13)C NMR, and MS (mass spectra). (4) The NIR probe displays selective detection of GSH in biological samples such as living cells and tissues.

  5. 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.

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

    PubMed

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

    2015-05-05

    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.

  7. Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

    PubMed

    Tous, G; Fausnaugh, J; Vieira, P; Stein, S

    1988-07-01

    A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

  8. Fluorescence probes of spectroscopic and dynamical aspects of molecular photoionization

    NASA Astrophysics Data System (ADS)

    Poliakoff, Erwin D.

    1988-11-01

    Studies were made of vibrationally resolved aspects of shape resonant excitation in the photoionization of N(2)0. This experiment was performed by generating dispersed fluorescence spectra from electronically excited photoions. These results are the first vibrationally resolved results on a polyatomic shape resonance. In vibrationally resolved measurements, different internuclear configurations are probed by sampling alternative vibrational levels of the ion. As a result, the continuum electron behavior can be mapped out most clearly, and the qualitative aspects of the electron ejection can be understood clearly. A central motivation for studying polyatomic shape resonances is that alternative vibrational modes may be explored, revealing facets that are nonexistent for diatomic systems, which are the only systems that have been characterized previously.

  9. PARASITIC PLANTS. Probing strigolactone receptors in Striga hermonthica with fluorescence.

    PubMed

    Tsuchiya, Yuichiro; Yoshimura, Masahiko; Sato, Yoshikatsu; Kuwata, Keiko; Toh, Shigeo; Holbrook-Smith, Duncan; Zhang, Hua; McCourt, Peter; Itami, Kenichiro; Kinoshita, Toshinori; Hagihara, Shinya

    2015-08-21

    Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/β hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga.

  10. 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.

  11. Data analysis considerations in probing single quantum dot fluorescence intermittency

    NASA Astrophysics Data System (ADS)

    Krogmeier, Jeffrey R.; Hwang, Jeeseong

    2005-04-01

    The fluorescence intermittency of single, bare, CdSe/ZnS quantum dots was probed using single molecule confocal microscopy and found to demonstrate power law kinetics. Various threshold values and line fitting parameters are employed in the data analysis and their effects on the extracted power law exponents, moff and mon, are presented. The threshold is found to be critical in determining moff while having no significant effect on mon. The mean plus 2σ threshold, calculated from the background noise in the measurement, results in a more negative moff slope in comparison to the mean plus 3σ or mean plus 4σ thresholds. This is likely due to the mean plus 2σ threshold lying within the background noise outliers which mimic short on events. In contrast, the mean plus 4σ threshold is above 99.99% of the background noise while adequately below the fluorescence signal. Additionally, it is found that fitting only the ten most probable data points rather than all the data points in the log-log probability density graphs results in no significant change in moff and mon.

  12. Mapping fast protein folding with multiple-site fluorescent probes.

    PubMed

    Prigozhin, Maxim B; Chao, Shu-Han; Sukenik, Shahar; Pogorelov, Taras V; Gruebele, Martin

    2015-06-30

    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

  13. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

  14. 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.

  15. 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.

  16. A fluorescent probe for imaging p53-MDM2 protein-protein interaction.

    PubMed

    Liu, Zhenzhen; Miao, Zhenyuan; Li, Jin; Fang, Kun; Zhuang, Chunlin; Du, Lupei; Sheng, Chunquan; Li, Minyong

    2015-04-01

    In this article, we describe a no-wash small-molecule fluorescent probe for detecting and imaging p53-MDM2 protein-protein interaction based on an environment-sensitive fluorescent turn-on mechanism. After extensive biological examination, this probe L1 exhibited practical activity and selectivity in vitro and in cellulo.

  17. 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.

  18. Rational Design of a Fluorescent Hydrogen Peroxide Probe Based on the Umbelliferone Fluorophore

    PubMed Central

    Du, Lupei; Li, Minyong; Zheng, Shilong; Wang, Binghe

    2008-01-01

    In this study, we report a novel water-soluble umbelliferone-based fluorescent probe for hydrogen peroxide. This probe shows very large increases (up to 100 fold) in fluorescent intensity upon reaction with hydrogen peroxide, and good selectivity over other reactive oxygen species (ROS). PMID:19081820

  19. Continuously tunable nucleic acid hybridization probes.

    PubMed

    Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

    2015-12-01

    In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

  20. 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.

  1. 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

  2. A highly sensitive ratiometric fluorescent probe for the detection of cytoplasmic and nuclear hydrogen peroxide.

    PubMed

    Wen, Ying; Liu, Keyin; Yang, Huiran; Li, Yi; Lan, Haichuang; Liu, Yi; Zhang, Xinyu; Yi, Tao

    2014-10-07

    As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H2O2) plays an important role in living systems. It is thus critical to monitor the changes in H2O2 in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H2O2 fluorescent probe (NP1) based on 1,8-naphthalimide and boric acid ester. In response to H2O2, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of NP1 toward H2O2 is estimated as 0.17 μM. It was capable of imaging endogenous H2O2 generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H2O2 produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications.

  3. In situ fluorescence labelling of jasmonic acid binding sites in plant tissues with cadmium-free quantum dots.

    PubMed

    Liao, Qiumei; Yu, Ying; Cao, Yujuan; Lin, Bixia; Wei, Jingjing

    2015-02-01

    The fluorescence labelling of plant hormone binding sites is an important analytical technique in research on the molecular mechanisms of plant hormone activities. The authors synthesised a jasmonic acid (JA)-conjugated ZnS:Mn quantum dot (QD) probe, with a cubic structure and average hydrodynamic sizes of about 17.0 nm. The maximum fluorescence emission of the probe was recorded at about 585 nm. The probe was used for fluorescence labelling of JA binding sites in mung bean seedling tissues. Analysis revealed that the probe exhibited high selectivity to JA binding sites and good performance in eliminating interference from background fluorescence in plant tissues. In addition, the probe did not exhibit any apparent biotoxicity, and is much more suitable than probes constructed from CdTe QDs for the analysis of biological samples.

  4. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

    PubMed Central

    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  5. Reaction-based two-photon probes for mercury ions: fluorescence imaging with dual optical windows.

    PubMed

    Rao, Alla Sreenivasa; Kim, Dokyoung; Wang, Taejun; Kim, Ki Hean; Hwang, Sekyu; Ahn, Kyo Han

    2012-05-18

    For fluorescent imaging of mercury ions in living species, two-photon probes with dual optical windows are in high demand but remain unexplored. Several dithioacetals were evaluated, and a probe was found, which, upon reaction with mercury species, yielded a two-photon dye; this conversion accompanies ratiometric emission changes with a 97-nm shift, enabling fluorescent imaging of both the probe and mercury ions in cells by one- and two-photon microscopy for the first time.

  6. A capillary-based probe for in situ detection of enhanced fluorescence signals

    NASA Astrophysics Data System (ADS)

    Long, F.; Xiao, R.; Zhu, A. N.; Shi, H. C.; Wang, S. Q.

    2013-07-01

    A simple, compact, and high sensitivity capillary-based probe for the in situ detection of fluorescence signals with high sensitivity is demonstrated. A home-made single-multi-mode fiber coupler that is coaxially aligned with the capillary-based probe provides for the transmission of excitation light and the collection and transmission of fluorescence. We propose a conceptually straightforward theoretical model to optimize the factors affecting the fluorescence-capture capability of the capillary-based probe. The fluorescence signal detected by fiber-optic spectroscopy non-linearly increases with the length of the capillary-based probe. In addition, the thicker the capillary tube wall is, the less the fluorescence signals determined are. The performance of the proposed probe is evaluated experimentally by measuring the fluorescence spectra of Cy5.5 dye and blue-green algae. The experimental results show that the proposed probe provides more than a ten-fold increase in fluorescence signal compared with direct measurements by a flat-tipped multi-mode fiber probe. The advantages of the capillary-based probe, which include its simple and compact structure, excellent light collection efficiency, requirement of small sample volume, and recoverability of samples, allow its wide application to in situ detection in the medical, forensic, biological, geological, and environmental fields with high sensitivity.

  7. High-contrast fluorescence sensing of aqueous Cu(I) with triarylpyrazoline probes: dissecting the roles of ligand donor strength and excited state proton transfer.

    PubMed

    Morgan, M Thomas; Bagchi, Pritha; Fahrni, Christoph J

    2013-03-07

    Cu(I)-responsive fluorescent probes based on a photoinduced electron transfer (PET) mechanism generally show incomplete fluorescence recovery relative to the intrinsic quantum yield of the fluorescence reporter. Previous studies on probes with an N-aryl thiazacrown Cu(I)-receptor revealed that the recovery is compromised by incomplete Cu(I)-N coordination and resultant ternary complex formation with solvent molecules. Building upon a strategy that successfully increased the fluorescence contrast and quantum yield of Cu(I) probes in methanol, we integrated the arylamine PET donor into the backbone of a hydrophilic thiazacrown ligand with a sulfonated triarylpyrazoline as a water-soluble fluorescence reporter. This approach was not only expected to disfavor ternary complex formation in aqueous solution but also to maximize PET switching through a synergistic Cu(I)-induced conformational change. The resulting water-soluble probe 1 gave a strong 57-fold fluorescence enhancement upon saturation with Cu(I) with high selectivity over other cations, including Cu(II), Hg(II), and Cd(II); however, the recovery quantum yield did not improve over probes with the original N-aryl thiazacrown design. Concluding from detailed photophysical data, including responses to acidification, solvent isotope effects, quantum yields, and time-resolved fluorescence decay profiles, the fluorescence contrast of 1 is compromised by inadequate coordination of Cu(I) to the weakly basic arylamine nitrogen of the PET donor and by fluorescence quenching via two distinct excited state proton transfer pathways operating under neutral and acidic conditions.

  8. Fluorescence characteristics of hydrophobic partial agonist probes of the cholecystokinin receptor.

    PubMed

    Harikumar, Kaleeckal G; Pinon, Delia I; Miller, Laurence J

    2006-04-01

    Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.

  9. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

  10. 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.

  11. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  12. Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format.

    PubMed

    Laitala, Ville; Ylikoski, Alice; Raussi, Hanna-Mari; Ollikka, Pia; Hemmilä, Ilkka

    2007-02-01

    We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.

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

    PubMed

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

    2017-02-24

    A novel adenine (Ad) fluorescence probe (Eu(III)-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 Eu(III)-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 Eu(III)-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 Eu(III)-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 Eu(III)-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 Eu(III)-dtpa-bis(guanine) at 570nm as a function of adenine (Ad) concentration in the range of 0.00-5.00×10(-5)molL(-1) was observed. The detection limit is about 4.70×10(-7)molL(-1).

  14. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes.

    PubMed

    Mecca, Carolina Z P; Fonseca, Fernando L A; Bagatin, Izilda A

    2016-11-05

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al(3+), Mg(2+), Zn(2+), and Cd(2+) salts (1-8); and characterized. The (1)H NMR spectra of complexes 1 and 5, containing Al(3+), were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

  15. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes

    NASA Astrophysics Data System (ADS)

    Mecca, Carolina Z. P.; Fonseca, Fernando L. A.; Bagatin, Izilda A.

    2016-11-01

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al3+, Mg2+, Zn2+, and Cd2+ salts (1-8); and characterized. The 1H NMR spectra of complexes 1 and 5, containing Al3+, were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

  16. Water-soluble BODIPY-based fluorescent probe for mitochondrial imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Sui, Binglin; Tang, Simon; Woodward, Adam W.; Kim, Bosung; Belfield, Kevin D.

    2016-03-01

    A new mitochondrial targeting fluorescent probe is designed, synthesized, characterized, and investigated. The probe is composed of three moieties, a BODIPY platform working as the fluorophore, two triphenylphosphonium (TPP) groups serving as mitochondrial targeting moiety, and two long highly hydrophilic polyethylene glycol (PEG) chains to increase its water solubility and reduce its cytotoxicity. As a mitochondria-selective fluorescent probe, the probe exhibits a series of desirable advantages compared with other reported fluorescent mitochondrial probes. It is readily soluble in aqueous media and emits very strong fluorescence. Photophysical determination experiments show that the photophysical properties of the probe are independent of solvent polarity and it has high quantum yield in various solvents examined. The probe also has good photostability and pH insensitivity over a broad pH range. Results obtained from cell viability tests indicate that the cytotoxicity of the probe is very low. Confocal fluorescence microscopy colocalization experiments reveal that this probe possesses excellent mitochondrial targeting ability and it is suitable for imaging mitochondria in living cells.

  17. Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Santo Domingo, Jorge W.; Kaufman, Michael G.; Klug, Michael J.; Tiedje, James M.

    1998-01-01

    Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The Bacteroides-Prevotella probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets. PMID:16349506

  18. A Simple and Effective Ratiometric Fluorescent Probe for the Selective Detection of Cysteine and Homocysteine in Aqueous Media.

    PubMed

    Na, Risong; Zhu, Meiqing; Fan, Shisuo; Wang, Zhen; Wu, Xiangwei; Tang, Jun; Liu, Jia; Wang, Yi; Hua, Rimao

    2016-08-05

    Biothiols such as cysteine (Cys) and homocysteine (Hcy) are essential biomolecules participating in molecular and physiological processes in an organism. However, their selective detection remains challenging. In this study, ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (NL) was synthesized as a ratiometric fluorescent probe for the rapid and selective detection of Cys and Hcy over glutathione (GSH) and other amino acids. The fluorescence intensity of the probe in the presence of Cys/Hcy increased about 3-fold at a concentration of 20 equiv. of the probe, compared with that in the absence of these chemicals in aqueous media. The limits of detection of the fluorescent assay were 0.911 μM and 0.828 μM of Cys and Hcy, respectively. ¹H-NMR and MS analyses indicated that an excited-state intramolecular proton transfer is the mechanism of fluorescence sensing. This ratiometric probe is structurally simple and highly selective. The results suggest that it has useful applications in analytical chemistry and diagnostics.

  19. 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.

  20. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    PubMed Central

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy. PMID:26794434

  1. 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.

  2. Novel pyrazoline-based selective fluorescent probe for the detection of hydrazine.

    PubMed

    Zheng, Xiao-Xin; Wang, Sheng-Qing; Wang, Hao-Yan; Zhang, Rong-Rong; Liu, Jin-Ting; Zhao, Bao-Xiang

    2015-03-05

    A novel pyrazoline-based fluorescent probe, 2-[4-(3,5-diphenyl-4,5-dihydro-pyrazol-1-yl)-benzylidene]-malononitrile, with a simple structure and low detection limit (6.16×10(-6)M) for the detection of hydrazine is designed and synthesized. The probe responds selectively to hydrazine over other molecules with marked fluorescence enhancement. The probe can detect hydrazine effectively at pH 5.0-9.0 with a special emission wavelength at 520nm. Moreover, the probe can be used to detect hydrazine from variety of natural source water.

  3. Environment-responsive fluorescent nucleoside analogue probe for studying oligonucleotide dynamics in a model cell-like compartment.

    PubMed

    Pawar, Maroti G; Srivatsan, Seergazhi G

    2013-11-21

    The majority of fluorescent nucleoside analogue probes that have been used in the in vitro study of nucleic acids are not suitable for cell-based biophysical assays because they exhibit excitation maxima in the UV region and low quantum yields within oligonucleotides. Therefore, we propose that the photophysical characterization of oligonucleotides labeled with a fluorescent nucleoside analogue in reverse micelles (RM), which are good biological membrane models and UV-transparent, could provide an alternative approach to studying the properties of nucleic acids in a cell-like confined environment. In this context, we describe the photophysical properties of an environment-sensitive fluorescent uridine analogue (1), based on the 5-(benzo[b]thiophen-2-yl)pyrimidine core, in micelles and RM. The emissive nucleoside, which is polarity- and viscosity-sensitive, reports the environment of the surfactant assemblies via changes in its fluorescence properties. The nucleoside analogue, incorporated into an RNA oligonucleotide and hybridized to its complementary DNA and RNA oligonucleotides, exhibits a significantly higher fluorescence intensity, lifetime, and anisotropy in RM than in aqueous buffer, which is consistent with the environment of RM. Collectively, our results demonstrate that nucleoside 1 could be utilized as a fluorescent label to study the function of nucleic acids in a model cellular milieu.

  4. 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.

  5. Kit for detecting nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2001-01-01

    A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

  6. Covalently bound fluorescent probes as reporters for hydroxyl radical penetration into liposomal membranes.

    PubMed

    Fortier, Chanel A; Guan, Bing; Cole, Richard B; Tarr, Matthew A

    2009-05-15

    The ability of hydroxyl radicals to penetrate into liposomal model membranes (dimyristoylphosphatidylcholine) has been demonstrated. Liposomes were prepared and then characterized by digital fluorescence microscopy and dynamic light scattering after extrusion to determine liposomal lamellarity, size, and shape. Hydroxyl radicals were generated in the surrounding aqueous medium using a modified Fenton reagent (hydrogen peroxide and Fe(2+)) with the water-soluble iron chelator EDTA. High and low doses of radical were used, and the low dose was achieved with physiologically relevant iron and peroxide concentrations. Fluorescent probes covalently bound to the membrane phospholipid were used, including two lipophilic pyrenyl probes within the membrane bilayer and one polar probe at the water-membrane interface. Radical reactions with the probes were monitored by following the decrease in fluorescence and by observing oxidation products via matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Differences in the probe position within the membrane were correlated with the reactivity of the probe to assess radical access to the site of the probe. For all probes, reaction rates increased with increasing temperature. Within the membrane bilayer, reaction rates were greater for the probe closest to the membrane-water interface. Cholesterol protected these probes from oxidation. Kinetic models, scavenger studies, and product identification studies indicated that hydroxyl radical reacted directly with the in-membrane probes without the mediation of a secondary radical.

  7. A Novel Coumarin-Based Fluorescent Probe for the Detection of Hydrazine Both in Aqueous Solution and Vapor State.

    PubMed

    Xiao, Yu-Hao; Xi, Gang; Zhao, Xuan-Xuan; Zhou, Shuai; Zhou, Ze-Quan; Zhao, Bao-Xiang

    2015-07-01

    A novel coumarin-based fluorescent probe CF was synthesized for the detection of hydrazine both in aqueous solution and vapor state with high sensitivity and selectivity. Upon addition of hydrazine, the solution of probe CF in MeCN-H2O (3/7, v/v, buffered CH3COOH/CH3COONa) at pH 5.0 exhibited a remarkable change in emission color from pale green to light blue, which could be recognized with naked eyes. Applied in weak acid condition, probe CF could detect hydrazine selectively with large amount of unknown environments according to the competing tests. Besides, with the limit of detection 8.32 ppb (2.6 × 10(-7) M), probe CF could well meet the request (10 ppb) of the U.S. Environmental Protection Agency (EPA).

  8. A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution.

    PubMed

    Liu, Xiaoqing; Savy, Alexandra; Maurin, Sylvie; Grimaud, Laurence; Darchen, François; Quinton, Damien; Labbé, Eric; Buriez, Olivier; Delacotte, Jérôme; Lemaître, Frédéric; Guille-Collignon, Manon

    2017-02-20

    In this work, Fluorescent False Neurotransmitter 102 (FFN102), a synthesized analogue of biogenic neurotransmitters, was demonstrated to show both pH-dependent fluorescence and electroactivity. To study secretory behaviors at the single-vesicle level, FFN102 was employed as a new fluorescent/electroactive dual probe in a coupled technique (amperometry and total internal reflection fluorescence microscopy (TIRFM)). We used N13 cells, a stable clone of BON cells, to specifically accumulate FFN102 into their secretory vesicles, and then optical and electrochemical measurements of vesicular exocytosis were experimentally achieved by using indium tin oxide (ITO) transparent electrodes. Upon stimulation, FFN102 started to diffuse out from the acidic intravesicular microenvironment to the neutral extracellular space, leading to fluorescent emissions and to the electrochemical oxidation signals that were simultaneously collected from the ITO electrode surface. The correlation of fluorescence and amperometric signals resulting from the FFN102 probe allows real-time monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms.

  9. Novel B,O-chelated fluorescent probe for nitric oxide imaging in Raw 264.7 macrophages and onion tissues.

    PubMed

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

    2013-10-24

    A novel fluorescent probe based on B,O-chelated dipyrromethene chromophore in far-visible and near-infrared spectral region (600-900 nm), boron chelated 8-(3,4-diaminophenyl)-3,5-bis(2-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indancene (BOPB), has been first developed for nitric oxide (NO) imaging. BOPB, a turn-on fluorescent probe, can react with NO rapidly under physiological condition. The reaction product of BOPB with NO, BOPB-T, emits bright red fluorescence at 643 nm when excited at 622 nm. Meanwhile, BOPB-T displays high fluorescent quantum yield of 0.21 and good photostability. The selectivity for NO over other reactive oxygen/nitrogen species and ascorbic acid has been investigated and BOPB has good specificity for the detection of NO. MTT assay shows that the toxicity of BOPB (below 10 μM) to living cells can be neglected. Based on these investigations, BOPB has been used for NO imaging in Raw 264.7 cells and onion tissues. Meanwhile, mechanical injury to onion tissues results in a brighter fluorescence around the wound, which indicates that more NO has been produced in plant tissues in response to external stimuli. Our studies illustrate that BOPB has advantages of high sensitivity, low background interference and little photo damage on fluorescence imaging of NO.

  10. Label-free silicon quantum dots as fluorescent probe for selective and sensitive detection of copper ions.

    PubMed

    Zhao, Jiangna; Deng, Jianhui; Yi, Yinhui; Li, Haitai; Zhang, Youyu; Yao, Shouzhuo

    2014-07-01

    In this work, label-free silicon quantum dots (SiQDs) were used as a novel fluorescence probe for the sensitive and selective detection of Cu(2+). The fluorescence of the SiQDs was effectively quenched by H2O2 from the reaction of ascorbic acid with O2, and hydroxyl radicals from Fenton reaction between H2O2 and Cu(+). The fluorescence intensity of SiQDs was quenched about 25% in 15 min after the addition of H2O2 (1mM). While the SiQDs was incubated with AA (1mM) and Cu(2+) (1 µM) under the same conditions, the fluorescence intensity of SiQDs decreased about 55%. Obviously, the recycling of Cu(2+) in the test system may lead to a dramatical decrease in the fluorescence of SiQDs. Under the optimized experimental conditions, the rate of fluorescence quenching of SiQDs was linearly dependent on the Cu(2+) concentration ranging from 25 to 600 nM with the limit of detection as low as 8 nM, which was much lower than that of existing methods. Moreover, the probe was successfully applied to the determination of Cu(2+) in different environmental water samples and human hair.

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

    DOEpatents

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    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.

  12. In-vivo concentration ratio estimation of two fluorescent probes for early detection of Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Harbater, Osnat; Gannot, Israel

    2015-03-01

    In-vivo measurement of the concentrations of biological compounds using fluorescence is one of the challenging biophotonic fields. These measurements are useful in diagnostic and treatment monitoring applications that use fluorescent probes which may bond to specific proteins and drugs. In some cases the relative concentration of two compounds is a sufficient biological indicator. For instance, it has been shown that the ratio between Amyloid-Beta and tau protein in the Cerebrospinal fluid (CSF) may predict the development of Alzheimer's disease (AD) several years before current diagnosis. We have previously suggested a system that could measure the concentration ratio of these two proteins in-vivo without the need to collect CSF samples. This system uses a miniature needle with an optical fiber which is coupled to a laser source and a detector. The fiber excites fluorescent probes which were injected and bond to the proteins in the CSF, and collects the fluorescence emission. Using the fluorescence intensity ratio, the concentration ratio between the proteins is estimated, and AD may be diagnosed. In this work we present the results of an in-vivo trial performed on mice. Miniature tubes containing two fluorescent probes in several concentration ratios were inserted into the mice in two locations: subcutaneously, and deeper in the abdomen. The fluorescent probes were excited and the fluorescence intensity was measured. The concentration ratios were extracted from the fluorescence intensities using a simple calibration curve. The extracted ratios are compared to the true ratios and the system's accuracy is estimated.

  13. Synthesis of Functional Fluorescent BODIPY‐based Dyes through Electrophilic Aromatic Substitution: Straightforward Approach towards Customized Fluorescent Probes

    PubMed Central

    Schoenmakers, Daniël C.; Veranič, Peter; Muševič, Igor

    2016-01-01

    Abstract Fluorescent materials are widely used in biological and material applications as probes for imaging or sensing; however, their customization is usually complicated without the support of an organic chemistry laboratory. Here, we present a straightforward method for the customization of BODIPY cores, which are among the most commonly used fluorescent probes. The method is based on the formation of a new C−C bond through Friedel–Crafts electrophilic aromatic substitution carried out at room temperature. The method presented can be used to obtain completely customized fluorescent materials in one or two steps from commercially available compounds. Examples of the preparation of fluorescent materials for cell staining and functionalization of silica colloids are also presented. PMID:27777837

  14. Synthesis and characterization of novel fluorescent nitrogen-containing bisphosphonate imaging probes for bone active drugs

    PubMed Central

    Sun, Shuting; Błażewska, Katarzyna M.; Kashemirov, Boris A.; Roelofs, Anke J.; Coxon, Fraser P.; Rogers, Michael J.; Ebetino, Frank H.; McKenna, Michael J.; McKenna, Charles E.

    2011-01-01

    Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed. PMID:21894242

  15. 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

  16. The synthesis of new fluorescent bichromophoric compounds as ratiometric pH probes for intracellular measurements.

    PubMed

    Saura, A Vanessa; Marín, María J; Burguete, M Isabel; Russell, David A; Galindo, Francisco; Luis, Santiago V

    2015-07-28

    Three different bichromophoric compounds (1-3) containing an aminomethyl anthracene moiety linked to a second chromophore (pyrene, 4-nitrobenzo-2-oxa-1,3-diazole (NBD) and dansyl) through a valine-derived pseudopeptidic spacer have been prepared and their fluorescent properties studied. The results obtained show that upon irradiation the photophysical behavior of these probes involves electronic energy transfer from the excited anthracene to the second chromophore and also intramolecular photoinduced electron transfer. The X-ray structure obtained for 3 reveals that the folding associated with the pseudopeptidic spacer favours a close proximity of the two chromophores. The emissive response of 3 is clearly dependent on the pH of the medium, hence this bichromophoric compound was shown to be an excellent ratiometric pH fluorescent sensor. The emission intensity due to the anthracene moiety exhibits a decrease at neutral-basic pH values that is concomitant with an increase in the intensity arising from the dansyl fluorophore. These properties make this compound a good candidate for biological pH sensing as has been confirmed by preliminary studies with RAW 264.7 macrophage cells imaged by means of confocal fluorescence microscopy with an average pH estimation of 5.4-5.8 for acidic organelles.

  17. A benzothiazole-based fluorescent probe for distinguishing and bioimaging of Hg(2+) and Cu(2).

    PubMed

    Gu, Biao; Huang, Liyan; Su, Wei; Duan, Xiaoli; Li, Haitao; Yao, Shouzhuo

    2017-02-15

    A new benzothiazole-based fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-(1,3- dithian-2-yl)phenol (BT) with two different reaction sites, a thioacetal group (site 1 for Hg(2+)), and O and N atoms of the benzothiazole dye (site 2 for Cu(2+)), was designed and synthesized. The probe BT showed ratiometric fluorescent response to Hg(2+) and fluorescence quenching behavior to Cu(2+), which induces naked-eye fluorescent color changes from green to blue and colorless, respectively. Moreover, it displayed highly sensitivity and selectivity toward Hg(2+) and Cu(2+) without interference from other metal ions. The sensing mechanisms were also confirmed by (1)H NMR titration, mass spectrum and Job's plot analyses. Finally, probe BT was successfully used for fluorescent imaging of Hg(2+) and Cu(2+) in living cells, demonstrating its potential applications in biological science.

  18. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

    PubMed

    Jin, Lei; Han, Zhou; Platisa, Jelena; Wooltorton, Julian R A; Cohen, Lawrence B; Pieribone, Vincent A

    2012-09-06

    Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.

  19. A single-wavelength-emitting ratiometric probe based on phototriggered fluorescence switching of graphene quantum dots.

    PubMed

    Qu, Zhi-bei; Zhang, Min; Zhou, Tianshu; Shi, Guoyue

    2014-10-13

    Ratiometric fluorescent probes are of great importance in research, because a built-in correction for environmental effects can be provided to reduce background interference. However, the traditional ratiometric fluorescent probes require two luminescent materials with different emission bands. Herein a novel ratiometric probe based on a single-wavelength-emitting material is reported. The probe works by regulating the luminescent property of graphene quantum dots with UV illumination as activator. The ratiometric sensor shows high sensitivity and specificity for iron ions. Moreover, the ratiometric sensor was successfully employed to monitor ferritin levels in Sprague Dawley rats with chemical-induced acute liver damage. The proposed single-wavelength ratiometric fluorescent probe may greatly broaden the applicability of ratiometric sensors in diagnostic devices, medical applications, and analytical chemistry.

  20. 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.

  1. Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria.

    PubMed

    Niu, Weifen; Guo, Lei; Li, Yinhui; Shuang, Shaomin; Dong, Chuan; Wong, Man Shing

    2016-02-02

    A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed on the basis of a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylate-functionalized merocyanine probe shows not only a mitochondria-targeting property but also highly selective detection and monitoring of Cys over other biothiols such as homocysteine (Hcy) and glutathione (GSH) and hydrogen sulfide (H2S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F518/F452), which are linearly proportional to Cys concentrations in the range of 0.5-40 μM. More importantly, the probe and its released fluorophore, merocyanine, exhibit strong two-photon excited fluorescence (TPEF) with two-photon action cross-section (Φσmax) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively, which is highly desirable for high contrast and brightness ratiometric two-photon fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150 μm by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.

  2. A fluorescent, genetically-encoded voltage probe capable of resolving action potentials.

    PubMed

    Barnett, Lauren; Platisa, Jelena; Popovic, Marko; Pieribone, Vincent A; Hughes, Thomas

    2012-01-01

    There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1-S4) of Ciona intestinalis voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ~1-2 ms) than any currently published fluorescent protein-based voltage probe. ElectricPk can follow the rise and fall of neuronal action potentials with a modest decrease in fluorescence intensity (~0.7% ΔF/F). The probe has a nearly linear fluorescence/membrane potential response to both hyperpolarizing and depolarizing steps. This is the first probe based on CiVSP that captures the rapid movements of the voltage sensor, suggesting that voltage probes designed with circularly permuted fluorescent proteins may have some advantages.

  3. Probes for biomolecules detection based on RET-enhanced fluorescence polarization.

    PubMed

    Ren, Dahai; Wang, Jun; Wang, Bin; You, Zheng

    2016-05-15

    Fluorescent probes based on the principle of resonance energy transfer (RET) or the principle of fluorescence polarization (FP) are already used to detect biomolecules independently. However, there were no in-depth studies about the impact of RET on FP. Also, very few studies gave a comprehensive analysis on how to effectively design such a fluorescent probe. Based on the principle of resonance energy transfer (RET), we constructed fluorescent probes (SA-488-sub-nanogold) using streptavidin labeled Alexa488 (SA-488), nanogold and biotinylated substrate peptide (biotin-subpeptide). The influence of the structure and the ingredients of the substrate peptide were discussed. After SA-488 was combined with the biotin-subpeptide and the nanogold, its fluorescence intensity (FI) would be suppressed due to the energy transfer, leading to an increase in its volume and mass. The suppression of the FI led to a decrease in SA-488's effective concentration, and the increase in the volume or mass prolonged the SA-488's rotational relaxation time. Both changes increased SA-488's polarization in the solution. Therefore, the FP performance of the probe is enhanced by the RET. Using the probe, trypsin and biotin were detected by the change in both fluorescence intensity and fluorescence polarization, showing higher reliability, higher sensitivity, and a lower detection limit.

  4. Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates

    PubMed Central

    Rice, J.; Sleigh, M. A.; Burkill, P. H.; Tarran, G. A.; O'Connor, C. D.; Zubkov, M. V.

    1997-01-01

    Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved. PMID:16535558

  5. 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.

  6. Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe

    PubMed Central

    Kilin, Vasyl; Glushonkov, Oleksandr; Herdly, Lucas; Klymchenko, Andrey; Richert, Ludovic; Mely, Yves

    2015-01-01

    To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N∗) and tautomer (T∗) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T∗ form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N∗ form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis. PMID:25992730

  7. Fluorescence lifetime imaging of membrane lipid order with a ratiometric fluorescent probe.

    PubMed

    Kilin, Vasyl; Glushonkov, Oleksandr; Herdly, Lucas; Klymchenko, Andrey; Richert, Ludovic; Mely, Yves

    2015-05-19

    To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.

  8. Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis.

    PubMed

    Lebar, Matthew D; May, Janine M; Meeske, Alexander J; Leiman, Sara A; Lupoli, Tania J; Tsukamoto, Hirokazu; Losick, Richard; Rudner, David Z; Walker, Suzanne; Kahne, Daniel

    2014-08-06

    The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both D-amino acids and D-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged D-amino acids. We exploited these observations to design a fluorescent D-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.

  9. A novel fluorescent probe for the detection of myeloperoxidase activity in atherosclerosis-associated macrophages

    PubMed Central

    Shepherd, Joanna; Hilderbrand, Scott A.; Waterman, Peter; Heinecke, Jay W.; Weissleder, Ralph; Libby, Peter

    2009-01-01

    SUMMARY The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl/OCl−) is implicated in the pathogenesis of atherosclerosis and other inflammatory states. We have synthesized a novel imaging probe, sulfonaphthoaminophenyl fluorescein (SNAPF), that selectively reacts to HOCl. SNAPF detects HOCl produced by stimulated MPO-expressing cells cultured from human whole blood, as well as HOCl from bone marrow (BM)-derived macrophages isolated from transgenic mice that express human MPO. Two lines of evidence indicate that SNAPF permits the in vivo imaging of HOCl production. First, we used this approach to demonstrate HOCl production by neutrophils in experimental murine peritonitis. Second, we detected HOCl production by MPO expressing cells in human atherosclerotic arteries. Thus, fluorescence reflectance imaging by SNAPF may provide a valuable non-invasive molecular imaging tool for implicating HOCl and MPO in the damage of inflamed tissues. PMID:18022561

  10. An 'activatable' aptamer-based fluorescence probe for the detection of HepG2 cells.

    PubMed

    Lai, Zongqiang; Tan, Juntao; Wan, Ruirong; Tan, Jie; Zhang, Zhenghua; Hu, Zixi; Li, Jieping; Yang, Wei; Wang, Yiwei; Jiang, Yafeng; He, Jian; Yang, Nuo; Lu, Xiaoling; Zhao, Yongxiang

    2017-05-01

    It is significant to develop a probe with sensitivity and specificity for the detection of cancer cells. The present study aimed to develop an 'activatable' aptamer-based fluorescence probe (AAFP) to detect cancer cells and frozen cancer tissue. This AAFP consisted of two fragments: aptamer TLS11a that targets HepG2 cells, and two short extending complementary DNA sequences with a 5'- and 3'-terminus that make the aptamer in hairpin structure a capable quencher to fluorophore. The ability of the AAFP to bind specifically to cancer cells was assessed using flow cytometry, fluorescence spectroscopy and fluorescence microscopy. Its ability to bind to frozen cancer tissue was assessed using fluorescence microscopy. As a result, in the absence of cancer cells, AAFP showed minimal fluorescence, reflecting auto-quenching. In the presence of cancer cells, however, AAFP showed a strong fluorescent signal. Therefore, this AAFP may be a promising tool for sensitive and specific detection of cancer.

  11. Tyrosine fluorescence probing of the surfactant-induced conformational changes of albumin.

    PubMed

    Zhdanova, Nadezda G; Shirshin, Evgeny A; Maksimov, Eugene G; Panchishin, Ivan M; Saletsky, Alexander M; Fadeev, Victor V

    2015-05-01

    Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.

  12. 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.

  13. Probing structure of blood plasma proteins with solvatochromic fluorescent probes based on Nile red and its derivatives

    NASA Astrophysics Data System (ADS)

    Kaler, Gregory V.; Ivanov, Andrei I.; Konev, Sergei V.

    1997-05-01

    Uncharged long-wave fluorescent probes, Nile red and its derivatives varying in lipophilicity, were used for probing hydrophobic binding sites of human serum albumin (HSA) and lipoproteins (LP) in norm and pathology. The synchro-scan fluorescence spectra (synchronous scanning of both excitation and emission wavelengths at constant (Delta) (lambda) ) of the probes were studied in HSA solutions and in whole blood plasma. The parameters of the spectra were sensitive to pH-induced conformational NyieldsB transition in HSA. In blood plasma, each of the probes displayed a two-component synchro-scan spectrum revealing two pools of the dye bound to HSA (longer wavelength) and LP (shorter wavelength). The probe distribution between LP and HSA was also sensitive to NyieldsB transition. The LP/HSA probe distribution ratio was shown to increase significantly in certain pathologies, due to either hypoalbuminemia or lowered ligand-binding capacity of HSA. Also, spectral shifts were observed in the band of albumin-bound probe. The determination of the distribution parameter may be proposed as an informative and feasible diagnostic test.

  14. 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.

  15. 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

  16. A cysteamine-selective two-photon fluorescent probe for ratiometric bioimaging.

    PubMed

    Sarkar, Avik R; Heo, Cheol Ho; Kim, Eunjin; Lee, Hyo Won; Singh, Hardev; Kim, Jeong Jin; Kang, Hyuk; Kang, Chulhun; Kim, Hwan Myung

    2015-02-11

    We report a two-photon fluorescent probe for ratiometric imaging of cysteamine in situ. This probe can detect the levels of endogenous cysteamine with statistical significance in live cells and brain hippocampal tissues, revealing that cysteamine is localized mainly in the perikaria of the pyramidal neurons and the granule cells.

  17. 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.

  18. A highly selective fluorescent probe based on Michael addition for fast detection of hydrogen sulfide.

    PubMed

    Gao, Baozhen; Cui, Lixia; Pan, Yong; Xue, Minjie; Zhu, Boyu; Zhang, Guomei; Zhang, Caihong; Shuang, Shaomin; Dong, Chuan

    2017-02-15

    A new 4-hydroxy-1,8-naphthalimide-based compound (probe 1) has been designed and synthesized. The colorimetric and fluorescent properties of probe 1 towards hydrogen sulfide (H2S) were investigated in detail. The results show that the probe 1 could selectively and sensitively recognize H2S rather than other reactive sulfur species. The reaction mechanism of this probe is an intramolecular cyclization caused by the Michael addition of H2S to give 4-hydroxy-1,8-naphthalimide. The intramolecular charge transfer of 4-hydroxy-1,8-naphthalimide is significant. Probe 1 quickly responded to H2S and showed a 75-fold fluorescence enhancement in 5min. Moreover, probe 1 could detect H2S quantitatively with a detection limit as low as 0.23μM.

  19. A highly selective fluorescent probe based on Michael addition for fast detection of hydrogen sulfide

    NASA Astrophysics Data System (ADS)

    Gao, Baozhen; Cui, Lixia; Pan, Yong; Xue, Minjie; Zhu, Boyu; Zhang, Guomei; Zhang, Caihong; Shuang, Shaomin; Dong, Chuan

    2017-02-01

    A new 4-hydroxy-1,8-naphthalimide-based compound (probe 1) has been designed and synthesized. The colorimetric and fluorescent properties of probe 1 towards hydrogen sulfide (H2S) were investigated in detail. The results show that the probe 1 could selectively and sensitively recognize H2S rather than other reactive sulfur species. The reaction mechanism of this probe is an intramolecular cyclization caused by the Michael addition of H2S to give 4-hydroxy-1,8-naphthalimide. The intramolecular charge transfer of 4-hydroxy-1,8-naphthalimide is significant. Probe 1 quickly responded to H2S and showed a 75-fold fluorescence enhancement in 5 min. Moreover, probe 1 could detect H2S quantitatively with a detection limit as low as 0.23 μM.

  20. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe

    NASA Astrophysics Data System (ADS)

    Beth, Albert H.; Cobb, Charles E.; Beechem, Joseph M.

    1992-04-01

    A spin-labeled derivative of eosin was chemically synthesized from 5-aminoeosin and the nitroxide spin label 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-carboxylic acid. Following determination of the chemical identity of the spin-labeled eosin (5-SLE) by FAB mass spectroscopy, its optical and magnetic resonance spectroscopic properties were characterized in aqueous solution and compared to a diamagnetic eosin derivative, 5-acetamido eosin (5- AcE). The visible light absorption maximum of 5-SLE was 518 nm, the same as for 5-AcE. The fluorescence quantum yield of 5-SLE was only reduced by approximately 10% relative to 5-AcE, and the fluorescence lifetime was marginally reduced relative to 5-AcE. The phosphorescence lifetime and yield for 5-SLE were very similar to those for 5-AcE. The phosphorescence yield of 5-SLE bound noncovalently to BSA was reduced by approximately 60% relative to 5-AcE, and the phosphorescence lifetime reduced from approximately 2.4 msec (5-AcE) to 1.6 msec (5-SLE). Reduction of the nitroxide moiety of the 5-SLE with sodium ascorbate resulted in minimal changes in the fluorescence and phosphorescence quantum yields and lifetimes. This indicated that the unpaired electron of the nitroxide spin label did not seriously affect the optical spectroscopic characteristics of the spin-labeled eosin molecule. The quantum yields and lifetimes of 5-SLE were still quite acceptable for time- resolved fluorescence and phosphorescence studies. The electron paramagnetic resonance (EPR) spectrum of 5-SLE in aqueous solution has a lineshape consistent with a molecule the size of 5-SLE undergoing rapid rotational reorientation. When bound to BSA, the EPR spectrum of 5-SLE was broadened to a near slow motion limit for EPR, as expected for the relatively slowly rotating protein-5-SLE complex. Time-resolved phosphorescence anisotropy and saturation transfer EPR (ST-EPR) experiments with samples of 5-SLE bound to BSA in solutions of varying glycerol concentrations at 2

  1. A new turn on coumarin-based fluorescence probe for Ga3 + detection in aqueous solution

    NASA Astrophysics Data System (ADS)

    Yan, Liqiang; Zhou, Yan; Du, Wenqi; Kong, Zhineng; Qi, Zhengjian

    2016-02-01

    The probe CT was synthesized and investigated as a novel label-free chemosensor for Ga3 + detection in water. Probe CT showed remarkable selectivity and sensitivity for Ga3 + in Tris-HCl aqueous buffer solution (pH 7.0). The chemosensor responded rapidly to Ga3 + with a 1:1 stoichiometry. Meanwhile, the unapparent changes of fluorescence lifetime decays suggest the turn-on process of probe CT by Ga3 + which appears to be a static mechanism.

  2. A fast-responsive fluorescent probe for sulfite and its bioimaging.

    PubMed

    Wang, Jiaoliang; Long, Liping; Xiao, Xiaoming

    2016-05-01

    A turn-on fluorescent probe Coumarin-SO2 based on a nucleophilic addition reaction was developed for the rapid detection of SO3(2-) in aqueous media. The probe Coumarin-SO2 displays excellent water solubility, fast response, highly sensitivity and highly selectivity over other biological related species. More importantly, living cell imaging experiments indicate the feasibility of using the probe for the detection of SO3(2-) in biological systems.

  3. 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

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

    PubMed

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

    2017-02-11

    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.

  5. Distribution of fluorescent probe molecules throughout the phospholipid membrane depth

    NASA Astrophysics Data System (ADS)

    Nemkovich, Nicolai A.; Rubinov, Anatoly N.; Savvidi, M. G.; Tomin, V. I.; Shcherbatska, Nina V.

    1991-05-01

    A method to determine the distribution function shape of the luminescent probe molecules over biological membrane depth is suggested. It is based on: - the unhomogeneous model of the probe ensemble which permits its selection alteration selectively of the position and distribution of excited probe molecules by the wavelength of the excitation light - the use of the nonradiative electronic energy transfer (NEET) mechanism from probe molecules in vesicules to the acceptor ones in a buffer solution. The distribution function of the donor-excited molecules for different wavelength of the excitation light was calculated. Using the spectra of probe molecules at different Ae and Lippert''s equation the values of the dielectric constant of the membrane e were obtained for different regions. 1.

  6. Two-photon fluorescent probe derived from naphthalimide for cysteine detection and imaging in living cells

    NASA Astrophysics Data System (ADS)

    Liu, Yanbin; Liu, Yuwen; Liu, Wei; Liang, Shucai

    2015-02-01

    A maleimide coupling naphthalimide was reported as new two-photon fluorescent (TPF) probe for cysteine (Cys). The probe was weakly fluorescent itself due to the donor-excited photoinduced electron transfer (d-PET). After reaction with Cys, d-PET process was blocked and fluorescence enhancement of the probe was observed at 470 nm. The d-PET principle was rationalized by theoretical calculations with density functional theory and time-dependent density functional theory. Thiol-maleimide addition between the probe and Cys was confirmed by 1H NMR and mass spectrum measurements. TPF analysis demonstrated a 24.7-fold emission increase of the probe induced by Cys upon excitation at 760 nm. The two-photon action cross-section of probe-Cys adduct at 760 nm reached 42 GM compared to 1.7 GM for free probe. The probe showed high sensitivity and selectivity to Cys over other potential interferences; especially it had the capability to discriminate Cys from glutathione and homocysteine. Through TPF imaging, the probe was successfully applied in the detection of Cys in living cells.

  7. 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

  8. Lanthanide based dual-emission fluorescent probe for detection of mercury (II) in milk.

    PubMed

    Tan, Hongliang; Li, Qian; Ma, Chanjiao; Song, Yonghai; Xu, Fugang; Chen, Shouhui; Wang, Li

    2015-01-15

    It is highly desirable to develop a simple and sensitive method for Hg(2+) detection because of the dangerous nature of Hg(2+). In this work, we prepared a dual-emission fluorescent probe for Hg(2+) detection by combining two lanthanide chelates with different emission wavelengths. Green-emitting terbium (Tb(3+)) chelates as reference signals were embedded into SiO2 nanoparticles and red-emitting europium (Eu(3+)) chelates as response units were covalently linked to the surface of silica shell. Upon the addition of Hg(2+), the fluorescence of Eu(3+) chelates can be selectively quenched, while the fluorescence of Tb(3+) chelates remained unchanged. As a kind of Hg(2+) nanosensor, the dual-emission fluorescent probe exhibited excellent selectivity to Hg(2+) and high sensitivity up to 7.07 nM detection limit. The Hg(2+) levels in drinking water and milk samples were determined by using the dual-emission fluorescent probe with satisfied recovery. Additionally, our probe has a long enough fluorescence lifetime, which can avoid the interference from autofluorescence of the biological samples. We envision that the proposed probe could find great potential applications for ultrasensitive time-resolved fluorometric assays and biomedical imaging in the future.

  9. A FRET-based fluorescent probe for mercury ions in water and living cells

    NASA Astrophysics Data System (ADS)

    Zhang, Bo; Ma, Pinyi; Gao, Dejiang; Wang, Xinghua; Sun, Ying; Song, Daqian; Li, Xuwen

    2016-08-01

    On the basis of fluorescence resonance energy transfer (FRET), a new rhodamine derivative (DRh) was synthesized as a ratiometric fluorescent probe for detecting Hg2 + in water and living cells samples. The recognition properties of the probe DRh with metal ions had been investigated in H2O/CH3CN (9:1, v/v; Tris-HCl 50 mmol L- 1; pH = 7.0) solution by the UV-Vis spectrophotometry and the fluorescence spectrophotometry. The results showed that the probe DRh exhibited the selective recognition of Hg2 +. Upon the addition of Hg2 +, the spirolactam ring of probe DRh was opened. The 1:1 stoichiometric structure between DRh and Hg2 + were supported by Job's plot, MS and DFT theoretical calculations. The linearly fluorescence intensity ratio (I582/I538) is proportional to the concentration of Hg2 + in the range 0-30 μmol L- 1. The limit of detection (LOD) of Hg2 + is 0.008 μmol L- 1 (base on S/N = 3). The present probe was applied to the determination of Hg2 + in neutral water samples and gave recoveries ranging from 104.5 to 107.9%. Furthermore, the fluorescent probe also can be applied as a bioimaging reagent for Hg2 + detection in HeLa cells.

  10. Novel use of poly(3,4-ethylenedioxythiophene) nanoparticles for fluorescent nucleic acid detection.

    PubMed

    Zhang, Yingwei; Liu, Sen; Wang, Lei; Luo, Yonglan; Tian, Jingqi; Asiri, Abdullah M; Al-Youbi, Abdulrahman O; Sun, Xuping

    2012-03-12

    In this paper, we demonstrate the novel use of poly(3,4-ethylene dioxythiophene) (PEDOT) nanoparticle as a very effective fluorescent sensing platform for the detection of nucleic acid sequences. The principle of the assay lies in the fact that the adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by PEDOT nanoparticle leads to substantial fluorescence quenching, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of the hybridized complex from PEDOT nanoparticle surface and subsequent recovery of fluorescence. A detection limit as low as 30 pM could be achieved in this sensing system. We also demonstrate its application for multiplexed detection of nucleic acid sequences. Furthermore, this sensing system can realize the detection of single-base mismatch even in multiplexed format. It is of importance to note that the successful use of this sensing platform in human blood serum system is also demonstrated.

  11. Reversible Fluorescent Probe for Selective Detection and Cell Imaging of Oxidative Stress Indicator Bisulfite.

    PubMed

    Zhang, Yajiao; Guan, Lingmei; Yu, Huan; Yan, Yehan; Du, Libo; Liu, Yang; Sun, Mingtai; Huang, Dejian; Wang, Suhua

    2016-04-19

    In this paper, we report a benzothiazole-functionalized cyanine fluorescence probe and demonstrate that it is selectively reactive to bisulfite, an intermediate indicator for oxidative stress. The selective reaction can be monitored by distinct ratiometric fluorescence variation favorable for cell imaging and visualization. The original probe can be regenerated in high yield through the elimination of bisulfite from the product by peroxides such as hydrogen peroxide, accompanied by fluorescence turning on at 590 nm, showing a potential application for the detection of peroxides. We successfully applied this probe for fluorescence imaging of bisulfite in cancer cells (MCF-7) treated with bisulfite and hydrogen peroxide as well as a selective detection limit of 0.34 μM bisulfite in aqueous solution.

  12. Noninvasive fluorescence excitation spectroscopy during application of 5-aminolevulinic acid in vivo.

    PubMed

    Juzenas, Petras; Juzeniene, Asta; Kaalhus, Olav; Iani, Vladimir; Moan, Johan

    2002-10-01

    The fluorescence of PpIX induced by topical application of 5-aminolevulinic acid (ALA) in normal mouse skin was studied noninvasively by means of a fibre optic probe. The fluorescence excitation spectrum of PpIX exhibits five distinct peaks at around 408. 510, 543, 583 and 633 nm under fluorescence monitoring at the second emission peak of PpIX (705 nm). The transmission of the excitation light is wavelength dependent: the long wavelength light (>600 nm) penetrates deeper into the tissues by a factor of 6 compared with the short wavelength light (<590 nm). Thus, the fluorescence excitation spectrum of PpIX measured on the surface of the skin can be used to estimate the depth of the penetration of topically applied ALA. The fluorescence excitation spectra calculated for the depth 1.1 mm obtained the best fit with the experimentally measured spectra after topical application of ALA.

  13. Photophysics of a Fluorescent Non-natural Amino Acid: p-Cyanophenylalanine

    PubMed Central

    Serrano, Arnaldo L.; Troxler, Thomas; Tucker, Matthew J.; Gai, Feng

    2010-01-01

    The non-natural amino acid p-cyanophenylalanine (PheCN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of PheCN in a series of solvents. It is found that the fluorescence lifetime of PheCN shows a linear dependence on the Kamlet-Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of PheCN fluorescence in protein conformational studies. PMID:20419080

  14. Confocal imaging with a fluorescent bile acid analogue closely mimicking hepatic taurocholate disposition.

    PubMed

    De Bruyn, Tom; Sempels, Wouter; Snoeys, Jan; Holmstock, Nico; Chatterjee, Sagnik; Stieger, Bruno; Augustijns, Patrick; Hofkens, Johan; Mizuno, Hideaki; Annaert, Pieter

    2014-06-01

    This study aimed to characterize the in vitro hepatic transport mechanisms in primary rat and human hepatocytes of the fluorescent bile acid derivative N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD), previously synthesized to study the activity of the bile salt export pump (BSEP). The fluorescent bile acid derivative exhibited saturable uptake kinetics in suspended rat hepatocytes. Hepatic uptake was inhibited in the presence of substrates/inhibitors of the organic anion transporting polypeptide (Oatp) family and Na(+) -taurocholate cotransporting peptide (Ntcp). Concentration-dependent uptake of the fluorescent bile acid was also saturable in Chinese hamster ovary cells transfected with rNtcp, hNTCP, OATP1B1, or OATP1B3. The fluorescent bile acid derivative was actively excreted in the bile canaliculi of sandwich-cultured rat and human hepatocytes (SCRH and SCHH), with a biliary excretion index (BEI) of 26% and 32%, respectively. In SCRH, cyclosporin A significantly decreased the BEI to 5%. Quantification by real-time confocal imaging further confirmed canalicular transport of the fluorescent bile acid derivative (BEI = 75%). We conclude that tauro-nor-THCA-24-DBD is a useful probe to study interference of drugs with NTCP/Ntcp- and BSEP/Bsep-mediated transport in fluorescence-based in vitro assays.

  15. Phenylboronic acid functionalized reduced graphene oxide based fluorescence nano sensor for glucose sensing.

    PubMed

    Basiruddin, S K; Swain, Sarat K

    2016-01-01

    Reduced graphene has emerged as promising tools for detection based application of biomolecules as it has high surface area with strong fluorescence quenching property. We have used the concept of fluorescent quenching property of reduced graphene oxide to the fluorescent probes which are close vicinity of its surface. In present work, we have synthesized fluorescent based nano-sensor consist of phenylboronic acid functionalized reduced graphene oxide (rGO-PBA) and di-ol modified fluorescent probe for detection of biologically important glucose molecules. This fluorescent graphene based nano-probe has been characterized by high resolution transmission electron microscope (HRTEM), Atomic force microscope (AFM), UV-visible, Photo-luminescence (PL) and Fourier transformed infrared (FT-IR) spectroscopy. Finally, using this PBA functionalized reduced GO based nano-sensor, we were able to detect glucose molecule in the range of 2 mg/mL to 75 mg/mL in aqueous solution of pH7.4.

  16. 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.

  17. Fluorescent probe for detection of Cu2+ using core-shell CdTe/ZnS quantum dots.

    PubMed

    Bian, Wei; Wang, Fang; Zhang, Hao; Zhang, Lin; Wang, Li; Shuang, Shaomin

    2015-11-01

    Core-shell CdTe/ZnS quantum dots capped with 3-mercaptopropionic acid (MPA) were successfully synthesized in aqueous medium by hydrothermal synthesis. These quantum dots have advantages compared to traditional quantum dots with limited biological applications, high toxicity and tendency to aggregate. The concentration of Cu(2+) has a significant impact on the fluorescence intensity of quantum dots (QDs), therefore, a rapid sensitive and selective fluorescence probe has been proposed for the detection of Cu(2+) in aqueous solution. Under optimal conditions, the fluorescence intensity of CdTe/ZnS QDs was linearly proportional to the concentration of Cu(2+) in the range from 2.5 × 10(-9) M to 17.5 × 10(-7) M with the limit of 1.5 × 10(-9) M and relative standard deviation of 0.23%. The quenching mechanism is static quenching with recoveries of 97.30-102.75%.

  18. A fluorescent probe for detection of histidine in cellular homogenate and ovalbumin based on the strategy of click chemistry.

    PubMed

    Qiu, Suyan; Miao, Miao; Wang, Tianxi; Lin, Zhenyu; Guo, Longhua; Qiu, Bin; Chen, Guonan

    2013-04-15

    A sensitive and selective fluorescent probe for histidine has been designed by taking the advantages of the click chemistry reaction (copper(I)-catalyzed azides and alkynes cycloaddition) and the inhibition of copper(II)-induced ascorbate oxidation by histidine. The fluorescence intensity decreases with the increasing of histidine concentration, and the value of F₀/F shows a good linear relationship with the concentration of histidine over the range of 0.5-100 μM with a detection limit of 76 nM, which is lower than those of many other fluorescent sensors. Moreover, the sensor has high specificity for histidine compared to some potential interferents, such as other amino acids and metal ions. In addition, the proposed sensor has been applied to determine histidine in cellular homogenate and ovalbumin samples with satisfactory results. The preferable simplicity, sensitivity and specificity for the detection of histidine indicate that the proposed sensor have potential prospect in environmental and biomedical analysis.

  19. High-fidelity hydrophilic probe for two-photon fluorescence lysosomal imaging.

    PubMed

    Wang, Xuhua; Nguyen, Dao M; Yanez, Ciceron O; Rodriguez, Luis; Ahn, Hyo-Yang; Bondar, Mykhailo V; Belfield, Kevin D

    2010-09-08

    The synthesis and characterization of a novel two-photon-absorbing fluorene derivative, LT1, selective for the lysosomes of HCT 116 cancer cells, is reported. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for two-photon fluorescence microscopy (2PFM) lysosomal imaging. The cytotoxicity of the probe was investigated to evaluate the potential of using this probe for live two-photon fluorescence biological imaging applications. Colocalization studies of the probe with commercial Lysotracker Red in HCT 116 cells demonstrated the specific localization of the probe in the lysosomes with an extremely high colocalization coefficient (0.96). A figure of merit was introduced to allow comparison between probes. LT1 has a number of properties that far exceed those of commercial lysotracker probes, including higher two-photon absorption cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. 2PFM was used to demonstrate lysosomal tracking with LT1.

  20. [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.

  1. Fluorescent diamond nanoparticle as a probe of intracellular traffic in primary neurons in culture

    NASA Astrophysics Data System (ADS)

    Le, Xuan Loc; Lepagnol-Bestel, Aude-Marie; Adam, Marie-Pierre; Thomas, Alice; Dantelle, Géraldine; Chang, Cheng-Chun; Mohan, Nitin; Chang, Huan-Cheng; Treussart, François; Simonneau, Michel

    2012-03-01

    Neurons display dendritic spines plasticity and morphology anomalies in numerous psychiatric and neurodegenerative diseases. These changes are associated to abnormal dendritic traffic that can be evidenced by fluorescence microscopy. As a fluorescent probe we propose to use fluorescent diamond nanoparticles with size of < 50 nm. Color centers embedded inside the diamond nanoparticles are perfectly photostable emitters allowing for long-term tracking. Nanodiamond carbon surface is also well suited for biomolecule functionalization to target specific cellular compartments. We show that fluorescent nanodiamonds can be spontaneously internalized in neurons in culture and imaged by confocal and Total Internal Reflection (TIRF) microscopy with a high signal over background ratio.

  2. Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes

    PubMed Central

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

    2009-01-01

    We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise. PMID:19413953

  3. Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes.

    PubMed

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

    2009-05-06

    We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.

  4. 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.

  5. A Smart Near-Infrared Fluorescence Probe for Selective Detection of Tau Fibrils in Alzheimer's Disease.

    PubMed

    Seo, Yujin; Park, Kwang-Su; Ha, Taewoong; Kim, Mi Kyoung; Hwang, Yu Jin; Lee, Junghee; Ryu, Hoon; Choo, Hyunah; Chong, Youhoon

    2016-11-16

    Development of a novel, tau-selective smart near-infrared fluorescence (NIRF) probe was attempted by combining the previously identified core scaffold 3,5-dimethoxy-N,N-dimethylaniline-4-yl moiety, with the characteristic donor-π-acceptor architecture of the smart NIRF Aβ probes DANIR-2c and MCAAD-3. A series of compounds (2 and 3) were prepared, which were identified as "turn-on" NIRF probes for the visual detection of tau aggregates and Aβ fibrils (λem = 650 nm, Stokes shifts = 70-110 nm). In particular, combination of the 3,5-dimethoxy-N,N-dimethylanilin-4-yl moiety and the donor part of MCAAD-3 endowed the resulting probes, 3g and 3h, with significant selectivity toward tau aggregates (selectivity for tau over Aβ = 5.7 and 3.8); they showed much higher fluorescence intensities upon binding to tau aggregates (FItau = 49 and 108) than when bound to Aβ fibrils (FIAβ = 9 and 28). Quantitative analysis of binding affinities and fluorescence properties of 3g and 3h revealed that microenvironment-sensitive molecular rotor-like behavior, rather than binding affinity to the target, is responsible for their selective turn-on fluorescence detection of tau fibrils. Selective fluorescent labeling of tau fibrils by 3g and 3h was further demonstrated by immunofluorescence staining of human Alzheimer's disease brain sections, which showed colocalization of the probes (3g and 3h) and phosphorylated tau antibody.

  6. Frequency-domain flow cytometry: fluorescence-lifetime-based sensing technology for analyzing cells and chromosomes labeled with fluorescent probes

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Crissman, Harry A.; Lehnert, Bruce E.; Lehnert, Nancy M.; Deka, Chiranjit

    1997-05-01

    A flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making frequency-domain, excited-state lifetime measurements on cells/chromosomes labeled with fluorescent probes, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence signals are processed by (1) low-pass filtering to obtain conventional FCM dc-excited signals and (2) phase-sensitive detection electronics to resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase-shifts and to quantify fluorescence lifetimes in real time. Processed signals are displayed as frequency distribution histograms and bivariate contour diagrams. Recent examples of biological applications include: (1) lifetime histograms recorded on autofluorescent human lung fibroblasts, murine thymus cells labeled with antibodies conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, and on cultured cells, nuclei, and chromosomes stained with DNA-binding fluorochromes and (2) phase-resolved, fluorescence signal- intensity histograms recorded on autofluorescent HLFs labeled with immunofluorescence markers and on murine thymus cells labeled with Red 613-antiThy 1.2 and propidium iodide (PI positive `dead' cells) to demonstrate the resolution of signals from highly overlapping emission spectra. This technology will increase the number of fluorescent markers usable in multilabeling studies and lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

  7. Determination of Levo-Rotatory Leucine by a Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Cheng, D.; Zhu, H.

    2015-03-01

    With a certain concentration of Michaelis buffer solution (pH 6.1), and in the presence of CTMAB, the fluorescence intensity of fluorescein, which is quenched by Pd 2+ , can be enhanced after adding a certain content of L-leucine. Thereby, we establish a fluorescence spectrometry method to detect the content of L-leucine, the added amount of which is proportional to the fluorescence enhancement. When the excitation slit width is 3 nm and the emission slit width is 5 nm, we obtain the following results: correlation coefficient R = 0.9981, linear range 0.125-1.375 mg/l, and detection limit 0.00028 mg/l.

  8. Self-quenching DNA probes based on aggregation of fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Schafer, Gabriela; Muller, Matthias; Hafner, Bernhard; Habl, Gregor; Nolte, Oliver; Marme, Nicole; Knemeyer, Jens-Peter

    2005-04-01

    Here we present a novel class of self-quenching, double-labeled DNA probes based on the formation of non fluorescent H-type dye dimers. We therefore investigated the aggregation behavior of the red-absorbing oxazine derivative MR121 and found a dimerization constant of about 3000 M-1. This dye was successfully used to develop hairpin-structured as well as linear self-quenching DNA probes that report the presence of the target DNA by an increase of the fluorescence intensity by a factor of 3 to 12. Generally fluorescence quenching of the hairpin-structure probes is more efficient compared to the linear probes, whereas the kinetic of the fluorescence increase is significantly slower. The new probes were used for the identification of different mycobacteria and their antibiotic resistant species. As a test system a probe for the identification of a DNA sequence specific for the Mycobacterium xenopi was synthesized differing from the sequence of the Mycobacterium fortuitum by 6 nucleotides. Furthermore we developed a method for the discrimination between the sequences of the wild type and an antibiotic resistant species of Mycobacterium tuberculosis. Both sequences differ by just 2 nucleotides and were detected specifically by the use of competing olignonucleotides.

  9. Fluorescent probes and fluorescence (microscopy) techniques--illuminating biological and biomedical research.

    PubMed

    Drummen, Gregor P C

    2012-11-28

    Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

  10. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  11. CO2 exploding cluster dynamics probed by XUV fluorescence

    NASA Astrophysics Data System (ADS)

    Negro, M.; Ruf, H.; Fabre, B.; Dorchies, F.; Devetta, M.; Staedter, D.; Vozzi, C.; Mairesse, Y.; Stagira, S.

    2014-07-01

    Clusters excited by intense laser pulses are a unique source of warm dense matter, that has been the subject of intensive experimental studies. The majority of these investigations concern atomic clusters, whereas the evolution of molecular clusters excited by intense laser pulses is less explored. In this work we trace the dynamics of C{{O}_{2}} clusters triggered by a few-cycle 1.45\\;\\mu m driving pulse through the detection of XUV fluorescence induced by a delayed 800 nm ignition pulse. Striking differences among fluorescence dynamics from different ionic species are observed.

  12. Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis

    PubMed Central

    Goswami, Linee; Pratihar, Sanjay; Dasgupta, Suman; Bhattacharyya, Pradip; Mudoi, Pronab; Bora, Jayanta; Bhattacharya, Satya Sundar; Kim, Ki Hyun

    2016-01-01

    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. PMID:27456167

  13. 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.

  14. Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis.

    PubMed

    Goswami, Linee; Pratihar, Sanjay; Dasgupta, Suman; Bhattacharyya, Pradip; Mudoi, Pronab; Bora, Jayanta; Bhattacharya, Satya Sundar; Kim, Ki Hyun

    2016-07-26

    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.

  15. 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.

  16. A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology.

    PubMed

    Hu, Ming-Hao; Chen, Xiao; Chen, Shuo-Bin; Ou, Tian-Miao; Yao, Meicun; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2015-11-25

    Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology.

  17. A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe1

    PubMed Central

    Messerli, Shanta M; Prabhakar, Shilpa; Tang, Yi; Shah, Khalid; Cortes, Maria L; Murthy, Vidya; Weissleder, Ralph; Breakefield, Xandra O; Tung, Ching-Hsuan

    2004-01-01

    Abstract Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin 1β-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-lacZ) expressing a catalytically active caspase-1-lacZ fusion protein; 3) inhibition of HGC-ICE-lacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo. PMID:15140398

  18. Hollow-core photonic crystal fiber probe for remote fluorescence sensing with single molecule sensitivity.

    PubMed

    Ghenuche, Petru; Rigneault, Hervé; Wenger, Jérôme

    2012-12-17

    Current optical fiber probes for fluorescence spectroscopy struggle with large luminescence background and low detection sensitivities that challenge the detection of fluorescent molecules at sub-micromolar concentration. Here we report the demonstration of a hollow-core photonic crystal fiber (HC-PCF) probe for remote fluorescence sensing with single molecule sensitivity down to nanomolar concentrations, where both the excitation and fluorescence beams are counter-propagating through the same fiber. A 20 μm polystyrene microsphere is used to efficiently excite and collect the fluorescence from the sample solution thanks to a photonic nanojet effect. Compared to earlier work with silica fibers, the new HC-PCF-microsphere probe achieves a 200x improvement of the signal-to-noise ratio for a single molecule detection event, and a 1000x reduction of the minimum detectable concentration. The device is implemented with fluorescence correlation spectroscopy to distinguish between molecules of similar fluorescence spectra based on the analysis of their translational diffusion properties, and provides similar performance as conventional confocal microscopes.

  19. An iminocoumarin benzothiazole-based fluorescent probe for imaging hydrogen sulfide in living cells.

    PubMed

    Zhang, Huatang; Xie, Yusheng; Wang, Ping; Chen, Ganchao; Liu, Ruochuan; Lam, Yun-Wah; Hu, Yi; Zhu, Qing; Sun, Hongyan

    2015-04-01

    Hydrogen sulfide (H2S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H2S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H2S under physiological condition. The fluorescent intensity of the probe was linearly related to H2S concentration in the range of 0-100 μM with a detection limit of 0.15 μM (3σ/slope). The probe also showed excellent selectivity towards H2S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H2S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H2S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H2S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H2S.

  20. LASER FLUORESCENCE EEM PROBE FOR CONE PENETROMETER POLLUTION ANALYSIS

    EPA Science Inventory

    A fiber optic LIF (Laser induced fluorescence) EEM (Excitation emission matrix) instrument for CPT deployment has been successfully developed and field tested. The system employs a Nd: YAG laser and Raman shifter as a rugged field portable excitation source. This excitation sou...

  1. Brain cancer probed by native fluorescence and stokes shift spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yan; Liu, Cheng-hui; He, Yong; Pu, Yang; Li, Qingbo; Wang, Wei; Alfano, Robert R.

    2012-12-01

    Optical biopsy spectroscopy was applied to diagnosis human brain cancer in vitro. The spectra of native fluorescence, Stokes shift and excitation spectra were obtained from malignant meningioma, benign, normal meningeal tissues and acoustic neuroma benign tissues. The wide excitation wavelength ranges were used to establish the criterion for distinguishing brain diseases. The alteration of fluorescence spectra between normal and abnormal brain tissues were identified by the characteristic fluorophores under the excitation with UV to visible wavelength range. It was found that the ratios of the peak intensities and peak position in both spectra of fluorescence and Stokes shift may be used to diagnose human brain meninges diseases. The preliminary analysis of fluorescence spectral data from cancer and normal meningeal tissues by basic biochemical component analysis model (BBCA) and Bayes classification model based on statistical methods revealed the changes of components, and classified the difference between cancer and normal human brain meningeal tissues in a predictions accuracy rate is 0.93 in comparison with histopathology and immunohistochemistry reports (gold standard).

  2. Boosting nanodiamond fluorescence: towards development of brighter probes.

    PubMed

    Havlik, Jan; Petrakova, Vladimira; Rehor, Ivan; Petrak, Vaclav; Gulka, Michal; Stursa, Jan; Kucka, Jan; Ralis, Jan; Rendler, Torsten; Lee, San-Yung; Reuter, Rolf; Wrachtrup, Joerg; Ledvina, Miroslav; Nesladek, Milos; Cigler, Petr

    2013-04-21

    A novel approach for preparation of ultra-bright fluorescent nanodiamonds (fNDs) was developed and the thermal and kinetic optimum of NV center formation was identified. Combined with a new oxidation method, this approach enabled preparation of particles that were roughly one order of magnitude brighter than particles prepared with commonly used procedures.

  3. Probing interaction of a fluorescent ligand with HIV TAR RNA

    NASA Astrophysics Data System (ADS)

    Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

    2017-02-01

    Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

  4. Probing interaction of a fluorescent ligand with HIV TAR RNA.

    PubMed

    Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

    2017-02-15

    Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

  5. Kinetics of intercalation of fluorescent probes in magnesium-aluminium layered double hydroxide within a multiscale reaction-diffusion framework

    NASA Astrophysics Data System (ADS)

    Saliba, Daniel; Al-Ghoul, Mazen

    2016-11-01

    We report the synthesis of magnesium-aluminium layered double hydroxide (LDH) using a reaction-diffusion framework (RDF) that exploits the multiscale coupling of molecular diffusion with chemical reactions, nucleation and growth of crystals. In an RDF, the hydroxide anions are allowed to diffuse into an organic gel matrix containing the salt mixture needed for the precipitation of the LDH. The chemical structure and composition of the synthesized magnesium-aluminium LDHs are determined using powder X-ray diffraction (PXRD), thermo-gravimetric analysis, differential scanning calorimetry, solid-state nuclear magnetic resonance (SSNMR), Fourier transform infrared and energy dispersive X-ray spectroscopy. This novel technique also allows the investigation of the mechanism of intercalation of some fluorescent probes, such as the neutral three-dimensional rhodamine B (RhB) and the negatively charged two-dimensional 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), using in situ steady-state fluorescence spectroscopy. The incorporation of these organic dyes inside the interlayer region of the LDH is confirmed via fluorescence microscopy, solid-state lifetime, SSNMR and PXRD. The activation energies of intercalation of the corresponding molecules (RhB and HPTS) are computed and exhibit dependence on the geometry of the involved probe (two or three dimensions), the charge of the fluorescent molecule (anionic, cationic or neutral) and the cationic ratio of the corresponding LDH. This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

  6. 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

  7. Detection of coalescing agents in water-borne latex emulsions using an environment sensitive fluorescent probe.

    PubMed

    Raja, Tanzeela Nazir; Brouwer, Albert M; Biemans, Koen; Nabuurs, Tijs; Tennebroek, Ronald

    2010-07-30

    In this paper we report the determination of partitioning of coalescing agents (organic co-solvents) in water-borne latex emulsions by means of a fluorescence method. An environment-sensitive fluorescent probe was copolymerized via emulsion polymerization. The presence of organic co-solvents inside the polymer particles is revealed by the photophysical properties of the probe. In particular, the position of the fluorescence emission maximum of co-polymerized can be used to measure the amount of coalescing agent present in the polymer particles. The spectral shifts are shown to be due to the softening of the matrix, rather than to solvation of the probe by the added co-solvent.

  8. Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors.

    PubMed

    Uri, Asko; Lust, Marje; Vaasa, Angela; Lavogina, Darja; Viht, Kaido; Enkvist, Erki

    2010-03-01

    Conjugates of adenosine mimics and d-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.

  9. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    PubMed Central

    Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

    2009-01-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

  10. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    PubMed Central

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-01-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

  11. Glutathione-functionalized graphene quantum dots as selective fluorescent probes for phosphate-containing metabolites

    NASA Astrophysics Data System (ADS)

    Liu, Jing-Jing; Zhang, Xiao-Long; Cong, Zhong-Xiao; Chen, Zhi-Tao; Yang, Huang-Hao; Chen, Guo-Nan

    2013-02-01

    Bright blue fluorescent glutathione-functionalized graphene quantum dots (GQDs@GSH) were prepared by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.6%. Futhermore, the obtained GQDs@GSH can be used as a probe to estimate the ATP level in cell lysates and human blood serum.Bright blue fluorescent glutathione-functionalized graphene quantum dots (GQDs@GSH) were prepared by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.6%. Futhermore, the obtained GQDs@GSH can be used as a probe to estimate the ATP level in cell lysates and human blood serum. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33794d

  12. CdTe quantum dot as a fluorescence probe for vitamin B12 in dosage form

    NASA Astrophysics Data System (ADS)

    Vaishnavi, E.; Renganathan, R.

    2013-11-01

    We here report the CdTe quantum dot (CdTe QDs)-based sensor for probing vitamin B12 derivatives in aqueous solution. In this paper, simple and sensitive fluorescence quenching measurements has been employed. The Stern-Volmer constant (KSV), quenching rate constant (kq) and binding constant (K) were rationalized from fluorescence quenching measurement. Furthermore, the fluorescence resonance energy transfer (FRET) mechanism was discussed. This method was applicable over the concentration ranging from 1 to 14 μg/mL (VB12) with correlation coefficient of 0.993. The limit of detection (LOD) of VB12 was found to be 0.15 μg/mL. Moreover, the present approach opens a simple pathway for developing cost-effective, sensitive and selective QD-based fluorescence sensors/probes for biologically significant VB12 in pharmaceutical sample with mean recoveries in the range of 100-102.1%.

  13. New fluorescence imaging probe with high spatial resolution for in vivo applications.

    PubMed

    Bonnans, V; Gharbi, T; Pieralli, C; Wacogne, B; Humbert, Ph

    2004-01-01

    A new fiberized fluorescence imaging probe is presented. This device can potentially be used for a wide range of biological or medical applications. By exploiting the chromatic aberrations of gradient index lenses, the excitation blue or near-UV excitation light is focused on the sample surface, while the red fluorescence signal is efficiently launched back to collecting fibers. The excitation fiber is single mode at the working wavelength so that a resolution of 5 microm is obtained over a scanning area of several square millimeters. Experimental fluorescence images are presented. They concern either self-fabricated fluorescent microsamples or views of leaves that constitute an example of biological tissues analysis. The probe can also be adapted for spectroscopic investigations.

  14. CdTe quantum dot as a fluorescence probe for vitamin B(12) in dosage form.

    PubMed

    Vaishnavi, E; Renganathan, R

    2013-11-01

    We here report the CdTe quantum dot (CdTe QDs)-based sensor for probing vitamin B12 derivatives in aqueous solution. In this paper, simple and sensitive fluorescence quenching measurements has been employed. The Stern-Volmer constant (KSV), quenching rate constant (kq) and binding constant (K) were rationalized from fluorescence quenching measurement. Furthermore, the fluorescence resonance energy transfer (FRET) mechanism was discussed. This method was applicable over the concentration ranging from 1 to 14μg/mL (VB12) with correlation coefficient of 0.993. The limit of detection (LOD) of VB12 was found to be 0.15μg/mL. Moreover, the present approach opens a simple pathway for developing cost-effective, sensitive and selective QD-based fluorescence sensors/probes for biologically significant VB12 in pharmaceutical sample with mean recoveries in the range of 100-102.1%.

  15. Highly selective "Off-On" fluorescent probe for histidine and its imaging in living cells.

    PubMed

    Chen, Tiantian; Yin, Liyan; Huang, Chusen; Qin, Yiqiao; Zhu, Weiping; Xu, Yufang; Qian, Xuhong

    2015-04-15

    A naphthalimide-based fluorescent probe CP has been synthesized with simple steps. It can selectively and sensitively recognize copper ions (Cu(2+)) in HEPES buffer (50mM, pH 7.2). The fluorescence intensity of CP is linearly proportional to the concentration of Cu(2+) ranging from 0-8.3μM (correlation coefficient R(2)=0.9808). The resulted complex CP@Cu can serve as a turn-on fluorescent probe for the detection of histidine and histidine rich proteins in broad pH application range. Upon the addition of histidine, the fluorescence intensity of CP@Cu exhibits a linear correlation with the concentration of histidine ranging from 0-200μM (correlation coefficient R(2)=0.9912). Moreover, CP@Cu has potential for imaging histidine in vitro experiments and has promise in real sample applications with great validity.

  16. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  17. Quantitatively mapping cellular viscosity with detailed organelle information via a designed PET fluorescent probe.

    PubMed

    Liu, Tianyu; Liu, Xiaogang; Spring, David R; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-24

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  18. Space-resolved fluorescence spectroscopic measurements with an optical fiber probe

    NASA Astrophysics Data System (ADS)

    Li, Enbang; Qiu, Hialin

    2008-12-01

    By monitoring of the emitted signal from a sample while varying the excitation wavelength, emission wavelength or both of them, fluorescence spectroscopy has become a powerful diagnostic technology. Fluorescence spectrometers can be used to measure and record the fluorescence spectra of a given sample, and have been successfully applied in different areas including biology, biochemistry, chemistry, medicine, environmental science, material science, food industry, and pharmaceutical industry. In order to increase the flexibility and applicability of conventional fluorescence spectrometers, we design an optic fiber probe for conducting the UV/Vis excitation light to a sample under study, and for collecting the fluorescence produced by the sample. Different excitation/emission fiber bundle arrangements have been fabricated and their performances have been evaluated and compared. Fiber adaptors which can be used for different commercial fluorescence spectrometers are also developed. In order to achieve space-resolved fluorescence spectroscopic measurements, we connect the fiber probe to a microscope which is mounted on a 3D traverse stage. Experiments and measurement results using the space-resolved fiber optic fluorescence spectrometer are presented in this paper.

  19. A coumarin-indole based colorimetric and 'turn on' fluorescent probe for cyanide

    NASA Astrophysics Data System (ADS)

    Xu, Yu; Dai, Xi; Zhao, Bao-Xiang

    2015-03-01

    A novel coumarin-indole based chemodosimeter with a simple structure was designed and prepared via a condensation reaction in high yield. The probe exhibited very high selectivity towards cyanide on both fluorescence and UV-vis spectra, which allowed it to quantitatively detect and imaging cyanide ions in organic-aqueous solution by either fluorescence enhancement or colorimetric changes. Confirmed by 1H NMR and HRMS spectra, the detection mechanism was proved to be related with the Michael addition reaction induced by cyanide ions, which blocked the intramolecular charge transfer (ICT) of the probe. Moreover, the probe was able to be utilized efficiently in a wide pH range (7.5-10) with negligible interference from other anions and a low detection limit of 0.51 μM. Application in 5 kinds of natural water source and accurate detection of cyanide in tap water solvent system also indicated the high practical significance of the probe.

  20. 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

  1. A coumarin-indole based colorimetric and "turn on" fluorescent probe for cyanide.

    PubMed

    Xu, Yu; Dai, Xi; Zhao, Bao-Xiang

    2015-03-05

    A novel coumarin-indole based chemodosimeter with a simple structure was designed and prepared via a condensation reaction in high yield. The probe exhibited very high selectivity towards cyanide on both fluorescence and UV-vis spectra, which allowed it to quantitatively detect and imaging cyanide ions in organic-aqueous solution by either fluorescence enhancement or colorimetric changes. Confirmed by (1)H NMR and HRMS spectra, the detection mechanism was proved to be related with the Michael addition reaction induced by cyanide ions, which blocked the intramolecular charge transfer (ICT) of the probe. Moreover, the probe was able to be utilized efficiently in a wide pH range (7.5-10) with negligible interference from other anions and a low detection limit of 0.51μM. Application in 5 kinds of natural water source and accurate detection of cyanide in tap water solvent system also indicated the high practical significance of the probe.

  2. 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.

  3. 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

  4. Synthesis of Fluorescent Probes based on Stilbenes and Diphenylacetylenes targeting β-Amyloid Plaques

    PubMed Central

    Parhi, Ajit K.; Kung, Mei-Ping; Ploessl, Karl; Kung, Hank F.

    2008-01-01

    Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in patients with Alzheimer’s disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a, 4a-diaza s-indacene, BODIPY) with the pharmacophore possessing either a stilbene or a diphenylacetylene moiety. Different polyethylene glycol chain lengths were used as linkers between the fluorophore and the pharmacophore to adjust the lipophilicity of these probes. These compounds exhibit strong fluorescence emission between 665–680 nm and have very high extinction coefficients comparable to the parent fluorophore, BODIPY dye. PMID:19122762

  5. A Novel Fluorescent Probe for the Highly Selective and Sensitive Detection of Palladium in Aqueous Medium.

    PubMed

    Ma, Zhiwei; Wang, Xiao; Sun, Yanling; Liu, Juntao; Tong, Yan; Liu, Zhijing

    2016-11-01

    Based on the Pd(0)-catalyzed Tsuji-Trost allylic oxidative insertion reaction, we developed a fluorescent probe PdL1 for sensing Pd(0). As expected, probe PdL1 exhibited high selectivity and excellent sensitivity in both absorbance and fluorescence detection of Pd(0) in CH3CH2OH/PBS (10 mM, pH = 7.4, 6:4, v/v) solution. The detection limit was calculated to be as low as 15 nM, which can meet the selective requirements for practical application.

  6. Targeting cancer cells with folic acid-iminoboronate fluorescent conjugates.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Chudasama, Vijay; Cordeiro, Carlos; Caddick, Stephen; Gois, Pedro M P

    2014-05-25

    Herein we present the synthesis of fluorescent 2-acetylbenzeneboronic acids that undergo B-N promoted conjugation with lysozyme and N-(2-aminoethyl) folic acid (EDA-FA), generating conjugates that are selectively recognized and internalized by cancer cells that over-express folic acid receptors.

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

    PubMed Central

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

    1990-01-01

    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. Images FIGURE 3 FIGURE 5 FIGURE 7 PMID:2291938

  8. Thermal Outlining using Focused Ultrasound (TOFU) with reversible temperature sensitive fluorescent probes

    NASA Astrophysics Data System (ADS)

    Kwong, Tiffany C.; Nouizi, Farouk; Lin, Yuting; Zhu, Yue; Sampathkumaran, Uma; Gulsen, Gultekin

    2016-03-01

    Optical imaging has long been hindered by the high absorption and scattering of light in biological tissue. This makes it difficult to probe beyond a few millimeters beneath the surface without sacrificing image resolution and quantitative accuracy. Strong scattering and the inherent nature of the inverse problem makes fluorescence diffuse optical tomography (FT) extremely challenging. To this end, multi-modality techniques that combine anatomical imaging with the functional optical information have been used to improve the resolution and accuracy of FT. Previously, we have reported on the feasibility of a new imaging method, "Thermal Outlining using Focused Ultrasound" (TOFU), which combines the sensitivity of FT with the resolution of focused ultrasound using temperature reversible fluorescent probes. In this method, the position of the temperature reversible fluorescent probes is localized by an increase in fluorescent signal when the hot spot of the focused ultrasound beam is scanned over the medium. This a priori information is then utilized to guide and constrain conventional reconstruction algorithm to recover the position and concentration of the probes more accurately. The small size of the focal spot (~1.4 mm) up to a depth of 6 cm, allows imaging the distribution of these temperature sensitive agents with not only high spatial resolution but also high quantitative accuracy in deep tissue. In this work, the performance of the system will be evaluated using simulation and phantoms to investigate the dependence that size of the fluorescent distribution has on the TOFU system performance.

  9. Rotationally resolved fluorescence as a probe of molecular photoionization dynamics

    NASA Astrophysics Data System (ADS)

    Kakar, Sandeep; Choi, Heung-Cheun; Poliakoff, E. D.

    1992-11-01

    We present rotationally resolved data for the v'=0 and v'=1 levels of N2+(B 2Σu+) produced via 2σu-1 photoionization of N2. The data are obtained over a broad photon energy range (19≤hνexc≤35 eV). This is made possible by using synchrotron radiation excitation in conjunction with dispersed fluorescence detection. The results exhibit both resonant and nonresonant effects.

  10. An ESIPT-based fluorescent probe for sensitive detection of hydrazine in aqueous solution.

    PubMed

    Zhou, Ji; Shi, Ruiyan; Liu, Jianxu; Wang, Rui; Xu, Yufang; Qian, Xuhong

    2015-05-21

    A fluorescent probe for sensitive detection of hydrazine based on an ESIPT mechanism and a substitution-cyclization-elimination cascade was developed. After the addition of hydrazine, an approximately 50-fold enhancement in fluorescence intensity at 465 nm was observed and the subsequent decrease at 375 nm was observed in 10 min with a detection limit of 0.147 μM. We also detected hydrazine in HeLa cells successfully.

  11. A ratiometric near-infrared fluorescent probe for hydrazine and its in vivo applications.

    PubMed

    Hu, Chong; Sun, Wen; Cao, Jianfang; Gao, Pan; Wang, Jingyun; Fan, Jiangli; Song, Fengling; Sun, Shiguo; Peng, Xiaojun

    2013-08-02

    Based on modulation of the conjugated polymethine π-electron system of a cyanine dye derivative, a ratiometric near-infared fluorescent probe (Cy7A) for hydrazine (N2H4) has been designed and synthesized. Cy7A can be selectively hydrazinolysized with great changes in its fluorescent excitation/emission profiles, which makes it possible to detect N2H4 in water samples and living cells and, for the first time, visualize N2H4 in living mice.

  12. A new boronic acid based fluorescent reporter for catechol.

    PubMed

    Wu, Zhongyu; Li, Minyong; Fang, Hao; Wang, Binghe

    2012-12-01

    Catechol skeleton widely exists in natural products and bioactive substances. Fluorescent reporters which could recognize catechol are very promising for the construction of chemosensors to detect catechol and its derivatives in biological environment. Herein, we reported a novel catechol reporter, 2-(4-boronophenyl)quinoline-4-carboxylic acid, which exhibits significant fluorescent property changes upon binding catechol containing molecules in an aqueous solution.

  13. 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.

  14. 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.

  15. Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe.

    PubMed

    Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin

    2015-05-01

    An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

  16. Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

    SciTech Connect

    Chotimarkorn, Chatchawan; Nagasaka, Reiko; Ushio, Hideki . E-mail: hushio@s.kaiyodai.ac.jp; Ohshima, Toshiaki; Matsunaga, Shigeki

    2005-12-16

    A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, {sup 1}H NMR, and {sup 13}C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.

  17. A SERS and fluorescence dual mode cancer cell targeting probe based on silica coated Au@Ag core-shell nanorods.

    PubMed

    Zong, Shenfei; Wang, Zhuyuan; Yang, Jing; Wang, Chunlei; Xu, Shuhong; Cui, Yiping

    2012-08-15

    We report a dual mode cancer cell targeting probe based on CdTe quantum dots (QDs) conjugated, silica coated Au@Ag core-shell nanorods (Au@Ag NRs), which can generate both surface enhanced Raman scattering (SERS) and fluorescence signals. In such a probe, folic acid (FA) is used as a targeting ligand for folate receptors (FRs) overexpressed cancer cells. To synthesize the probe, Au@Ag NRs were first prepared to serve as the SERS substrates by coating an Ag shell on the gold nanorods. Then the Au@Ag NRs were labeled with 4-mercaptobenzoic acid (4MBA) to generate SERS signals, followed by being coated with a silica shell through a modified Stöber method. Finally, CdTe QDs and FA were conjugated to the silica coated Au@Ag NRs by the carbodiimide chemistry to yield fluorescence and the targeting ability, respectively. To validate the targeting capability of the probe, in vitro experiments were conducted, using HeLa cells with overexpressed FRs as the model target cells and MRC-5 cells with a low folate receptor expression level as the negative control. Both the fluorescence imaging and the SERS mapping results confirmed that the proposed probe can be used as an efficient cancer cell targeting agent. This kind of multifunctional probe has great potential in the diagnosis and therapeutics of cancerous diseases due to its specific targeting and multiplex imaging abilities, especially in the simultaneous tracking of multiple components in a hybrid bio-system.

  18. 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.

  19. Novel coumarin-based fluorescent probe for selective detection of Cu(II)

    NASA Astrophysics Data System (ADS)

    Bekhradnia, Ahmadreza; Domehri, Elham; Khosravi, Masome

    2016-01-01

    We report an efficient and convenient method for preparing nitro-3-carboxamide coumarin derivatives, proposed as novel fluorescent chemosensor, through microwave irradiation. This compound can be used as fluorescent probe for Cu2+ with selectivity over other metal ions in aqueous solution. The fluorescence of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide(3) is the highest in the presence of Cu2+, with stronger excitation at λ = 320 nm than for the other cations tested.

  20. A fluorescent benzothiazole probe with efficient two-photon absorption

    NASA Astrophysics Data System (ADS)

    Echevarria, Lorenzo; Moreno, Iván; Camacho, José; Salazar, Mary Carmen; Hernández, Antonio

    2012-11-01

    In this work, we report the two-photon absorption of 2-[4-(dimethylamino)phenyl]-1,3-benzothiazole-6-carbonitrile (DBC) in DMSO solution pumping at 779 nm with a 10 ns pulse laser-Nd:YAG system. The obtained two-photon absorption cross-section in DBC (407 ± 18 GM) is considerably high. Because DBC is a novel compound and have high values of fluorescence quantum yield, this result is expected to have an impact in biomolecules detection, diagnosis and treatment of cancer. Similar structures have previously been reported to show remarkable antitumour effects.

  1. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    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.

  2. Multicolor imaging of hydrogen peroxide level in living and apoptotic cells by a single fluorescent probe.

    PubMed

    Wen, Ying; Xue, Fengfeng; Lan, Haichuang; Li, Zhenhua; Xiao, Shuzhang; Yi, Tao

    2017-05-15

    To understand the entangled relationship between reactive oxygen species (ROS) and apoptosis, there is urgent need for simultaneous dynamic monitoring of these two important biological events. In this study, we have developed a fluorescent probe, pep4-NP1, which can simultaneously detect H2O2 and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H2O2 fluorescence reporter (NP1) and Cy5 fluorescent chromophore connected by a caspase 3 specific recognition peptide. The detecting strategy was realized through a controllable fluorescence resonance energy transfer (FRET) process between NP1 and Cy5 of pep4-NP1, after reaction with H2O2, which was verified by molecular calculation and in vitro spectral studies. In the absent of caspase 3, the accumulation of H2O2 induces red fluorescence of pep4-NP1 centered at 663nm in living cells due to the existence of FRET. In contrast, FRET is inhibited in apoptotic cells due to cleavage of the peptide spacer of pep4-NP1 by over-expressed caspase 3. Consequently, green fluorescence (555nm) predominated when labelling production of H2O2 in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H2O2 and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H2O2 between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.

  3. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

    PubMed Central

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-01-01

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

  4. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes.

    PubMed

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-03-02

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application.

  5. [Development of near-infrared fluorescent probes for in-vivo imaging].

    PubMed

    Kojima, Hirotatsu

    2008-11-01

    The number of reports on new techniques in molecular imaging has been recently increasing because of their usefulness in biological, medical, and clinical research. Fluorescence imaging methods are generally superior in terms of sensitivity, selectivity and ease of use. Cyanine dyes have been employed as fluorescent labels in fluorescence imaging studies of biological mechanisms. In particular, tricarbocyanines have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region around 650-900 nm is relatively poorly absorbed by biomolecules, and so can penetrate deeply into tissues. There is also less autofluorescence in this region. In addition to cyanine dyes for straightforward fluorescence labeling, we successfully developed cyanine dyes whose fluorescence intensity changes upon specific reaction with nitric oxide, which is an important signaling molecule involved in the regulation of a wide range of physiological and pathophysiological mechanisms, and many disorders. Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal ions. Upon addition of zinc ion, a red shift of the absorbance maximum was observed. Namely, DIPCY can work as a ratiometric fluorescent sensor for zinc ion in the NIR region. Moreover, we have recently developed several pH probes based on the amine-substituted tricarbocyanine fluorophore. We could measure pH with these fluorescent probes by a ratiometric monitoring method.

  6. DBD dyes as fluorescence lifetime probes to study conformational changes in proteins.

    PubMed

    Wawrzinek, Robert; Ziomkowska, Joanna; Heuveling, Johanna; Mertens, Monique; Herrmann, Andreas; Schneider, Erwin; Wessig, Pablo

    2013-12-16

    Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

  7. Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

    PubMed Central

    Gudmand, M.; Fidorra, Matthias; Bjørnholm, T.; Heimburg, T.

    2009-01-01

    The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems. PMID:19486682

  8. Fluorescence probe for cervical examination during various reproductive states

    NASA Astrophysics Data System (ADS)

    Glassman, Wenling S.; Liao, Qin-Ping; Shi, Shao-Qing; Goodrum, Linda; Olson, Gayle; Martin, Elizabeth; Saade, George; Garfield, Robert E.

    1997-05-01

    These studies represent further investigations that have been done utilizing the fluorescence from pyridinoline, one of the major crosslinks of type I and III collagen, to evaluate cervical connective tissue changes during various female reproductive periods. Based on our previous studies, a prototype instrument has been constructed. The instrument was specifically designed for the purpose of vaginal examination of cervical connective tissue by measuring light induced fluorescence directly from the surface of the external os of the cervix. The studies were carried out on nonpregnant rats, rats during gestation at different periods, rats at different times during postpartum, and rats during preterm birth after being treated with antiprogesterone drugs. A study has also been done on humans during pregnancy and postpartum. The results parallel previous investigations that have used various invasive methods to analyze cervical extensibility, cervical collagen content and collagenase. In consideration of the important role of the collagen fibers and their turnover in the process of cervical function during pregnancy (softening or ripening at term), this method could be a useful tool for evaluating treatment strategies of the cervix. Moreover, the instrument could serve as a device for the non-invasive estimation of cervical status in the clinic and the diagnosis of the changes in the cervix during the preparation for labor.

  9. 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

  10. Rapidly responsive and highly selective fluorescent probe for sulfite detection in real samples and living cells.

    PubMed

    Li, Hongda

    2015-10-15

    Sulfites (HSO3(-) or SO3(-)) have very significant toxicity in the environment and in the system. However, developing specific identification of sulfite probes is still very important. In this paper, a highly selective colorimetric and fluorescent probe (HHC) was synthesized to detect HSO3(-) in real samples and living cells. Sensing performance and preponderance are listed as follows. First, probe HHC showed remarkable selectivity for HSO3(-) over varieties of other species, including cysteine, glutathione, S(2-), CN(-), and reactive oxygen species, mainly because of the introduction of the electron-poor C=C double bond for HSO3(-). Second, probe HHC has great molar absorptivity, allowing it to act as a visual detection of probe for HSO3(-). Third, the fluorescence intensities of HHC linearly correlate with the concentration of HSO3(-), with a detection limit of 6.8 nm. Finally, our proposed probe can be applied to the visually determination of trace HSO3(-) in real samples and living HeLa cells with high precision. We hope that our proposed probe will greatly benefit biological sciences when biological researchers survey the role of HSO3(-) in biological systems.

  11. Development of near-infrared fluorescent probes for nitric oxide and zinc ion

    NASA Astrophysics Data System (ADS)

    Kojima, Hirotatsu; Kiyose, Kazuki; Sasaki, Eita; Nishimatsu, Hiroaki; Hirata, Yasunobu; Nagano, Tetsuo

    2007-02-01

    In fluorescence imaging studies of biological mechanisms, cyanine dyes have been employed as fluorescent labels. In particular, tricarbocyanines have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region around 650-900 nm can penetrate deeply into tissues. We successfully developed two types of cyanine dyes whose fluorescence properties change upon specific reaction with nitric oxide (NO) or zinc ion. The mechanism of fluorescence modulation of the NO probes involves photoinduced electron transfer, and the fluorescent intensity can change at the same wavelengths. We synthesized a series of amine-substituted tricarbocyanines in order to examine the correlation between the electron-donating ability of the amine and the fluorescence peak wavelength. We found that changing the electron-donating ability of the amine substituent altered the absorption and emission wavelengths. Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal ions. DIPCY can work as a ratiometric fluorescent sensor for zinc ion. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities. Thus, we have established two mechanisms for modulating the fluorescence properties of cyanines.

  12. A dendritic single-molecule fluorescent probe that is monovalent, photostable and minimally blinking

    NASA Astrophysics Data System (ADS)

    Yang, Si Kyung; Shi, Xinghua; Park, Seongjin; Ha, Taekjip; Zimmerman, Steven C.

    2013-08-01

    Single-molecule fluorescence techniques have emerged as a powerful approach to understanding complex biological systems. However, a challenge researchers still face is the limited photostability of nearly all organic fluorophores, including the cyanine and Alexa dyes. We report a new, monovalent probe that emits in the far-red region of the visible spectrum with properties desirable for single-molecule optical imaging. This probe is based on a ring-fused boron-dipyrromethene (BODIPY) core that is conjugated to a polyglycerol dendrimer (PGD). The dendrimer makes the hydrophobic fluorophore water-soluble. This probe exhibits excellent brightness, with an emission maximum of 705 nm. We have observed strikingly long and stable emission from individual PGD-BODIPY probes, even in the absence of anti-fading agents such as Trolox, a combined oxidizing-reducing agent often used in single-molecule studies for improving the photostability of common imaging probes. These interesting properties greatly simplify use of the fluorophore.

  13. Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy.

    PubMed

    Daniels, Charlisa R; Reznik, Carmen; Kilmer, Rachel; Felipe, Mary Jane; Tria, Maria Celeste R; Kourentzi, Katerina; Chen, Wen-Hsiang; Advincula, Rigoberto C; Willson, Richard C; Landes, Christy F

    2011-11-01

    The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe-PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe-PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.

  14. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

    Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.

  15. Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

    PubMed Central

    Ma, Yongmin; de Groot, Herbert; Liu, Zudong; Hider, Robert C.; Petrat, Frank

    2005-01-01

    A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline–iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4±1.3 μM. PMID:16336208

  16. Nitroreductase-triggered activation of a novel caged fluorescent probe obtained from methylene blue.

    PubMed

    Bae, Jungeun; McNamara, Louis E; Nael, Manal A; Mahdi, Fakhri; Doerksen, Robert J; Bidwell, Gene L; Hammer, Nathan I; Jo, Seongbong

    2015-08-18

    A near-infrared fluorescent probe based on methylene blue (p-NBMB) was developed for the detection of nitroreductase. Conjugating methylene blue with a p-nitrobenzyl moiety enables it to be activated by nitroreductase-catalyzed 1,6-elimination, resulting in the release of an active methylene blue fluorophore.

  17. Spatially Resolved Analysis of Amines Using a Fluorescence Molecular Probe: Molecular Analysis of IDPs

    NASA Technical Reports Server (NTRS)

    Clemett, S. J.; Messenger, S.; Thomas-Keprta, K. L.; Wentworth, S. J.; Robinson, G. A.; McKay, D. S.

    2002-01-01

    Some Interplanetary Dust Particles (IDPs) have large isotope anomalies in H and N. To address the nature of the carrier phase, we are developing a procedure to spatially resolve the distribution of organic species on IDP thin sections utilizing fluorescent molecular probes. Additional information is contained in the original extended abstract.

  18. Cy3 in AOT reverse micelles II. Probing intermicellar interactions using fluorescence correlation spectroscopy.

    PubMed

    McPhee, Jeffrey T; Scott, Eric; Levinger, Nancy E; Van Orden, Alan

    2011-08-11

    Cyanine-3 (Cy3) fluorescent dye molecules confined in sodium di-2-ethylhexyl sulfosuccinate (AOT) reverse micelles were examined using dynamic light scattering and fluorescence correlation spectroscopy to probe the kinetics of Cy3 dye and reverse micelle aggregation. This study explored a range of reverse micelle sizes, defined as w(0) = [H(2)O]/[AOT], in which the occupation number ranged from one Cy3 molecule per ∼10(5) to ∼10(6) reverse micelles. These measurements reveal that in the smallest reverse micelle, w(0) = 1, the Cy3 molecules aggregate to form H-aggregate dimers, and the Cy3 dimerization is accompanied by the formation of a transient dimer between reverse micelles. Transient reverse micelle dimer particles are only observed in the small fraction of Cy3-labeled reverse micelles probed by fluorescence correlation spectroscopy and are not observed in the bulk solution probed by dynamic light scattering. Furthermore, fluorescence correlation spectroscopy makes it possible to probe the size and shape of these dimers, revealing prolate ellipsoid-shaped particles with twice the volume and surface area of a single reverse micelle.

  19. Intrasequence GFP in class I MHC molecules, a rigid probe for fluorescence anisotropy measurements of the membrane environment.

    PubMed

    Rocheleau, Jonathan V; Edidin, Michael; Piston, David W

    2003-06-01

    Fluorescence anisotropy measurements can elucidate the microenvironment of a membrane protein in terms of its rotational diffusion, interactions, and proximity to other proteins. However, use of this approach requires a fluorescent probe that is rigidly attached to the protein of interest. Here we describe the use of one such probe, a green fluorescent protein (GFP) expressed and rigidly held within the amino acid sequence of a major histocompatibility complex (MHC) class I molecule, H2L(d). We contrast the anisotropy of this GFP-tagged MHC molecule, H2L(d)GFPout, with that of an H2L(d) that was GFP-tagged at its C-terminus, H2L(d)GFPin. Both molecules fold properly, reach the cell surface, and are recognized by specific antibodies and T-cell receptors. We found that polarized fluorescence images of H2L(d)GFPout in plasma membrane blebs show intensity variations that depend on the relative orientation of the polarizers and the membrane normal, thus demonstrating that the GFP is oriented with respect to the membrane. These variations were not seen for H2L(d)GFPin. Before transport to the membrane surface, MHC class I associates with the transporter associated with antigen processing complex in the endoplasmic reticulum. The intensity-dependent steady-state anisotropy in the ER of H2L(d)GFPout was consistent with FRET homotransfer, which indicates that a significant fraction of these molecules were clustered. After MCMV-peptide loading, which supplies antigenic peptide to the MHC class I releasing it from the antigen processing complex, the anisotropy of H2L(d)GFPout was independent of intensity, suggesting that the MHC proteins were no longer clustered. These results demonstrate the feasibility and usefulness of a GFP moiety rigidly attached to the protein of interest as a probe for molecular motion and proximity in cell membranes.

  20. Molecular engineering of two-photon fluorescent probes for bioimaging applications.

    PubMed

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

    2017-03-22

    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.

  1. Fluorescent probe ABM and estimation of immune state in patients with different pathologies (review article).

    PubMed

    Kalnina, Inta; Bruvere, Ruta; Zvagule, Tija; Gabruseva, Natalija; Klimkane, Laura; Kirilova, Elena; Meirovics, Imants; Kizane, Gunta

    2010-01-01

    The fluorescent probe ABM (3-aminobenzanthrone derivative) one of the fluorescent probes synthesized in Riga Technical University proved to be an excellent, independent model for studying cell membranes. In our work we have investigated the possibility of using the fluorescent probe ABM for detection of immune state in patients with different pathologies. There is a strong correlation among all studied ABM spectral parameters, immunological characteristics, clinical and laboratory investigations of the all observed patients groups. The obtained results suggest that ABM spectral parameters in cell suspension reflect the alterations of the cellular mechanisms of immunity. Therefore fluorescent method could be used as preliminary screening test in immune diagnostics instead of more expensive, time consuming methods (subset detection, radioisotope method etc.) used as routine in clinics. Spectral parameters of ABM reflect a wide range of interrelated (interdependent) characteristics of cells (physico-chemical state and microviscosity of membrane, proliferating and lipid metabolic activity of cells, distribution of cells among subsets). The observed change of the studied parameters reflects alterations of the cellular mechanisms of immunity which is a main focus for its application as preliminary screening test in immune diagnostics. The fluorescence based method is sensitive, less expensive and time consuming, technically simple and convenient.

  2. 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.

  3. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between L-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH = 4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that L-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of L-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  4. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe.

    PubMed

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between l-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH=4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that l-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of l-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  5. Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

    PubMed Central

    Yun, Bo; Azad, Mohammad A. K.; Nowell, Cameron J.; Nation, Roger L.; Thompson, Philip E.; Roberts, Kade D.

    2015-01-01

    Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. PMID:26392495

  6. MUC1 aptamer based near infrared fluorescence probes for tumor diagnosis

    NASA Astrophysics Data System (ADS)

    Zhao, Juan; Ma, Yuxiang; Cui, Sisi; Cao, Jie; Achilefu, Samuel; Gu, Yueqing

    2013-02-01

    Mucin 1 (MUC1) is a cell surface mucin broadly expressed in mucosal tissues. The aberrant expression of MUC1 under-glycosylated forms has been reported in various carcinomas of the epithelium, such as breast, pancreatic and ovarian cancers. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology, aptamers previously selected against MUC1 glycoprotein with high affinities and specificities. In this study, we developed two targeted near-infrared fluorescent probes for tumor in-vivo diagnostics using a MUC1 aptamer(APT) as targeted ligand and near-infrared fluorescent dye (ICG-Der-02) as labelling. MUC1 aptamer conjugated ICG-Der-02 (APT-ICG-Der-02) displayed a great selectivity to MUC1 positive cell line MCF7 and MCF7 xenograft-bearing nude mice. To improve the high targeting of the probe to the tumor cells, PEG, with high biocompatibility, non immunogenicity and long circulation, was conjugated to the probe .The new probe (APT-PEG-ICG-Der-02) showed better tumour uptake and clearance, and also displayed a great selectivity to MCF7 tumor-bearing nude mice. Data obtained demonstrate a high potential of the targeted near-infrared fluorescent probes in cancer early diagnosis.

  7. Coexistence of probe conformations in lipid phases-a polarized fluorescence microspectroscopy study.

    PubMed

    Urbančič, Iztok; Ljubetič, Ajasja; Arsov, Zoran; Strancar, Janez

    2013-08-20

    Several well-established fluorescence methods depend on environment-sensitive probes that report about molecular properties of their local environment. For reliable interpretation of experiments, careful characterization of probes' behavior is required. In this study, bleaching-corrected polarized fluorescence microspectroscopy with nanometer spectral peak position resolution was applied to characterize conformations of two alkyl chain-labeled 7-nitro-2-1,3-benzoxadiazol-4-yl phospholipids in three model membranes, representing the three main lipid phases. The combination of polarized and spectral detection revealed two main probe conformations with their preferential fluorophore dipole orientations roughly parallel and perpendicular to membrane normal. Their peak positions were separated by 2-6 nm because of different local polarities and depended on lipid environment. The relative populations of conformations, estimated by a numerical model, indicated a specific sensitivity of the two probes to molecular packing with cholesterol. The coexistence of probe conformations could be further exploited to investigate membrane organization below microscopy spatial resolution, such as lipid rafts. With the addition of polarized excitation or detection to any environment-sensitive fluorescence imaging technique, the conformational analysis can be directly applied to explore local membrane complexity.

  8. 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence in Meningioma: Qualitative and Quantitative Measurements In Vivo

    PubMed Central

    Valdes, Pablo A.; Bekelis, Kimon; Harris, Brent T.; Wilson, Brian C.; Leblond, Frederic; Kim, Anthony; Simmons, Nathan E.; Erkmen, Kadir; Paulsen, Keith D.; Roberts, David W.

    2014-01-01

    BACKGROUND The use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence has shown promise as a surgical adjunct for maximizing the extent of surgical resection in gliomas. To date, the clinical utility of 5-ALA in meningiomas is not fully understood, with most descriptive studies using qualitative approaches to 5-ALA-PpIX. OBJECTIVE To assess the diagnostic performance of 5-ALA-PpIX fluorescence during surgical resection of meningioma. METHODS ALA was administered to 15 patients with meningioma undergoing PpIX fluorescence-guided surgery at our institution. At various points during the procedure, the surgeon performed qualitative, visual assessments of fluorescence by using the surgical microscope, followed by a quantitative fluorescence measurement by using an intra-operative probe. Specimens were collected at each point for subsequent neuropathological analysis. Clustered data analysis of variance was used to ascertain a difference between groups, and receiver operating characteristic analyses were performed to assess diagnostic capabilities. RESULTS Red-pink fluorescence was observed in 80% (12/15) of patients, with visible fluorescence generally demonstrating a strong, homogenous character. Quantitative fluorescence measured diagnostically significant PpIX concentrations (CPpIx) in both visibly and nonvisibly fluorescent tissues, with significantly higher CPpIx in both visibly fluorescent (P < .001) and tumor tissue (P = .002). Receiver operating characteristic analyses also showed diagnostic accuracies up to 90% for differentiating tumor from normal dura. CONCLUSION ALA-induced PpIX fluorescence guidance is a potential and promising adjunct in accurately detecting neoplastic tissue during meningioma resective surgery. These results suggest a broader reach for PpIX as a biomarker for meningiomas than was previously noted in the literature. PMID:23887194

  9. A 4-methylumbelliferone-based fluorescent probe for the sensitive detection of captopril.

    PubMed

    Wang, Liping; Yang, Xiao-Feng; Zhao, Minglei

    2009-07-01

    A highly sensitive fluorogenic probe for captopril, 4-methylumbelliferyl-2, 4-dinitrobenzenesulfonate (4-MUDNBS), was designed and synthesized. 4-MUDNBS is a nonfluorescent compound and was synthesized via the one-step reaction of 4-methylumbelliferone (4-MU) with 2,4-dinitrobenzenesulfonyl chloride. Upon mixing with captopril in basic solution, the 2,4-dinitrobenzenesulfonyl group of 4-MUDNBS was efficiently removed and highly fluorescent 4-MU was released, hence leading to the dramatic fluorescence increase of the reaction solution. The fluorescence intensity is linear with captopril concentration in the range 3.0-500 ng mL(-1) with a detection limit of 2.2 ng mL(-1) (3sigma). The effect of substituents on the benzenesulfonyl moiety of the probe is discussed, and the presence of electronegative groups is favorable for the thiolate-induced cleavage reaction. The proposed method has been successfully applied to the captopril determination in pharmaceutical preparations.

  10. Hydrogen sulfide and resolution of acute inflammation: A comparative study utilizing a novel fluorescent probe.

    PubMed

    Dufton, Neil; Natividad, Jane; Verdu, Elena F; Wallace, John L

    2012-01-01

    Hydrogen sulfide is an essential gasotransmitter associated with numerous pathologies. We assert that hydrogen sulfide plays an important role in regulating macrophage function in response to subsequent inflammatory stimuli, promoting clearance of leukocyte infiltrate and reducing TNF-α levels in vivo following zymosan-challenge. We describe two distinct methods of measuring leukocyte hydrogen sulfide synthesis; methylene blue formation following zinc acetate capture and a novel fluorescent sulfidefluor probe. Comparison of these methods, using pharmacological tools, revealed they were complimentary in vitro and in vivo. We demonstrate the application of sulfidefluor probe to spectrofluorimetry, flow cytometry and whole animal imaging, to monitor the regulation of hydrogen sulfide synthesis in vivo during dynamic inflammatory processes. Both methodologies revealed that granulocyte infiltration negatively affects hydrogen sulfide synthesis. Our report offers an insight into the profile of hydrogen sulfide synthesis during inflammation and highlight opportunities raised by the development of novel fluorescent hydrogen sulfide probes.

  11. Hydrogen sulfide and resolution of acute inflammation: A comparative study utilizing a novel fluorescent probe

    PubMed Central

    Dufton, Neil; Natividad, Jane; Verdu, Elena F.; Wallace, John L.

    2012-01-01

    Hydrogen sulfide is an essential gasotransmitter associated with numerous pathologies. We assert that hydrogen sulfide plays an important role in regulating macrophage function in response to subsequent inflammatory stimuli, promoting clearance of leukocyte infiltrate and reducing TNF-α levels in vivo following zymosan-challenge. We describe two distinct methods of measuring leukocyte hydrogen sulfide synthesis; methylene blue formation following zinc acetate capture and a novel fluorescent sulfidefluor probe. Comparison of these methods, using pharmacological tools, revealed they were complimentary in vitro and in vivo. We demonstrate the application of sulfidefluor probe to spectrofluorimetry, flow cytometry and whole animal imaging, to monitor the regulation of hydrogen sulfide synthesis in vivo during dynamic inflammatory processes. Both methodologies revealed that granulocyte infiltration negatively affects hydrogen sulfide synthesis. Our report offers an insight into the profile of hydrogen sulfide synthesis during inflammation and highlight opportunities raised by the development of novel fluorescent hydrogen sulfide probes. PMID:22787557

  12. Gaseous ammonia fluorescence probe based on cellulose acetate modified microstructured optical fiber

    NASA Astrophysics Data System (ADS)

    Peng, Lirong; Yang, Xinghua; Yuan, Libo; Wang, Lili; Zhao, Enming; Tian, Fengjun; Liu, Yanxin

    2011-09-01

    In this article, we report a novel fluorescent ammonia gas probe based on microstructured optical fiber (MOF) which is modified with eosin-doped cellulose acetate film. This probe was fabricated by liquid fluxion coating process. Polymer solution doped with eosin was directly inhaled into 18 array holes of MOF and then formed matrix film in them. The sensing properties of the optical fiber sensor to gaseous ammonia at room temperature were investigated. The sensing probe showed different fluorescence intensity at 576 nm to different concentrations of trace ammonia in carrier gas of nitrogen. The response range was 50-400 ppm, with short response time within 500 ms. Furthermore, the response range could be tailored through CTAB co-entrapment process in the sensing film. These test results demonstrated that low cost, simple structured fiber optic sensors for detecting ammonia gas samples could be developed based on MOF.

  13. NanoSOSG: a Nanostructured Fluorescent Probe for the Detection of Intracellular Singlet Oxygen.

    PubMed

    Ruiz-González, Rubén; Bresolí-Obach, Roger; Gulías, Òscar; Agut, Montserrat; Savoie, Huguette; Boyle, Ross W; Nonell, Santi; Giuntini, Francesca

    2017-02-02

    A biocompatible fluorescent nanoprobe for singlet oxygen ((1) O2 ) detection in biological systems was designed, synthesized, and characterized, that circumvents many of the limitations of the molecular probe Singlet Oxygen Sensor Green(®) (SOSG). This widely used commercial singlet oxygen probe was covalently linked to a polyacrylamide nanoparticle core using different architectures to optimize the response to (1) O2 . In contrast to its molecular counterpart, the optimum SOSG-based nanoprobe, which we call NanoSOSG, is readily internalized by E. coli cells and does not interact with bovine serum albumin. Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellularly generated (1) O2 with an increase in fluorescence.

  14. Fluorescein aldehyde with disulfide functionality as a fluorescence turn-on probe for cysteine and homocysteine in HEPES buffer.

    PubMed

    Lee, Heejin; Kim, Hae-Jo

    2013-08-14

    We developed a fluorescein aldehyde probe with disulfide functionality for the fluorescence detection of biologically important thiols. The probe displayed highly selective responses to cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) due to the rapid ring formation reaction of Cys and Hcy with the aldehyde group of the probe and the concomitant cleavage of the disulfide group followed by subsequent intramolecular cyclization. The fluorescent probe also exhibited a highly sensitive fluorescence turn-on response to Hcy with a detection limit of 2.4 μM Hcy in HEPES buffer.

  15. Supercharged fluorescent protein as a versatile probe for the detection of glycosaminoglycans in vitro and in vivo.

    PubMed

    Wang, Wenshuang; Han, Naihan; Li, Ruijuan; Han, Wenjun; Zhang, Xiaoru; Li, Fuchuan

    2015-09-15

    Glycosaminoglycans (GAGs) are linear acidic heteropolysaccharides that are ubiquitously expressed in animal tissues and participate in various life processes. To date, the detection and visualization of GAGs in complex biological samples and living organisms remain a challenge because of the lack of powerful biocompatible probes. In this study, a superpositively charged green fluorescent protein (ScGFP) was shown great potential in GAG detection for the first time. First, on the basis of the phenomenon of GAGs dose-dependently inhibiting the fluorescence quenching of ScGFP by graphene oxide, a simple and highly sensitive signal-on homogeneous platform was established for detecting and quantifying GAGs, even in complex samples such as heparin in citrated plasma and oversulfated chondroitin sulfate in heparin. Furthermore, ScGFP with excellent stability and biocompatibility could be easily used as a highly sensitive and selective probe to visualize different types of GAGs in vitro and in vivo through combination with specific GAG-degrading enzymes. This study introduces a versatile probe for GAG detection, which is easy to prepare and which shows a high practical value in basic research and medical applications.

  16. A novel ratiometric fluorescent probe based on 1, 8-naphthalimide for the detection of Ho3 + and its bioimaging

    NASA Astrophysics Data System (ADS)

    Zhang, Huifang; Liu, Tao; Yin, Caixia; Wen, Yin; Chao, Jianbin; Zhang, Yongbin; Huo, Fangjun

    2017-03-01

    A ratiometric fluorescent probe for the detection of Ho3 + in DMSO-aqueous medium was designed and synthesized based on 1, 8-naphthalimide. The probe displayed response to Ho3 + with a fluorescence decrease at 512 nm and enhancement at 480 nm, accompanying with a distinct fluorescence change from bright yellow-green to cyan. Besides, the probe exhibited a lower detection limit (6 × 10- 8 M) and could be used in intracellular fluorescence imaging. To the best of the knowledge, it was the first ratiometric fluorescent probe for Ho3 + detection. This probe was expected to be a useful tool for further elucidating the roles of Ho3 + in materials, biology and environment.

  17. In vivo inflammation imaging using a CB2R-targeted near infrared fluorescent probe.

    PubMed

    Zhang, Shaojuan; Shao, Pin; Ling, Xiaoxi; Yang, Ling; Hou, Weizhou; Thorne, Steve H; Beaino, Wissam; Anderson, Carolyn J; Ding, Ying; Bai, Mingfeng

    2015-01-01

    Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund's Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases.

  18. Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe.

    PubMed

    Zhang, Hongge; Wang, Minjuan; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2011-05-15

    A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F(0)=2.73 C (μM)+1.14 (R=0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N=3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.

  19. Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding.

    PubMed

    Yan, Xiu-Fang; Chen, Zeng-Ping; Cui, Yin-Yin; Hu, Yuan-Liang; Yu, Ru-Qin

    2016-05-19

    PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFMGRP) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFMGRP has been tested on the quantitative determination of free Ca(2+) in both simulated and real turbid media using a Ca(2+) sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFMGRP could realize precise and accurate quantification of free Ca(2+) in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca(2+) bound Rhod-2. The average relative predictive error value of QFMGRP for the test simulated turbid samples was 5.9%, about 2-4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca(2+) bound Rhod-2 and eosin B. The recovery rates of QFMGRP for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry.

  20. Biomolecular interactions probed by fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Lange, Daniela Charlotte

    2000-09-01

    This thesis describes how a physical phenomenon, Fluorescence Resonance Energy Transfer (FRET), can be exploited for the study of interactions between biomolecules. The physical basis of this phenomenon is discussed and it is described how some of its characteristics can be exploited in measurement. A recently introduced method, photobleaching FRET microscopy, was implemented and its image analysis refined to suit our biological context. Further, a new technique is proposed, which combines FRET with confocal laser scanning microscopy to optimize resolution and to allow for 3D-studies in living cells. The first part of this thesis presents the application of FRET to the study of oligomerization of G-protein coupled receptors (GPCRs), which was performed at the Fraser Laboratories at McGill University in Montreal. It is demonstrated how FRET microscopy allowed us to circumvent problems of traditional biochemical approaches and provided the first direct evidence for GPCR oligomerization in intact cells. We found that somatostatin receptors (SSTRs) functionally interact by forming oligomers with their own kind, with different SSTR isoforms, and even with distantly related GPCRs, such as dopamine receptors, the latter of which is breaking with the dogma that GPCRs would only pair up with their own kind. The high sensitivity of the FRET technique allowed us to characterize these interactions under more physiological conditions, which lead to the observation that oligomerization is induced by receptor agonist. We further studied the differential effects of agonists and antagonists on receptor oligomerization, leading to a model for the molecular mechanism underlying agonist/antagonist function and receptor activation. The second part was carried out at the Neurobiology Laboratory of the VA Medical Center in Newington, CT. The objective was to further our understanding of Niemann- Pick type C disease, which is characterized by a defect in intracellular cholesterol

  1. Determination of total plasma hydroperoxides using a diphenyl-1-pyrenylphosphine fluorescent probe.

    PubMed

    Santas, Jonathan; Guardiola, Francesc; Rafecas, Magda; Bou, Ricard

    2013-03-01

    Plasma hydroperoxides (HPs) are widely accepted to be good indicators of oxidative stress. By means of the method proposed here, which uses diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe, all types of plasma HP were determined. The limits of detection and quantification of the method were 0.08 and 0.25 nmol of cumene hydroperoxide (CHP) equivalents in 40 μl of plasma, respectively. The method is satisfactory in terms of precision (5.3% for 14.5 μM CHP eq., n=8), and the recoveries were 91% and 92% after standard additions of 26 and 52 μM CHP, respectively. The selectivity of the proposed method is higher than 96%. Moreover, optimization of the reaction conditions and the addition of ethylenediaminetetraacetic acid (EDTA) disodium salt and 2,6-di-tert-butyl-4-methylphenol (BHT) prevented the formation of HP artifacts during the analysis. Therefore, the proposed method is useful for simple and quantitative determination of total plasma HPs.

  2. Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

    PubMed

    Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

    2017-05-15

    The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R(2)=0.99) in aqueous solutions, 2.98nM (R(2)=0.98) in 1% serum samples, and 3.43nM (R(2)=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.

  3. 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.

  4. Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

    PubMed Central

    2016-01-01

    Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

  5. A mechanosynthesized, sequential, cyclic fluorescent probe for mercury and iodide ions in aqueous solutions.

    PubMed

    Chen, Shangwen; Wang, Pipi; Jia, Chunmei; Lin, Qiang; Yuan, Wenbing

    2014-12-10

    A fluorescent Hg(2+)-selective chemosensor, 2,5-dimethoxybenzaldehyde thiosemicarbazone (1), was quantitatively prepared by grinding 2,5-dimethoxybenzaldehyde and thiosemicarbazide together in a ball mill for 15min. The excitation and emission maxima of compound 1 are 347 and 450nm, respectively. The reaction of this ligand with Hg(2+) was investigated by FT-IR, (1)H NMR, and fluorescence titration. Results show that the composition of the resulting Hg complex 1-Hg is 2:1 1:Hg, and that the S and imino N atoms serve as the binding sites of the ligand to the Hg(2+) ions. Coordination-assisted fluorescence quenching results show that compound 1 exhibits a highly selective fluorescence response to trace amounts of Hg(2+) in water. More importantly, the resulting complex 1-Hg can be used as a turn-on fluorescence probe for I(-) at a detection limit of 8.4×10(-8)M. Thus, compound 1 is a relatively stable, sequential, cyclic fluorescent probe for Hg(2+) and I(-).

  6. Luminol as a fluorescent acid-base indicator.

    PubMed

    Erdey, L; Buzás, I; Vigh, K

    1966-03-01

    The acid and base dissociation constants of luminol are determined at various ionic strengths. The transition interval occurs at pH 7.7-9.0, therefore luminol is a fluorescent indicator for the titration of strong and weak acids and strong bases. Its value as an indicator is established by titrating milk, red wine and cherry juice.

  7. Probing peroxisome dynamics and biogenesis by fluorescence imaging.

    PubMed

    Jauregui, Miluska; Kim, Peter K

    2014-03-03

    Peroxisomes are the most recently discovered classical organelles, and only lately have their diverse functions been truly recognized. Peroxisomes are highly dynamic structures, changing both morphologically and in number in response to both extracellular and intracellular signals. This metabolic organelle came to prominence due to the many genetic disorders caused by defects in its biogenesis or enzymatic functions. There is now growing evidence that suggests peroxisomes are involved in lipid biosynthesis, innate immunity, redox homeostasis, and metabolite scavenging, among other functions. Therefore, it is important to have available suitable methods and techniques to visualize and quantify peroxisomes in response to various cellular signals. This unit includes a number of protocols that will enable researchers to image, qualify, and quantify peroxisome numbers and morphology-with both steady-state and time-lapse imaging using mammalian cells. The use of photoactivatable fluorescent proteins to detect and measure peroxisome biogenesis is also described. Altogether, the protocols described here will facilitate understanding of the dynamic changes that peroxisomes undergo in response to various cellular signals.

  8. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-07-01

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the

  9. Probing Field-Induced Tissue Polarization Using Transillumination Fluorescent Imaging

    PubMed Central

    Caldwell, Bryan J.; Wellner, Marcel; Mitrea, Bogdan G.; Pertsov, Arkady M.; Zemlin, Christian W.

    2010-01-01

    Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2–6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model. PMID:20923639

  10. Selective fluorescent detection of aspartic acid and glutamic acid employing dansyl hydrazine dextran conjugate.

    PubMed

    Nasomphan, Weerachai; Tangboriboonrat, Pramuan; Tanapongpipat, Sutipa; Smanmoo, Srung

    2014-01-01

    Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1 × 10(-4) M to 25 × 10(-3) M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.

  11. pHLIP-FIRE, a cell insertion-triggered fluorescent probe for imaging tumors demonstrates targeted cargo delivery in vivo.

    PubMed

    Karabadzhak, Alexander G; An, Ming; Yao, Lan; Langenbacher, Rachel; Moshnikova, Anna; Adochite, Ramona-Cosmina; Andreev, Oleg A; Reshetnyak, Yana K; Engelman, Donald M

    2014-11-21

    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.

  12. Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes

    NASA Astrophysics Data System (ADS)

    Afonina, Irina A.; Lokhov, Sergey G.; Belousov, Yevheniy S.; Reed, Michael W.; Lukhtanov, Eugeny A.; Shishkina, Irina G.; Gorn, Vladimir V.; Sanders, Silvia M.; Walburger, David K.; Hoekstra, Merl F.; Vermeulen, Nicolaas M. J.

    2002-06-01

    Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described. These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 order of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, final load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new Tm prediction software to design robust 5'-MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the Tm of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo(3,4- d)pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.

  13. An aqueous red emitting fluorescent fluoride sensing probe exhibiting a large Stokes shift and its application in cell imaging.

    PubMed

    Hou, Peng; Chen, Song; Wang, Hongbo; Wang, Jianxiu; Voitchovsky, Kislon; Song, Xiangzhi

    2014-01-11

    A novel red emitting fluorescent probe exhibiting a 143 nm Stokes shift for the detection of fluoride ions in an aqueous solution was developed. The probe displays a rapid response, high selectivity and good sensitivity towards F(-). Application of the probe for the selective detection of intracellular F(-) has been successfully demonstrated in living cells.

  14. Water-soluble Hantzsch ester as switch-on fluorescent probe for efficiently detecting nitric oxide

    NASA Astrophysics Data System (ADS)

    Wang, Hui-Li; Liu, Fu-Tao; Ding, Ai-Xiang; Ma, Su-Fang; He, Lan; Lin, Lan; Lu, Zhong-Lin

    2016-12-01

    A water soluble Hantzsch ester derivative of coumarin, DHPS, was synthesized and successfully applied in the fluorescent sensing nitric oxide (NO) in aqueous solution. The fluorescence of probe DHPS is extremely weak, while its fluorescence was greatly switched on upon the addition of NO solution and showed high selectivity and sensitivity to NO. The limitation of the detection was calculated to be 18 nM. The NO-induced aromatization of dihydropyridine in DHPS to pyridine derivative (PYS) proved to be the switching mechanism for the fluorescent sensing process, which was confirmed through spectra characterization and computation study. Cytotoxicity assay demonstrated both DHPS and PYS are biocompatible, the DHPS was successfully applied to track the endogenously produced NO in the RAW 264.7 cells.

  15. Discovery of a Novel Fluorescent Probe for the Sensitive Detection of β-Amyloid Deposits

    PubMed Central

    Ren, Wenming; Xu, Mingming; Liang, Steven H.; Xiang, Huaijiang; Tang, Li; Zhang, Minkui; Ding, Dejun; Li, Xin; Zhang, Haiyan; Hu, Youhong

    2016-01-01

    Here we reported the development of the first photoinduced electron transfer (PeT) probe (1) to directly locate β-amyloid aggregates (Aβ plaques) in the brain without the need of post-washing procedures. The probe showed a high affinity for Aβ aggregates with a Kd value of 3.5 nM. It is weakly emissive by itself with its fluorescence quenched by electron transfer from PeT donor to the excited fluorophore. But selective binding to Aβ plaques would attenuate the PeT process and restore the fluorescence, therefore facilitating the tracking of Aβ plaques. The probe is advantageous in that its fluorescence is environment-less-sensitive and no washing procedure is required to provide high contrast fluorescent signal when applied to stain brain tissues. As a proof of concept, its application has been exemplified by staining Aβ plaques in slices of brain tissue from double transgenic (APP/PS1) mice of Alzheimer’s disease. PMID:26313423

  16. Red emission fluorescent probes for visualization of monoamine oxidase in living cells

    PubMed Central

    Li, Ling-Ling; Li, Kun; Liu, Yan-Hong; Xu, Hao-Ran; Yu, Xiao-Qi

    2016-01-01

    Here we report two novel red emission fluorescent probes for the highly sensitive and selective detection of monoamine oxidase (MAO) with large Stokes shift (227 nm). Both of the probes possess solid state fluorescence and can accomplish the identification of MAO on test papers. The probe MAO-Red-1 exhibited a detection limit down to 1.2 μg mL−1 towards MAO-B. Moreover, the cleavage product was unequivocally conformedby HPLC and LCMS and the result was in accordance with the proposed oxidative deamination mechanism. The excellent photostability of MAO-Red-1 was proved both in vitro and in vivo through fluorescent kinetic experiment and laser exposure experiment of confocal microscopy, respectively. Intracellular experiments also confirmed the low cytotoxity and exceptional cell imaging abilities of MAO-Red-1. It was validated both in HeLa and HepG2 cells that MAO-Red-1 was capable of reporting MAO activity through the variation of fluorescence intensity. PMID:27499031

  17. Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe.

    PubMed

    Nuthanakanti, Ashok; Boerneke, Mark A; Hermann, Thomas; Srivatsan, Seergazhi G

    2017-03-01

    Comprehensive understanding of the structure-function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation-sensitive fluorophore and an anomalous X-ray diffraction label (5-selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand-induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

  18. A multi-emissive fluorescent probe for the discrimination of glutathione and cysteine.

    PubMed

    Liu, Xue-Liang; Niu, Li-Ya; Chen, Yu-Zhe; Yang, Yunxu; Yang, Qing-Zheng

    2017-04-15

    Glutathione (GSH) and cysteine (Cys) play different roles in biological systems, thus the discrimination between them is of great importance. Herein we report a multi-emissive fluorescent probe for the selective detection of GSH and Cys. The probe was composed of covalently linked BODIPY and coumarin fluorophores. The BODIPY fluorophore was designed to react with GSH and Cys and generate different products with distinct photophysical properties, and the coumarin fluorophore acted as an internal standard. The probe exhibited green emission in aqueous solution. Upon addition of Cys, it yielded nitrogen-substituted BODIPY with weak fluorescence and free coumarin with blue emission. In the presence of glutathione, it generated mono- and di-sulfur substituted BODIPY and coumarin, resulting in various emission colors at different concentrations of GSH. Interestingly, the solution exhibited white fluorescence at GSH concentration of 0.4mM. The probe was capable of detecting and imaging GSH and Cys in living HeLa cells, indicating its significant potential in biological applications.

  19. Cell flow analysis with a two-photon fluorescence fiber probe

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Baker, James R., Jr.; Norris, Theodore B.

    2010-11-01

    We report the use of a sensitive double-clad fiber (DCF) probe for in situ cell flow velocity measurements and cell analysis by means of two-photon excited fluorescence correlation spectroscopy (FCS). We have demonstrated the feasibility to use this fiber probe for in vivo two-photon flow cytometry previously. However, because of the viscosity of blood and the non-uniform flow nature in vivo, it is problematic to use the detected cell numbers to estimate the sampled blood volume. To precisely calibrate the sampled blood volume, it is necessary to conduct real time flow velocity measurement. We propose to use FCS technique to measure the flow velocity. The ability to measure the flow velocities of labeled cells in whole blood has been demonstrated. Our two-photon fluorescence fiber probe has the ability to monitor multiple fluorescent biomarkers simultaneously. We demonstrate that we can distinguish differently labeled cells by their distinct features on the correlation curves. The ability to conduct in situ cell flow analysis using the fiber probe may be useful in disease diagnosis or further comprehension of the circulation system.

  20. A Fluorescent Probe for Detecting Mycobacterium tuberculosis and Identifying Genes Critical for Cell Entry

    PubMed Central

    Yang, Dong; Ding, Feng; Mitachi, Katsuhiko; Kurosu, Michio; Lee, Richard E.; Kong, Ying

    2016-01-01

    The conventional method for quantitating Mycobacterium tuberculosis (Mtb) in vitro and in vivo relies on bacterial colony forming unit (CFU) enumeration on agar plates. Due to the slow growth rate of Mtb, it takes 3–6 weeks to observe visible colonies on agar plates. Imaging technologies that are capable of quickly quantitating both active and dormant tubercle bacilli in vitro and in vivo would accelerate research toward the development of anti-TB chemotherapies and vaccines. We have developed a fluorescent probe that can directly label the Mtb cell wall components. The fluorescent probe, designated as DLF-1, has a strong affinity to the D-Ala-D-Ala unit of the late peptidoglycan intermediates in the bacterial cell wall. We demonstrate that DLF-1 is capable of detecting Mtb in both the actively replicating and dormant states in vitro at 100 nM without inhibiting bacterial growth. The DLF-1 fluorescence signal correlated well with CFU of the labeled bacteria (R2 = 1 and 0.99 for actively replicating and dormant Mtb, respectively). DLF-1 can also quantitate labeled Mtb inside of cells. The utility of DLF-1 probe to quantitate Mtb was successfully applied to identify genes critical for cell invasion. In conclusion, this novel near infrared imaging probe provides a powerful new tool for enumerating Mtb with potential future use in bacterial virulence study. PMID:28066347

  1. Spectral-fluorescent study of the interaction of the polymethine dye probe Cyan 2 with chondroitin-4-sulfate

    NASA Astrophysics Data System (ADS)

    Tatikolov, Alexander S.; Akimkin, Timofey M.; Panova, Ina G.; Yarmoluk, Sergiy M.

    2017-04-01

    The noncovalent interaction of the polymethine dye probe 3,3‧,9-trimethylthiacarbocyanine iodide (Cyan 2) with chondroitin-4-sulfate (C4S) in buffer solutions with different pH and in water in the absence of buffers has been studied by spectral-fluorescent methods. It has been shown that in all media studied, at relatively high concentrations, the dye is bound to C4S mainly as a monomer, which is accompanied by a steep rise of fluorescence (the intermediate formation of dye aggregates on the biopolymer is also observed). From the dependence of the fluorescence quantum yield on the concentration of C4S, the parameters of binding of the dye monomer to C4S were obtained: the effective binding constant K, the number of the monomeric C4S units n per one dye monomer bound to C4S, and the fluorescence quantum yield of the bound dye monomer Φfb. The dependence of Φfb (and K) on рН of the medium is not monotonic: it has a minimum in the region of neutral pH and a growth in the regions of acid and basic pH. This can be explained by changing the charge of a C4S macromolecule as a function of pH and related conformational alterations in the biopolymer, which can affect the rigidity of a dye molecule and the energy of its interaction with the biopolymer.

  2. A new near-infrared absorption and fluorescent probe based on bombesin for molecular imaging

    NASA Astrophysics Data System (ADS)

    Kujala, Naresh; Zhai, Huifang; Smith, Charles; Prasanphanich, Adam; Sieckman, Gary; Hoffman, Timothy; Volkert, Wynn; Ma, Lixin; Yu, Ping

    2009-02-01

    We have developed a series of new dye bombesin conjugates for site-specific absorption and fluorescence imaging of human prostate and breast cancers. Bombesin (BBN), an amphibian analog to the endogenous ligand, binds to the gastrin releasing peptide (GRP) receptors with high specificity and affinity. Previously, we developed an Alexa Fluor 680-GGG-BBN peptide conjugate which demonstrated high binding affinity and specificity for breast cancer cells in the in vitro and in vivo tests (Ref: Ma et al., Molecular Imaging, vol. 6, no. 3, 2007: 171-180). This probe can not be used as an absorption probe in near-infrared imaging because its absorption peak is in the visible wavelength range. In addition, site specific longer wavelength fluorescent probe is desired for in vivo molecular imaging because long wavelength photons penetrate deeper into tissue. The new absorption and fluorescent probe we developed is based on the last eight-residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), and labeled with AlexaFluor750 through a chemical linker, beta-alanine. The new probe, Alexa Fluor 750-BetaAla-BBN(7-14)NH2, exhibits optimal pharmacokinetics for specific targeting and optical imaging of the GRP receptor over-expressing cancer cells. Absorption spectrum has been measured and showed absorption peaks at 690nm, 720nm and 735nm. Fluorescent band is located at 755nm. In vitro and in vivo investigations have demonstrated the effectiveness of the new conjugates to specifically target human cancer cells overexpressing GRP receptors and tumor xenografts in severely compromised immunodeficient mouse model.

  3. 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

  4. Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes.

    PubMed

    Uno, Kakishi; Sasaki, Taeko; Sugimoto, Nagisa; Ito, Hideto; Nishihara, Taishi; Hagihara, Shinya; Higashiyama, Tetsuya; Sasaki, Narie; Sato, Yoshikatsu; Itami, Kenichiro

    2017-01-17

    Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope.

  5. Bichromophoric hemicyanine dyes as fluorescence probes applied for monitoring of the photochemically initiated polymerization

    NASA Astrophysics Data System (ADS)

    Kabatc, Janina; Bajorek, Agnieszka; Dobosz, Robert

    2011-01-01

    The spectroscopic properties of series homodimmeric hemicyanine dyes based on ( p-N,N-dimethylaminostyryl)benzothiazolium, ( p-N,N-dimethylaminostyryl)benzoxazolium, ( p-N,N-dimethylaminostyryl)-2,3,3-trimethyl-3H-indolium residues were determined. The absorption and fluorescence spectra of the dyes under study were measured in different polarity solvents at room temperature. On the basis of the solvatochromic behavior the ground state ( μ g) and excited state ( μ e) dipole moments of bis-(N,N-dimethylaminostyryl) derivatives were evaluated. The dipole moments ( μ g and μ e) were estimated from solvatochromic shifts of absorption and fluorescence spectra as function of dielectric constant ( ɛ) and refractive index ( n) of applied solvents. The absorption and fluorescence spectra are only slightly affected by the solvent polarity. The analysis of solvatochromic behavior of the fluorescence spectra as a function of Δ f ( ɛ, n) revealed that the emission occurs from a high polarity excited state. The large dipole moment changes along with the red-shifted fluorescence, as the solvent polarity is increased, demonstrates the formation of an intramolecular charge transfer state (ICT). Six bichromophoric hemicyanine dyes, possessing benzothiazole, benzoxazole or indolinium group linked by 5 or 10 methylene groups were evaluated as fluorescence probes applied for monitoring of the polymerization process. The study on the changes in fluorescence intensity and spectroscopic shift of studied compounds were carried out during photochemically initiated polymerization of 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol triacrylate (TMPTA).

  6. Studies of benzothiazole and benzoselenazole squaraines as fluorescent probes for albumins detection.

    PubMed

    Volkova, Kateryna D; Kovalska, Vladyslava B; Losytskyy, Mykhaylo Yu; Bento, Artur; Reis, Lucinda V; Santos, Paulo F; Almeida, Paulo; Yarmoluk, Sergiy M

    2008-09-01

    Series of squaraine benzothiazole and benzoselenazole dyes were studied as possible fluorescent probes for the detection of proteins, particularly albumins. It was shown that majority of the studied squaraines give significant fluorescent response on the human serum albumin (HSA) and bovine serum albumin presence. For squaraine dyes with N-hexyl pendent groups (P-1, P-2, P-3, P-5) about 100-540-fold fluorescence intensity increase upon albumins addition was observed. At the same time in presence of other proteins, namely insulin, avidin from hen egg white, immunoglobulin G (IgG), carbonic anhydrase fluorescence enhancement values were considerably lower -up to 43 times in IgG presence. It was noted that generally, squaraines with long N-hexyl pendent groups demonstrate higher emission increase values upon proteins addition comparing with their analogues with short N-ethyl tails. It was shown that fluorescence intensity enhancement for benzothiazole squaraine dye P-3, relates linearly to the HSA concentration over the wide range-from 0.2 to 500 microg/ml. Together with noticeable selectivity of this dye to albumins, existence of wide dynamic range gives possibility to propose P-3 dye as probe for HSA quantification.

  7. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

    NASA Technical Reports Server (NTRS)

    Yu, F. P.; McFeters, G. A.

    1994-01-01

    Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

  8. Magnetically engineered Cd-free quantum dots as dual-modality probes for fluorescence/magnetic resonance imaging of tumors.

    PubMed

    Ding, Ke; Jing, Lihong; Liu, Chunyan; Hou, Yi; Gao, Mingyuan

    2014-02-01

    Magnetically engineered Cd-free CuInS2@ZnS:Mn quantum dots (QDs) were designed, synthesized, and evaluated as potential dual-modality probes for fluorescence and magnetic resonance imaging (MRI) of tumors in vivo. The synthesis of Mn-doped core-shell structured CuInS2@ZnS mainly comprised three steps, i.e., the preparation of fluorescent CuInS2 seeds, the particle surface coating of ZnS, and the Mn-doping of the ZnS shells. Systematic spectroscopy studies were carried out to illustrate the impacts of ZnS coating and the following Mn-doping on the optical properties of the QDs. In combination with conventional fluorescence, fluorescence excitation, and time-resolved fluorescence measurements, the structure of CuInS2@ZnS:Mn QDs prepared under optimized conditions presented a Zn gradient CuInS2 core and a ZnS outer shell, while Mn ions were mainly located in the ZnS shell, which well balanced the optical and magnetic properties of the resultant QDs. For the following in vivo imaging experiments, the hydrophobic CuInS2@ZnS:Mn QDs were transferred into water upon ligand exchange reactions by replacing the 1-dodecanethiol ligand with dihydrolipoic acid-poly(ethylene glycol) (DHLA-PEG) ligand. The MTT assays based on HeLa cells were carried out to evaluate the cytotoxicity of the current Cd-free CuInS2@ZnS:Mn QDs for comparing with that of water soluble CdTe QDs. Further in vivo fluorescence and MR imaging experiments suggested that the PEGylated CuInS2@ZnS:Mn QDs could well target both subcutaneous and intraperitoneal tumors in vivo.

  9. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

    PubMed

    Jin, Qiang; Feng, Lei; Wang, Dan-Dan; Dai, Zi-Ru; Wang, Ping; Zou, Li-Wei; Liu, Zhi-Hong; Wang, Jia-Yue; Yu, Yang; Ge, Guang-Bo; Cui, Jing-Nan; Yang, Ling

    2015-12-30

    In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

  10. A fluorescent probe for intracellular cysteine overcoming the interference by glutathione.

    PubMed

    Tian, Minggang; Guo, Fuqiang; Sun, Yuming; Zhang, Weijia; Miao, Fang; Liu, Yong; Song, Guofen; Ho, Cheuk-Lam; Yu, Xiaoqiang; Sun, Jing Zhi; Wong, Wai-Yeung

    2014-08-28

    Cysteine (Cys) plays important roles in many physiological processes of eukaryotic cells and its detection in cells is of fundamental significance. However, glutathione (GSH), homocysteine, N-acetyl-L-cysteine and other thiols greatly hamper the detection of Cys. In particular, GSH strongly interferes with the detection of cellular Cys (30–200 μM) due to its high intracellular concentration (1–10 mM). In this work, an off–on fluorescent probe (HOTA) for the detection of Cys is presented. This probe possesses both excellent sensitivity and satisfactory selectivity for cellular Cys detection: with the addition of 200 μM Cys, the fluorescence intensity of the probe (10 μM) enhanced 117-fold and the detection limit was calculated to be 13.47 μM, which is lower than the cellular Cys concentration; the probe also selectively detected 30–200 μM cysteine over 1–10 mM glutathione. Consequently, cell imaging experiments were performed with probe HOTA. Furthermore, the results of the thiol-blocking and GSH synthesis inhibiting experiments confirmed that the intracellular emission mainly originates from the interaction between Cys and HOTA.

  11. Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field.

    PubMed

    Yi, Xiaomin; Wang, Fuli; Qin, Weijun; Yang, Xiaojian; Yuan, Jianlin

    2014-01-01

    Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer detection with high sensitivity and multi-detection capability. Due to convenient modification by conjugating with moieties of interests, NIRF probes are ideal candidates for cancer targeted imaging. Additionally, the combinatory application of NIRF imaging and other imaging modalities that can delineate anatomical structures extends fluorometric determination of biomedical information. Moreover, nanoparticles loaded with NIRF dyes and anticancer agents contribute to the synergistic management of cancer, which integrates the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment. Appropriate probe design with targeting moieties can retain the original properties of NIRF and pharmacokinetics. In recent years, great efforts have been made to develop new NIRF probes with better photostability and strong fluorescence emission, leading to the discovery of numerous novel NIRF probes with fine photophysical properties. Some of these probes exhibit tumoricidal activities upon light radiation, which holds great promise in photothermal therapy, photodynamic therapy, and photoimmunotherapy. This review aims to provide a timely and concise update on emerging NIRF dyes and multifunctional agents. Their potential uses as agents for cancer specific imaging, lymph node mapping, and therapeutics are included. Recent advances of NIRF dyes in clinical use are also summarized.

  12. Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field

    PubMed Central

    Yi, Xiaomin; Wang, Fuli; Qin, Weijun; Yang, Xiaojian; Yuan, Jianlin

    2014-01-01

    Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer detection with high sensitivity and multi-detection capability. Due to convenient modification by conjugating with moieties of interests, NIRF probes are ideal candidates for cancer targeted imaging. Additionally, the combinatory application of NIRF imaging and other imaging modalities that can delineate anatomical structures extends fluorometric determination of biomedical information. Moreover, nanoparticles loaded with NIRF dyes and anticancer agents contribute to the synergistic management of cancer, which integrates the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment. Appropriate probe design with targeting moieties can retain the original properties of NIRF and pharmacokinetics. In recent years, great efforts have been made to develop new NIRF probes with better photostability and strong fluorescence emission, leading to the discovery of numerous novel NIRF probes with fine photophysical properties. Some of these probes exhibit tumoricidal activities upon light radiation, which holds great promise in photothermal therapy, photodynamic therapy, and photoimmunotherapy. This review aims to provide a timely and concise update on emerging NIRF dyes and multifunctional agents. Their potential uses as agents for cancer specific imaging, lymph node mapping, and therapeutics are included. Recent advances of NIRF dyes in clinical use are also summarized. PMID:24648733

  13. Water-soluble and fluorescent dendritic perylene bisimides for live-cell imaging.

    PubMed

    Gao, Baoxiang; Li, Hongxia; Liu, Hongmei; Zhang, Licui; Bai, Qianqian; Ba, Xinwu

    2011-04-07

    We prepared dendritic perylene bisimide probes with triblock structures: perylene bisimides fluorescence cores, branched oligo(glutamic acid)s and polyethylene glycol chains. These probes showed good water solubility, low cytotoxicity and strong fluorescence in live cells.

  14. Dependence upon conditions of the properties of specifically located fluorescent probes on wheat germ calmodulin

    NASA Astrophysics Data System (ADS)

    Steiner, Robert F.; Waldron, Richard; Juminaga, D.

    1992-04-01

    The single tyrosine, Tyr-139, of wheat germ calmodulin provides an intrinsic fluorescent probe to monitor Ca2+-binding domain 4, while the single cysteine, Cys-27, provides a site for the attachment of an extrinsic fluorescent label to monitor the N-terminal lobe. This has resulted in a means of comparing the response of the N- and C- terminal regions to pH, ionic strength, and Ca2+ level. Ca2+ ligation decreases the mobility sensed by Tyr-139 at neutral pH, while a shift in pH to 5.2 results in a further decrease.

  15. Spectral and photophysical properties of intramolecular charge transfer fluorescence probe: 4'-Dimethylamino-2,5-dihydroxychalcone

    NASA Astrophysics Data System (ADS)

    Xu, Zhicheng; Bai, Guan; Dong, Chuan

    2005-12-01

    The spectral and photophysical properties of a new intramolecular charge transfer (ICT) probe, namely 4'-dimethylamino-2,5-dihydroxychalcone (DMADHC) were studied in different solvents by using steady-state absorption and emission spectroscopy. Whereas the absorption spectrum undergoes minor change with increasing polarity of the solvents, the fluorescence spectrum experiences a distinct bathochromic shift in the band position and the fluorescence quantum yield increases reaching a maximum before decrease with increasing the solvent polarity. The magnitude of change in the dipole moment was calculated based on the Lippert-Mataga equation. These results give the evidence about the intramolecular charge transfer character in the emitting singlet state of this compound.

  16. Cyanines as new fluorescent probes for DNA detection and two-photon excited bioimaging.

    PubMed

    Feng, Xin Jiang; Wu, Po Lam; Bolze, Frédéric; Leung, Heidi W C; Li, King Fai; Mak, Nai Ki; Kwong, Daniel W J; Nicoud, Jean-François; Cheah, Kok Wai; Wong, Man Shing

    2010-05-21

    A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.

  17. 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.

  18. Development of a coumarin-furan conjugate as Zn2 + ratiometric fluorescent probe in ethanol-water system

    NASA Astrophysics Data System (ADS)

    Li, Chao-rui; Li, Si-liang; Yang, Zheng-yin

    2017-03-01

    In this study, a novel coumarin-derived compound bearing the furan moiety called 7-diethylamino-3-formylcoumarin (2‧-furan formyl) hydrazone (1) has been designed, synthesized and evaluated as a Zn2 + ratiometric fluorescent probe in ethanol-water system. This probe 1 showed good selectivity and high sensitivity towards Zn2 + over other metal ions investigated, and a decrease in fluorescence emission intensity at 511 nm accompanied by an enhancement in fluorescence emission intensity at 520 nm of this probe 1 was observed in the presence of Zn2 + in ethanol-water (V : V = 9 : 1) solution, which provided ratiometric fluorescence detection of Zn2 +. Additionally, the ratiometric fluorescence response of 1 to Zn2 + was nearly completed within 0.5 min, which suggested that this probe 1 could be utilized for sensing and monitoring Zn2 + in environmental and biological systems for real-time detection.

  19. Genetically encoded fluorescent coumarin amino acids

    DOEpatents

    Wang, Jiangyun [San Diego, CA; Xie, Jianming [San Diego, CA; Schultz, Peter G [La Jolla, CA

    2012-06-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  20. Genetically encoded fluorescent coumarin amino acids

    DOEpatents

    Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

    2010-10-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

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

    PubMed

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

    2012-09-01

    We describe the development of a rhodamine chromene-based turn-on fluorescence probe to monitor the intracellular Cu(2+) 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 Cu(2+) under physiological conditions with high sensitivity and selectivity, and facilitates naked-eye detection of Cu(2+). The fluorescence intensity was remarkably increased upon the addition of Cu(2+) within 1 or 2 min, while the other sixteen metal ions caused no significant effect.

  2. Probing of cosolvents in polymer latex materials by using solvatochromic fluorescence.

    PubMed

    Brouwer, Albert M; Raja, Tanzeela N; Biemans, Koen; Nabuurs, Tijs; Tennebroek, Ronald

    2008-01-01

    The process of film formation is of great importance for the application of organic coatings. In waterborne coatings, organic cosolvents are still indispensable, but regulations force the industry to reduce their amounts. Here we describe a method that uses the solvatochromic fluorescence of a probe molecule copolymerized in an emulsion polymerization process with different monomers to shed light on the partitioning of cosolvents in polymer latex materials. The formulation of the latex with organic cosolvents that are not very water soluble leads to a quantifiable redshift of the emission of the probe. The transfer of the cosolvent upon mixing of cosolvent-containing and cosolvent-free compartments can also be monitored.

  3. Small quinolinium-based enzymatic probes via blue-to-red ratiometric fluorescence.

    PubMed

    Wang, Pan; Du, Jiajun; Liu, Huijing; Bi, Guoqiang; Zhang, Guoqing

    2016-02-21

    A small fluorescence ratiometric probe consisting of a single dye species, N-methyl-6-hydroxyquinolinium (MHQ), and coupled enzymatic substrates, exhibits a dramatic colour change (deep blue to red) and possesses a huge response ratio (over 2000 fold) upon specific recognition of target enzymes. Such dramatic responses are attributed to the excited-state proton transfer processes of MHQ molecules in water. Here the detection of β-galactosidase and porcine pancreatic lipase is successfully demonstrated and this class of molecules has the potential to be developed as a "naked-eye" probe in vitro.

  4. Two-Photon Ratiometric Fluorescence Probe with Enhanced Absorption Cross Section for Imaging and Biosensing of Zinc Ions in Hippocampal Tissue and Zebrafish.

    PubMed

    Li, Wanying; Fang, Bingqing; Jin, Ming; Tian, Yang

    2017-02-21

    Zinc ion (Zn(2+)) not only plays an important function in the structural, catalytic, transcription, and regulatory of proteins, but is also an essential ionic signal to regulate brain neurotransmitters pass process. In this work, we designed and synthesized an intramolecular charge transfer-based ratiometric two-photon fluorescence probe, P-Zn, for imaging and biosensing of Zn(2+) in live cell, hippocampal tissue, and zebrafish. The developed probe demonstrated high two-photon absorption cross section (δ) of 516 ± 77 GM, which increased to 958 ± 144 GM after the probe was coordinated with Zn(2+). Furthermore, this P-Zn probe quickly recognized Zn(2+) with high selectivity, over other metal ions, amino acids, and reactive oxygen species. More interestingly, the initial emission peak of the present probe at 465 nm decreased with a new peak increased at 550 nm, leading to the ratiometric determination of Zn(2+) with high accuracy. Finally, this two-photon fluorescence probe with high temporal resolution and remarkable analytical performance, as well as low-cytotoxicity, was successfully applied in imaging of live cells, hippocampal tissues, and zebrafishes. The present P-Zn probe combined with FLIM provided accurate mapping of Zn(2+) distribution at single-cell level. More interestingly, the two-photon spectroscopic results demonstrated that the level of Zn(2+) in hippocampal tissue of mouse with AD was higher than that in normal mouse brain.

  5. Polyamide fluorescent probes for visualization of repeated DNA sequences in living cells.

    PubMed

    Nozeret, Karine; Loll, François; Escudé, Christophe; Boutorine, Alexandre S

    2015-03-02

    DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.

  6. A new Schiff base based on vanillin and naphthalimide as a fluorescent probe for Ag+ in aqueous solution

    NASA Astrophysics Data System (ADS)

    Zhou, Yanmei; Zhou, Hua; Ma, Tongsen; Zhang, Junli; Niu, Jingyang

    2012-03-01

    A new Schiff base based on vanillin and naphthalimide was designed and synthesized as fluorescent probe. The probe showed high selectivity for Ag+ over other metal ions such as Pb2+, Na+, K+, Cd2+, Ba2+, Cr3+, Zn2+, Cu2+, Ni2+, Ca2+, Al3+ and Mg2+ in aqueous solution. A new fluorescence emission was observed at 682 nm in the presence of Ag+ ion. The fluorescence intensity quenched with increasing the concentration of Ag+ at 682 nm. The method of job's plot confirmed the 1:2 complex between Ag+ and probe, and the mechanism was proposed.

  7. A new pyrazoline-based probe of quenched fluorescent reversible recognition for Cu2 + and its application in cells

    NASA Astrophysics Data System (ADS)

    Zhang, Ying-Peng; Dong, Yu-Ying; Yang, Yun-Shang; Guo, Hui-Chen; Cao, Bi-xia; Sun, Shi-Qi

    2017-04-01

    A new pyrazoline-based probe D was designed and synthesized, which can be used as a highly sensitive, selective and reversible recognizing fluorescent to detect Cu2 +. The recognition properties of this compound was investigated by UV-vis absorption and fluorescence spectrophotometry. The results showed that the probe D forms a 1:1 complex with Cu2 + and displayed a linear fluorescence response to Cu2 + with a detection limit of 1.94 × 10- 7 M. In addition, the probe have a good biocompatibility in living cells.

  8. Fluorescence microscopy studies of a peripheral-benzodiazepine-receptor-targeted molecular probe for brain tumor imaging

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Vernier, P. Thomas; Manning, H. Charles; Salemi, Sarah; Li, Aimin; Craft, Cheryl M.; Gundersen, Martin A.; Bornhop, Darryl J.

    2003-10-01

    This study investigates the potential of a new multi-modal lanthanide chelate complex for specifically targeting brain tumor cells. We report here results from ongoing studies of up-take, sub-cellular localization and binding specificity of this new molecular imaging probe. Fluorescence microscopy investigations in living rat C6 glioma tumor cells demonstrate that the new imaging agent has affinity for glioma cells and binds to mitochondria.

  9. Probing the dynamics of photosynthetic membranes with fluorescence recovery after photobleaching.

    PubMed

    Mullineaux, Conrad W; Sarcina, Mary

    2002-06-01

    In the past few years, there has been remarkable progress in knowledge of the structures and organization of the protein complexes of photosynthetic membranes. However, static structures do not tell the whole story. Photosynthetic membranes, like other biological membranes, are dynamic systems. Recent technological advances are making it increasingly easy to probe the dynamics of photosynthetic membranes using fluorescence recovery after photobleaching. Here we explain the potential and the limitations of the technique.

  10. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

    PubMed Central

    Tanpure, Arun A.; Srivatsan, Seergazhi G.

    2015-01-01

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential. PMID:26202965

  11. Photolysis of Cyclopiazonic Acid to Fluorescent Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), which is produced by certain species of Aspergillus and Penicillium, can co-occur with aflatoxins under certain conditions. A large proportion of A. flavus strains can produce CPA and it has been found as a natural contaminant in cheeses, corn, rice, peanuts, millet and fe...

  12. Photolysis of Cyclopiazonic Acid to Fluorescent Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA) is a mycotoxin produced by some of the same species of fungi that produce the more widely known aflatoxins. As a consequence it has been found previously that CPA and the aflatoxins may co-occur in commodities under certain conditions. CPA, which is a substituted indole, h...

  13. Determination of torasemide by fluorescence quenching method with some dihalogenated fluorescein dyes as probes

    NASA Astrophysics Data System (ADS)

    Cui, Zhiping; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Hu, Xiaoli; Tian, Jing

    2013-10-01

    A novel fluorescence quenching method for the determination of torasemide (TOR) with some dihalogenated fluorescein dyes as fluorescence probes was developed. In acidulous medium, TOR could interact with some dihalogenated fluorescein dyes such as dichlorofluorescein (DCF), dibromofluorescein (DBF) and diiodofluorescein (DIF) to form binary complexes, which could lead to fluorescence quenching of above dihalogenated fluorescein dyes. The maximum fluorescence emission wavelengths were located at 532 nm (TOR-DCF), 535 nm (TOR-DBF) and 554 nm (TOR-DIF). The relative fluorescence intensities (ΔF = F0 - F) were proportional to the concentration of TOR in certain ranges. The detection limits were 4.8 ng mL-1 for TOR-DCF system, 9.8 ng mL-1 for TOR-DBF system and 35.1 ng mL-1 for TOR-DIF system. The optimum reaction conditions, influencing factors were studied; and the effect of coexisting substances was investigated owing to the highest sensitivity of TOR-DCF system. In addition, the reaction mechanism, composition and structure of the complex were discussed by quantum chemical calculation and Job's method. The fluorescence quenching of dihalogenated fluorescein dyes by TOR was a static quenching process judging from the effect of temperature and the Stern-Volmer plots. The method was satisfactorily applied to the determination of TOR in tablets and human urine samples.

  14. Determination of torasemide by fluorescence quenching method with some dihalogenated fluorescein dyes as probes.

    PubMed

    Cui, Zhiping; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Hu, Xiaoli; Tian, Jing

    2013-10-01

    A novel fluorescence quenching method for the determination of torasemide (TOR) with some dihalogenated fluorescein dyes as fluorescence probes was developed. In acidulous medium, TOR could interact with some dihalogenated fluorescein dyes such as dichlorofluorescein (DCF), dibromofluorescein (DBF) and diiodofluorescein (DIF) to form binary complexes, which could lead to fluorescence quenching of above dihalogenated fluorescein dyes. The maximum fluorescence emission wavelengths were located at 532 nm (TOR-DCF), 535 nm (TOR-DBF) and 554 nm (TOR-DIF). The relative fluorescence intensities (ΔF=F0-F) were proportional to the concentration of TOR in certain ranges. The detection limits were 4.8 ng mL(-1) for TOR-DCF system, 9.8 ng mL(-1) for TOR-DBF system and 35.1 ng mL(-1) for TOR-DIF system. The optimum reaction conditions, influencing factors were studied; and the effect of coexisting substances was investigated owing to the highest sensitivity of TOR-DCF system. In addition, the reaction mechanism, composition and structure of the complex were discussed by quantum chemical calculation and Job's method. The fluorescence quenching of dihalogenated fluorescein dyes by TOR was a static quenching process judging from the effect of temperature and the Stern-Volmer plots. The method was satisfactorily applied to the determination of TOR in tablets and human urine samples.

  15. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Crisci, Alfonso; Giordano, Flavio; Rossari, Susanna; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-07-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  16. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  17. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-08-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events.

  18. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    PubMed Central

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-01-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events. PMID:27574102

  19. Multimodal fiber probe spectroscopy for tissue diagnostics applications: a combined Raman-fluorescence approach

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2014-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnosing melanocytic lesions in a good agreement with common routine histology. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. Further investigations were performed on colon and brain tissue samples in order to have a benchmark for diagnosing a broader range of tissue lesions and malignancies. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  20. Combined fiber probe for fluorescence lifetime and Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Wachsmann-Hogiu, Sebastian; Marple, Eric; Urmey, Kirk; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-03-01

    Raman spectroscopy has been proven to have tremendous potential as biomedical analytical tool for spectroscopic disease diagnostics. The use of fiberoptic coupled Raman spectroscopy systems can enable in-vivo characterization of suspicious lesions. However, Raman spectroscopy has the drawback of rather long acquisition times of several hundreds of milliseconds which makes scanning of larger regions quite challenging. By combining Raman spectroscopy with a fast imaging technique this problem can be alleviate in part. Fluorescence lifetime imaging (FLIm) offers a great potential for such a combination. FLIm can allow for fast tissue area pre-segmentation and location of the points for Raman spectra acquisition. Here, we introduce an optical fiber probe combining FLIm and Raman spectroscopy with an outer diameter of 2 mm. Fluorescence is generated via excitation with a fiber laser at 355 nm. The fluorescence emission is spectrally resolved using a custom-made wavelength-selection module (WSM). The Raman excitation power at 785 nm was set to 50 mW for the in-vivo measurements to prevent sample drying. The lateral probe resolution was determined to be <250 μm for both modalities. This value was taken as step size for several raster scans of different tissue types which were conducted to show the overlap of both modalities under realistic conditions. Finally the probe was used for in vivo raster scans of a rat's brain and subsequently to acquire FLIm guided Raman spectra of several tissues in and around the craniotomy.

  1. Salicylic acid interferes with GFP fluorescence in vivo.

    PubMed

    de Jonge, Jennifer; Hofius, Daniel; Hennig, Lars

    2017-03-29

    Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.

  2. 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 pKa 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.

  3. Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly

    PubMed Central

    Yaroslavsky, Anastasia I.; Smolina, Irina V.

    2013-01-01

    SUMMARY We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific. PMID:23521801

  4. A highly selective fluorescent probe based on coumarin for the imaging of N2H4 in living cells

    NASA Astrophysics Data System (ADS)

    Chen, Song; Hou, Peng; Wang, Jing; Liu, Lei; Zhang, Qi

    2017-02-01

    A turn-on fluorescence probe for highly sensitive and selective detection of N2H4 was developed based on hydrazine-triggered a substitution- cyclization-elimination cascade. Upon the treatment with N2H4, probe 1, 4-methyl-coumarin-7-yl bromobutanoate, displayed a remarkable fluorescence enhancement (25-fold) with a maximum at 450 nm. This probe can quantitatively detect N2H4 with a extremely low detection limit as 7 × 10- 8 M. Moreover, cell imaging experiments have indicated that probe 1 has potential ability to detect and image N2H4 in biological systems.

  5. Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

    PubMed Central

    Brunet, J E; Vargas, V; Gratton, E; Jameson, D M

    1994-01-01

    Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered. PMID:8161698

  6. Development of background-free tame fluorescent probes for intracellular live cell imaging

    PubMed Central

    Alamudi, Samira Husen; Satapathy, Rudrakanta; Kim, Jihyo; Su, Dongdong; Ren, Haiyan; Das, Rajkumar; Hu, Lingna; Alvarado-Martínez, Enrique; Lee, Jung Yeol; Hoppmann, Christian; Peña-Cabrera, Eduardo; Ha, Hyung-Ho; Park, Hee-Sung; Wang, Lei; Chang, Young-Tae

    2016-01-01

    Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging. PMID:27321135

  7. Design and Synthesis of Near-infrared Fluorescent Probes for Imaging of Biological Nitroxyl

    PubMed Central

    Tan, Yi; Liu, Ruochuan; Zhang, Huatang; Peltier, Raoul; Lam, Yun-Wah; Zhu, Qing; Hu, Yi; Sun, Hongyan

    2015-01-01

    Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO), has recently been identified as an interesting and important signaling molecule in biological systems. However, research on its biosynthesis and bioactivities are hampered by the lack of versatile HNO detection methods applicable to living cells. In this report, two new near-infrared (NIR) probes were designed and synthesized for HNO imaging in living cells. One of the probes was found to display high sensitivity towards HNO, with up to 67-fold of fluorescence increment after reaction with HNO. The detection limit was determined to be as low as 0.043 μM. The probe displayed high selectivity towards HNO over other biologically related species including metal ions, reactive oxygen species, reactive nitrogen species and reactive sulfur species. Furthermore, the probe was shown to be suitable for imaging of exogenous and endogenous HNO in living cells. Interestingly, the probe was found to be mainly localized in lysosomes. We envision that the new NIR probe described here will serve as a useful tool for further elucidation of the intricate roles of HNO in living cells. PMID:26584764

  8. A colorimetric and fluorescence enhancement anion probe based on coumarin compounds

    NASA Astrophysics Data System (ADS)

    Zhao, Limin; Liu, Ge; Zhang, Baofeng

    2016-12-01

    In this paper, anion probe 1 was designed and synthesized by using phenprocoumon containing acyl hydrazine with p-nitro azo salicylaldehyde reaction Dickson et al. (2008) Dickson et al. (2008) [1]. In the anion probe 1, the nitro moiety is a signaling group and the phenolic hydroxyl moiety is anion binding site. Then the anion probe 1 was characterized by mass spectra (MS) and infrared spectra (IR). The binding properties of the anion probe 1 for anions such as F-, AcO-, H2PO4-, OH-, Cl-, Br- and I- were investigated by ultraviolet-visible (UV-Vis) spectra and fluorescence spectra Shao et al. (2008) Shao et al. (2008) [2]. Furthermore, the color of anion probe 1 after addition of F-, AcO-, H2PO4- and OH- in DMSO changed from yellow to blue, while no obvious color changes were observed by addition of other tested anions. Accordingly, the anion probe 1 could sense visually F-, AcO-, H2PO4- and OH- without resorting to any spectroscopic instrumentation Amendola et al. (2010) Amendola et al. (2010) [3].

  9. Development of background-free tame fluorescent probes for intracellular live cell imaging.

    PubMed

    Alamudi, Samira Husen; Satapathy, Rudrakanta; Kim, Jihyo; Su, Dongdong; Ren, Haiyan; Das, Rajkumar; Hu, Lingna; Alvarado-Martínez, Enrique; Lee, Jung Yeol; Hoppmann, Christian; Peña-Cabrera, Eduardo; Ha, Hyung-Ho; Park, Hee-Sung; Wang, Lei; Chang, Young-Tae

    2016-06-20

    Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as 'tame' probes, and novel tools for live cell intracellular imaging.

  10. Fluorescence Probe for Detecting CCG Trinucleotide Repeat DNA Expansion and Slip-Out.

    PubMed

    Shibata, Tomonori; Nakatani, Kazuhiko

    2016-09-15

    Trinucleotide repeat expansion in genomic DNA causes severe neurodegenerative diseases. Fluorescence probes that bind to trinucleotide repeats have potential as diagnostic tools of trinucleotide repeat disorders. Here, we report a novel tricyclic ligand that binds to CCG trinucleotide repeat DNA. The expansion of the aromatic ring system of the 2-amino-1,8-naphthyridine chromophore from the bicyclic to the tricyclic improved the binding ability to the CCG/CCG motif without losing the selectivity and emissive character. The fluorescence sensitively decreased in response to binding to the CCG trinucleotide repeat. The degree of quenching depended on the number of CCG repeats. In addition, the fluorescence detection was applicable to CCG slip-out DNA.

  11. β-Carboline-functionalized dithioacetal as Hg2+-selective fluorescence probe in water

    NASA Astrophysics Data System (ADS)

    Li, Na; Dai, Jiang-Kun; Du, Hong-Tao; Yuan, Mao-Sen; Zhang, Ji-Wen; Wang, Jun-Ru

    2015-02-01

    A novel sensing system based on the β-carboline core has been designed and synthesized for Hg2+ detection in water. We have demonstrated that a straight forward methodology can provide rapid, sensitive and selective recognition (cross-contamination experiments) for Hg2+ over a wide pH range. The vivid fluorescence change from blue to colorless can be clearly discriminated by the naked eye. Furthermore, there is a good negative correlation between the fluorescent intensity and the concentration of Hg2+ in the range 1.0 × 10-6 M-7.0 × 10-6 M. β-Carboline as a fluorophore synthesized via this route also provides a new strategy for the design of novel fluorescence probes and fluorochromes.

  12. Enhancing Photoinduced Electron Transfer Efficiency of Fluorescent pH-Probes with Halogenated Phenols

    PubMed Central

    2014-01-01

    Photoinduced electron transfer (PET), which causes pH-dependent quenching of fluorescent dyes, is more effectively introduced by phenolic groups than by amino groups which have been much more commonly used so far. That is demonstrated by fluorescence measurements involving several classes of fluorophores. Electrochemical measurements show that PET in several amino-modified dyes is thermodynamically favorable, even though it was not experimentally found, underlining the importance of kinetic aspects to the process. Consequently, the attachment of phenolic groups allows for fast and simple preparation of a wide selection of fluorescent pH-probes with tailor-made spectral properties, sensitive ranges, and individual advantages, so that a large number of applications can be realized. Fluorophores carrying phenolic groups may also be used for sensing analytes other than pH or molecular switching and signaling. PMID:25126834

  13. A novel fiber optic biosensor for nitric oxide determination based on vicinal diaminobenzozcridine fluorescent probe

    NASA Astrophysics Data System (ADS)

    Ding, Liyun; Huang, Lanfen; Huang, Jun; Zhong, Yunming; Fan, Dian

    2010-04-01

    A novel fiber optic biosensor for the determination of nitric oxide based on vicinal diaminobenzozcridine (VDABA) fluorescent probe was designed and fabricated. The reaction conditions between VDABA and NO, which include concentration of VDABA, temperature and pH, were studied in-depth. The sensitivity of VDABA for NO detection under the optimum conditions and its optical properties were also investigated. The fluorescence responses were concentration-dependent and a good linear relationship (R2=0.9863) was observed over the range 1.8×10-6 to 9×10-6 mol/L NO, the regression equation was F = 3.8889[NO] (mol/L)+217.2. Besides, a complex sensitive film embedding VDABA in cellulose acetate (CA) was prepared, and a fiber optic NO biosensor was fabricated using this film. Then the change of fluorescence phase shift of this biosensor was studied preliminarily by means of the lock-in technology.

  14. 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

    2017-03-16

    In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO2-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@SiO2 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@SiO2-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.

  15. 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.

  16. A combined Raman-fluorescence spectroscopic probe for tissue diagnostics applications

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Cosci, Alessandro; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2013-06-01

    We designed and developed two different optical fibre probes for combined Raman and fluorescence spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multimodal approach. Two laser diodes, respectively emitting in the UV (378 nm) and in the visible (445 nm), were used for fluorescence spectroscopy. An additional laser diode emitting in the NIR (785 nm) was used for Raman spectroscopy. Laser light was delivered to the tissue under examination through a multimode optical fibre located in the centre of the fibre bundle probe. The surrounding 24 optical fibres were used for collection of the signal of interest and for delivering light to a common detection unit. Both fluorescence and Raman spectra were acquired on a cooled CCD camera, connected to a spectrograph. The device was successfully used for diagnosing melanocytic lesions in a good agreement with common routine histology. Additional measurements were performed on other human tissue samples, such as colon tissue and brain tissue in order to test the capability of the device for diagnosing a broader range of tissue lesions and malignancies. The system has the potential to improve diagnostic capabilities on a broad range of tissues and to be used for endoscopic inspections in the near future.

  17. 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.

  18. A cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing.

    PubMed

    Ke, Guoliang; Zhu, Zhi; Wang, Wei; Zou, Yuan; Guan, Zhichao; Jia, Shasha; Zhang, Huimin; Wu, Xuemeng; Yang, Chaoyong James

    2014-09-10

    Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid-DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid-DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid-DNA probe showed sensitive and reversible response to pH change in the range of 6.0-8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid-DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid-DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

  19. Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV.

    PubMed

    Lai, Koon Siew; Ho, Nan-Hui; Cheng, Jonathan D; Tung, Ching-Hsuan

    2007-01-01

    Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.

  20. Selective Fluorescence Probes for Dipeptidyl Peptidase Activity - Fibroblast Activation Protein and Dipeptidyl Peptidase IV

    PubMed Central

    Lai, Koon Siew; Ho, Nan-Hui; Cheng, Jonathan D.; Tung, Ching-Hsuan

    2008-01-01

    Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, Fibroblast activation protein (FAP) and Dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion and metastasis. We here validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery. PMID:17489551

  1. 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

  2. Toward a Chemical Marker for Inflammatory Disease: A Fluorescent Probe for Membrane-Localized Thioredoxin

    PubMed Central

    2015-01-01

    Thioredoxin (Trx) is a redox-active protein that plays a key role in mitigating the effects of oxidative stress. The secretion of Trx on the plasma membrane has been suggested as a distinctive feature of inflammation. However, selective monitoring of membrane-associated Trx activity has proved challenging because of the ubiquity of Trx action in cells. Here, we report a Trx-specific probe that allows visualization of Trx activity associated with the membranes via fluorescence microscopy. The ability of this probe to act as a possible screening tool for agents that modulate Trx secretion was demonstrated in HeLa cells under oxidative stress conditions and in a cellular hepatosteatosis model. Control experiments serve to confirm that the response seen for the present probe is due to Trx and that it is selective over various potentially competing metabolites, including thiol-containing small molecules and test proteins. PMID:24840911

  3. Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

    PubMed Central

    Larin, Z; Fricker, M D; Maher, E; Ishikawa-Brush, Y; Southern, E M

    1994-01-01

    We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories. Images PMID:7937078

  4. Membrane effects of N-terminal fragment of apolipoprotein A-I: a fluorescent probe study.

    PubMed

    Trusova, Valeriya; Gorbenko, Galyna; Girych, Mykhailo; Adachi, Emi; Mizuguchi, Chiharu; Sood, Rohit; Kinnunen, Paavo; Saito, Hiroyuki

    2015-03-01

    The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity.

  5. Fluorescence pH probe based on microstructured polymer optical fiber.

    PubMed

    Yang, X H; Wang, L L

    2007-12-10

    A kind of optical pH sensor was demonstrated that is based on a pH-sensitive fluorescence dye-doped (eosin) cellulose acetate (CA) thin-film modified microstructured polymer optical fiber (MPOF). It was obtained by directly inhaling an eosin-CA-acetic acid mixed solution into array holes in a MPOF and then removing the solvent (acetic acid). The sensing film showed different fluorescence intensities to different pH solutions in a pH range of 2.5-4.5. Furthermore, the pH response range could be tailored through doping a surfactant, hexadecyl trimethyl ammonium bromide (CTAB), in the sensing film.

  6. Designer nucleic acids to probe and program the cell.

    PubMed

    Krishnan, Yamuna; Bathe, Mark

    2012-12-01

    Recent advances in nucleic acid sequencing, structural, and computational technologies have resulted in dramatic progress in our understanding of nucleic acid structure and function in the cell. This knowledge, together with the predictable base-pairing of nucleic acids and powerful synthesis and expression capabilities now offers the unique ability to program nucleic acids to form precise 3D architectures with diverse applications in synthetic and cell biology. The unique modularity of structural motifs that include aptamers, DNAzymes, and ribozymes, together with their well-defined construction rules, enables the synthesis of functional higher-order nucleic acid complexes from these subcomponents. As we illustrate here, these highly programmable, smart complexes are increasingly enabling researchers to probe and program the cell in a sophisticated manner that moves well beyond the use of nucleic acids for conventional genetic manipulation alone.

  7. Concise and Efficient Fluorescent Probe via an Intromolecular Charge Transfer for the Chemical Warfare Agent Mimic Diethylchlorophosphate Vapor Detection.

    PubMed

    Yao, Junjun; Fu, Yanyan; Xu, Wei; Fan, Tianchi; Gao, Yixun; He, Qingguo; Zhu, Defeng; Cao, Huimin; Cheng, Jiangong

    2016-02-16

    Sarin, used as chemical warfare agents (CWAs) for terrorist attacks, can induce a number of virulent effects. Therefore, countermeasures which could realize robust and convenient detection of sarin are in exigent need. A concise charge-transfer colorimetric and fluorescent probe (4-(6-(tert-butyl)pyridine-2-yl)-N,N-diphenylaniline, TBPY-TPA) that could be capable of real-time and on-site monitoring of DCP vapor was reported in this contribution. Upon contact with DCP, the emission band red-shifted from 410 to 522 nm upon exposure to DCP vapor. And the quenching rate of TBPY-TPA reached up to 98% within 25 s. Chemical substances such as acetic acid (HAc), dimethyl methylphosphonate (DMMP), pinacolyl methylphosphonate (PAMP), and triethyl phosphate (TEP) do not interfere with the detection. A detection limit for DCP down to 2.6 ppb level is remarkably achieved which is below the Immediately Dangerous to Life or Health concentration. NMR data suggested that a transformation of the pyridine group into pyridinium salt via a cascade reaction is responsible for the sensing process which induced the dramatic fluorescent red shift. All of these data suggest TBPY-TPA is a promising fluorescent sensor for a rapid, simple, and low-cost method for DCP detection, which could be easy to prepare as a portable chemosensor kit for its practical application in real-time and on-site monitoring.

  8. 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.

  9. RGD-conjugated Two-photon Absorbing Near-IR Emitting Fluorescent Probes for Tumor Vasculature Imaging

    PubMed Central

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

    2015-01-01

    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. PMID:26351137

  10. 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.

  11. Targetable fluorescent probe for monitoring exogenous and endogenous NO in mitochondria of living cells.

    PubMed

    Yu, Haibo; Zhang, Xinfu; Xiao, Yi; Zou, Wei; Wang, Liping; Jin, Liji

    2013-08-06

    Nitric oxide (NO) is a ubiquitous cellular messenger molecule in the cardiovascular, nervous, and immune systems. Mitochondrion is the main area where endogenous NO is synthesized by inducible NOS enzymes in mammalian cells. Thus, real-time monitoring NO in mitochondria is very meaningful for NO chemical biology. Although a variety of fluorescent probes for NO have been successfully developed, they are not suited for detecting mitochondrial NO because none of them can specifically localize in mitochondria. Herein, Mito-Rh-NO, the first mitochondria-targetable "turn-on" fluorescent probe for NO, has been developed through attaching a triphenylphosphonium to a rhodamine spirolactam. The characteristics of this probe are as following: (1) Mito-Rh-NO exhibits high sensitivity toward NO. In solution, Mito-Rh-NO responds to NO by significant fluorescence enhancement up to 60-fold, and its NO detection limit is as low as 4.0 nM. (2) The NO sensing of Mito-Rh-NO is highly selective, which will not interfere with the other reactive oxygen and nitrogen species. (3) Mito-Rh-NO has a low cytotoxic effect: after being treated with 10 μM Mito-Rh-NO for 24 h, the survival rate is higher than 90%. (4) Mito-Rh-NO specifically localizing in mitochondria: colocalization experiment of Mito-Rh-NO and Rh 123, a typical mitotracker, shows the merged fluorescent microcopy image with a high Pearson's colocalization coefficient 0.92 and overlap coefficient 0.99. (5) Mito-Rh-NO demonstrates high applicability for real-time monitoring of mitochondrial NO in live cells. Both the exogenous NO released by the donor NOC13 and endogenous NO generated in cells under stimulation have been visualized under confocal microscopy.

  12. A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.

    PubMed

    Enkvist, Erki; Viht, Kaido; Bischoff, Nils; Vahter, Jürgen; Saaver, Siiri; Raidaru, Gerda; Issinger, Olaf-Georg; Niefind, Karsten; Uri, Asko

    2012-11-21

    Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

  13. Chlorin p 6 as a fluorescent probe for the investigation of surfactant-cyclodextrin interactions

    NASA Astrophysics Data System (ADS)

    Mishra, Padmaja P.; Adhikary, Ramkrishna; Lahiri, Priyanka; Datta, Anindya

    2006-02-01

    Cyclodextrins (CD) are often projected as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity / lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p 6, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between α-CD and β-CD with the neutral surfactants TX 100 and CTAB. The fluorophore is found to be a sensitive one for the study of the interaction of α- and β-CD with the surfactants Tritron X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). It is found that contrary to earlier reports, a complex between α-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p 6, which does not bind to the CDs, but not with a fluorophore like TNS, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and α-CD as compared to that between TNS and CD.

  14. An Activatable Near Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

    PubMed Central

    Li, Jinbo; Chen, Kai; Liu, Hongguang; Cheng, Kai; Yang, Meng; Zhang, Jiping; Cheng, Jonathan D.; Zhang, Yan; Cheng, Zhen

    2012-01-01

    Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAPα expressing tumors. PMID:22812530

  15. Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts.

    PubMed Central

    Millard, P J; Roth, B L; Thi, H P; Yue, S T; Haugland, R P

    1997-01-01

    A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy. PMID:9212436

  16. Measurement of fluorescent probes concentration ratio in the cerebrospinal fluid for early detection of Alzheimer's disease

    NASA Astrophysics Data System (ADS)

    Harbater, Osnat; Gannot, Israel

    2014-03-01

    The pathogenic process of Alzheimer's Disease (AD), characterized by amyloid plaques and neurofibrillary tangles in the brain, begins years before the clinical diagnosis. Here, we suggest a novel method which may detect AD up to nine years earlier than current exams, minimally invasive, with minimal risk, pain and side effects. The method is based on previous reports which relate the concentrations of biomarkers in the Cerebrospinal Fluid (CSF) (Aβ and Tau proteins) to the future development of AD in mild cognitive impairment patients. Our method, which uses fluorescence measurements of the relative concentrations of the CSF biomarkers, replaces the lumbar puncture process required for CSF drawing. The process uses a miniature needle coupled trough an optical fiber to a laser source and a detector. The laser radiation excites fluorescent probes which were prior injected and bond to the CSF biomarkers. Using the ratio between the fluorescence intensities emitted from the two biomarkers, which is correlated to their concentration ratio, the patient's risk of developing AD is estimated. A theoretical model was developed and validated using Monte Carlo simulations, demonstrating the relation between fluorescence emission and biomarker concentration. The method was tested using multi-layered tissue phantoms simulating the epidural fat, the CSF in the sub-arachnoid space and the bone. These phantoms were prepared with different scattering and absorption coefficients, thicknesses and fluorescence concentrations in order to simulate variations in human anatomy and in the needle location. The theoretical and in-vitro results are compared and the method's accuracy is discussed.

  17. A highly sensitive fluorescence probe for metallothioneins based on tiron-copper complex

    NASA Astrophysics Data System (ADS)

    Xiao, Xilin; Xue, Jinhua; Liao, Lifu; Huang, Mingyang; Zhou, Bin; He, Bo

    2015-06-01

    The fabrication of tiron-copper complex as a novel fluorescence probe for the sensitive directly detection of metallothioneins at nanomolar levels was demonstrated. In Britton-Robinson (B-R) buffer (pH 7.50), the interaction of bis(tiron)copper(II) complex cation [Cu(tiron)2]2+ and metallothioneins enhanced the fluorescence intensity of the system. The fluorescence enhancement at 347 nm was proportional to the concentration of metallothioneins. The mechanism was studied and discussed in terms of the fluorescence spectra. Under the optimal experimental conditions, at 347 nm, there was a linear relationship between the fluorescence intensity and the concentration of the metallothioneins in the range of 8.80 × 10-9-7.70 × 10-7 mol L-1, with a correlation coefficient of r = 0.995 and detection limit 2.60 × 10-9 mol L-1. The relative standard deviation was 0.77% (n = 11), and the average recovery 94.4%. The method proposed was successfully reliable, selective and sensitive in determining of trace metallothioneins in fish visceral organ samples with the results in good agreement with those obtained by HPLC.

  18. New naphthalimide modified polyethylenimine nanoparticles as fluorescent probe for DNA detection

    NASA Astrophysics Data System (ADS)

    Liang, Shucai; Yu, Hui; Xiang, Jin; Yang, Wang; Chen, Xiaohui; Liu, Yanbin; Gao, Chen; Yan, Guoping

    2012-11-01

    A new naphthalimide modified polyethylenimine (PEI) nanoparticles (called NPEI-NPs) was synthesized and applied as fluorescent probe for rapid, selective and sensitive fluorometric detection of trace DNA. The synthesis involved the covalent modification of PEI with 4-butylamino-N-carboxymethyl-1,8-naphthalimide(BACMN) for getting amphiphilic polymer. Then the amphiphilic polymer was self-assembled in water to give the NPEI-NPs. NPEI-NPs was soluble in water and emitted fluorescence at 545 nm with exciting at 460 nm. The fluorescence spectra resulting from the interaction between NPEI-NPs and DNA indicated that the fluorescence of NPEI-NPs increased in the present of DNA. Therefore, a fluorescence enhancement method was developed for the determination of trace fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA). Under the optimal conditions, the calibration curves were linear over the concentration ranges of 0.05-2.8 μg/mL for fsDNA and 0.08-3.0 μg/mL for ctDNA. The detection limits for fsDNA and ctDNA were 1.6 and 2.0 ng/mL, respectively. The proposed method has been employed to quantify DNA in synthetic samples with the satisfactory results.

  19. Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

    PubMed

    Zeng, Xiaodan; Zhang, Xiaoling; Yang, Wen; Jia, Hongying; Li, Yamin

    2012-05-01

    An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

  20. Isoindoline nitroxide-labeled porphyrins as potential fluorescence-suppressed spin probes.

    PubMed

    Liu, F; Zou, T J; Tan, Z L; Chen, S; Wu, Z H; Yan, G P; Zhang, Q; Liang, S C; Yang, J

    2017-02-07

    A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane and 5-(4'-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO) using the Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these compounds were characterized and their in vitro properties were evaluated. The electrochemical assay demonstrated that these isoindoline nitroxide-labeled porphyrins had similar electrochemical and redox properties to 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. The in vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated that the reduced isoindoline nitroxide-labeled porphyrins eliminated fluorescence suppression and displayed strong red fluorescence imaging in HepG2 cells. Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as biological spin probes for fluorescence imaging and EPR spectroscopy.

  1. Effect of pH on the fluorescence characteristics of some flavones probes.

    PubMed

    Voicescu, Mariana; Ionescu, Sorana; Gatea, Florentina

    2014-04-05

    The photophysical properties such as electronic absorption, molar extinction coefficient, emission spectra, fluorescence quantum yield and lifetime of three different hydroxyflavones (a typical model of flavonols) such as: 3-HF, 3,6-diHF and 3,7-diHF, have been studied in the pH range from 2.5 to 9.2. Both electronic absorption and fluorescence spectra are sensitive to pH. The fluorescence quantum yield at pH 7.4 of the mentioned flavones probes have been determined. The fluorescence lifetime of different emissive species (Normal, Tautomer and Anion forms) as pH dependence have been also estimated. The effect of pH on the intramolecular excited state proton transfer process (ESIPT) has been discussed. The normal and tautomeric forms change as a function of pH, the normal one being more sensitive. The position of the -OH group on the second aromatic ring in the flavonol's structure has been also discussed. The results have relevance to compounds which have photoreactions accompanied by dual fluorescence.

  2. A new series of fluorescent indicators for super acids.

    PubMed

    Shih, I-Chih; Yeh, Yu-Shan; Wu, I-Che; Chen, You-Hua; Shen, Jiun-Yi; Chen, Yi-An; Ho, Mei-Lin; Chou, Pi-Tai

    2015-01-01

    The photophysical properties of fluorescent Hammett acidity indicator derived from 3,4,5,6-tetrahydrobis(pyrido[3,2-g]indolo)[2,3-a:3',2'-j]acridine (1a), 6-bis(pyrido[3,2-g]indol-2'-yl)pyridine (1b) and their analogues have been investigated in sulfuric acid solutions by means of absorption, fluorimetry, relaxation dynamics and computational approach. These new indicators undergo a reversible protonation process in the Hammett acidity range of H0 < 0, accompanied by a drastic increase of the bright blue-green (1a) or yellow (1b) fluorescence intensity upon increasing the acidity. For 1a in H2 SO4 , the emission yield increases as large as 200 folds from pH = -0.41 to the Hammett acidity range of -5.17, the results of which are rationalized by a much increase of the steric hindrance upon third protonation toward the central pyridinic site, together with their accompanied changes of electronic configuration from charge transfer to a delocalized ππ* character in the lowest lying excited state. The combination of 1a and 1b renders a wide and linear range of H0 measurement from -1.2 to -5.1 detected by highly intensive fluorescence.

  3. 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.

  4. A gold-based nanobeacon probe for fluorescence sensing of organophosphorus pesticides.

    PubMed

    Dou, Xiaowen; Chu, Xianfeng; Kong, Weijun; Luo, Jiaoyang; Yang, Meihua

    2015-09-03

    A nanomaterials-based novel molecular beacon has attracted growing attentions in fluorescent assays as many nanomaterials possess excellent quenching efficiency. In this work, a gold-based nanobeacon probe was established to detect organophosphorus pesticides for the first time. The constructed gold-based nanobeacon acted as a signal indicator and could display the decreasing of the intensity in the presence of targets, which competitively bound to single strand DNA. To achieve a high sensitive probe, some parameters including solution pH, temperature and reaction time were investigated and optimized. The gold-based nanobeacon probe assay was proved to be rapid and sensitive to achieve a detection limit of 0.035 μM for isocarbophos, 0.134 μM for profenofos, 0.384 μM for phorate and 2.35 μM for omethoate, respectively. The prepared nanobeacon effectively reduced the background and improved the detection sensitivity and selectivity. The probe is stable, easy to operate and does not need sophisticated instruments. These features makes the probe feasible for screening trace organophosphorus pesticides in real samples.

  5. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer

    PubMed Central

    Whitley, Melodi Javid; Cardona, Diana M.; Lazarides, Alexander L.; Spasojevic, Ivan; Ferrer, Jorge M.; Cahill, Joan; Lee, Chang-Lung; Snuderl, Matija; Blazer, Dan G.; Hwang, E. Shelley; Greenup, Rachel A.; Mosca, Paul J.; Mito, Jeffrey K.; Cuneo, Kyle C.; Larrier, Nicole A.; O’Reilly, Erin K.; Riedel, Richard F.; Eward, William C.; Strasfeld, David B.; Fukumura, Dai; Jain, Rakesh K.; Lee, W. David; Griffith, Linda G.; Bawendi, Moungi G.; Kirsch, David G.; Brigman, Brian E.

    2016-01-01

    Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes. PMID:26738797

  6. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer.

    PubMed

    Whitley, Melodi Javid; Cardona, Diana M; Lazarides, Alexander L; Spasojevic, Ivan; Ferrer, Jorge M; Cahill, Joan; Lee, Chang-Lung; Snuderl, Matija; Blazer, Dan G; Hwang, E Shelley; Greenup, Rachel A; Mosca, Paul J; Mito, Jeffrey K; Cuneo, Kyle C; Larrier, Nicole A; O'Reilly, Erin K; Riedel, Richard F; Eward, William C; Strasfeld, David B; Fukumura, Dai; Jain, Rakesh K; Lee, W David; Griffith, Linda G; Bawendi, Moungi G; Kirsch, David G; Brigman, Brian E

    2016-01-06

    Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

  7. Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

    PubMed

    Kurzawa, Laetitia; Pellerano, Morgan; Coppolani, J B; Morris, May C

    2011-01-01

    Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

  8. Cyclodextrin-clicked silica/CdTe fluorescent nanoparticles for enantioselective recognition of amino acids

    NASA Astrophysics Data System (ADS)

    Zhou, Jie; Liu, Yun; Zhang, Zhixing; Yang, Sha; Tang, Jian; Liu, Wei; Tang, Weihua

    2016-03-01

    Fluorescent sensors based on semiconductor quantum dots (QDs) have been immensely investigated for achiral molecular recognition. For chiral discrimination of amino acids (AAs), we herein report a versatile fluorescent sensor, i.e., CdTe QDs encapsulated with cyclodextrin (CD) clicked silica via layer-by-layer modification. The as-obtained hybrid molecular recognition platform exhibited excellent chirality sensing of AAs at micromolar concentrations in water. By taking advantage of the inclusion complexation of CD and the optical properties of the QD core, chiral discrimination was realized on the basis of the different binding energies of the CD-AA enantiomer complexes, as revealed using density-functional theory calculation. The fluorescent probe exhibited linearly enhanced photoluminescence with increased concentration of d-histidine at 0-60 μM and l-histidine at 0-20 μM. These water-soluble fluorescent sensors using a chiral host with a covalently linked chromophore may find applications in the robust sensing of a wide range of achiral and chiral molecules in water.Fluorescent sensors based on semiconductor quantum dots (QDs) have been immensely investigated for achiral molecular recognition. For chiral discrimination of amino acids (AAs), we herein report a versatile fluorescent sensor, i.e., CdTe QDs encapsulated with cyclodextrin (CD) clicked silica via layer-by-layer modification. The as-obtained hybrid molecular recognition platform exhibited excellent chirality sensing of AAs at micromolar concentrations in water. By taking advantage of the inclusion complexation of CD and the optical properties of the QD core, chiral discrimination was realized on the basis of the different binding energies of the CD-AA enantiomer complexes, as revealed using density-functional theory calculation. The fluorescent probe exhibited linearly enhanced photoluminescence with increased concentration of d-histidine at 0-60 μM and l-histidine at 0-20 μM. These water

  9. 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.

  10. Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

    PubMed

    Albani, J R; Sillen, A; Plancke, Y D; Coddeville, B; Engelborghs, Y

    2000-07-24

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor

  11. A Zn2+-specific fluorescent molecular probe for the selective detection of endogenous cyanide in biorelevant samples.

    PubMed

    Divya, Kizhumuri P; Sreejith, Sivaramapanicker; Balakrishna, Bugga; Jayamurthy, Purushothaman; Anees, Palappuravan; Ajayaghosh, Ayyappanpillai

    2010-09-07

    A Zn(2+)-specific molecular probe 3 was developed for the selective detection of CN(-) under aqueous conditions. The fluorescent Zn(2+) complex of 3 upon CN(-) addition generates a bright blue fluorescence that allows the detection of the latter and is useful for the screening of natural products with and without endogenous cyanide content.

  12. Fluorescence probing investigation of the mechanism of formation of MSU-type mesoporous silica prepared in fluoride medium.

    PubMed

    Lesaint, Cédric; Lebeau, Bénédicte; Marichal, Claire; Patarin, Joël; Zana, Raoul

    2005-09-13

    The mechanism of formation of a MSU-type siliceous material from tetraethyl orthosilicate (TEOS) in the presence of the nonionic surfactant tergitol T-15-S-12, sulfuric acid, and sodium fluoride has been investigated using mainly fluorescence probing techniques and, to a lesser extent, dynamic light scattering (DLS) and 29Si NMR spectroscopy. The tergitol micelles present in the systems obtained by progressively generating the reaction mixture giving rise to the mesostructured material by adding to an appropriate tergitol solution sulfuric acid, TEOS, and NaF were characterized by fluorescence probing (micelle aggregation number, micropolarity, and microviscosity) and also by dynamic light scattering (apparent micelle diameter). 29Si NMR experiments were also performed on selected systems after hydrolysis of the TEOS. The fluorescence probing techniques were also used to follow the changes of micelle characteristics with time during the evolution of the full reaction mixture from a limpid solution to a system containing a minor amount of condensed siliceous material. The synthesized solid material was characterized by X-ray diffraction and nitrogen adsorption-desorption analyses. The micelle aggregation number N was found to change only little, and the micropolarity remained constant when going from the tergitol solution to the full reaction mixture. The results of DLS measurements agree with this finding. Besides, while the condensation of silica took place after addition of NaF, the N value increased only very little with time up to the point where a small amount of mesostructured material precipitated out. These results indicate that the interaction between tergitol micelles and the siliceous species formed in the system by the hydrolysis of TEOS and also between micelles and the growing siliceous species must be very weak. As in our previous studies of the mechanism of formation of MCM41-type material from sodium silicate in the presence of

  13. Fluorescent probe for high-throughput screening of membrane protein expression

    PubMed Central

    Backmark, A E; Olivier, N; Snijder, A; Gordon, E; Dekker, N; Ferguson, A D

    2013-01-01

    Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture. PMID:23776061

  14. Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane

    NASA Astrophysics Data System (ADS)

    Mohapatra, Monalisa; Mishra, Ashok K.

    2012-03-01

    Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (<= 1 mM) concentrations of the bile salts. The incorporation and location of fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.

  15. Mapping hydrogen sulfide in rats with a novel azo-based fluorescent probe.

    PubMed

    Li, Xin; Cheng, Juan; Gong, Yanling; Yang, Bo; Hu, Yongzhou

    2015-03-15

    We report herein a reaction-based fluorescent switch-on sulfide sensor, azo3, for the quantification of endogenous sulfides in rat tissues. The sensor was exploited based on the novel azo-sulfide chemistry and designed by locking the rhodol fluorophore into its nonfluorescent form with an azo group. However, the azo group would undergo a specific and biocompatible reaction with sulfides, triggering significant fluorescence increasements which were linear to the concentrations of sulfides. Azo3 distinguished by its high sensitivity (148-fold fluorescent switch-on response), good selectivity (22-fold more selective towards sulfides than other bio-thiol species) and low detection limit (500nM). Moreover, the azo3-based assay for biological sulfides displayed the unique advantage of being insusceptible to ultraviolet (UV) irradiation. Azo3 has been successfully applied to the quantification of endogenous sulfides in rat plasma and tissues including heart, brain, liver, spleen, lung and kidney. In addition to providing azo3 as a valuable tool to analyze sulfides in biological samples, we also discussed the influences of the electron effect on the sensitivity of the probes, which would shed some light on the design of future reaction-based probes.

  16. Sensing for intracellular thiols by water-insoluble two-photon fluorescent probe incorporating nanogel.

    PubMed

    Guo, Xudong; Zhang, Xin; Wang, Shuangqing; Li, Shayu; Hu, Rui; Li, Yi; Yang, Guoqiang

    2015-04-15

    A novel "turn-on" two-photon fluorescent probe containing a π-conjugated triarylboron luminogen and a maleimide moiety DMDP-M based on the photo-induced electron transfer (PET) mechanism for biothiol detection was designed and synthesized. By simply loading the hydrophobic DMDP-M on a cross-linked Pluronic(®) F127 nanogel (CL-F127), a probing system DMDP-M/CL-F127 was established, which shows quick response, high selectivity and sensitivity to cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous phase. The DMDP-M/CL-F127 system presented the fastest response to Cys with a rate constant of 0.56 min(-1), and the detection limit to Cys was calculated to be as low as 0.18 μM. The DMDP-M/CL-F127 system has been successfully applied to the fluorescence imaging of biothiols in NIH/3T3 fibroblasts either with single-photon or two-photon excitation because of its high biocompatibility and cell-membrane permeability. The present work provides a general, simple and efficient strategy for the application of hydrophobic molecules to sensing biothiols in aqueous phase, and a novel sensing system for intracellular biothiols fitted for both single-photon and two-photon fluorescence imaging.

  17. Silica micro/nanospheres for theranostics: from bimodal MRI and fluorescent imaging probes to cancer therapy

    PubMed Central

    Walia, Shanka

    2015-01-01

    Summary Nano-theranostics offer remarkable potential for future biomedical technology with simultaneous applications for diagnosis and therapy of disease sites. Through smart and careful chemical modifications of the nanoparticle surface, these can be converted to multifunctional tiny objects which in turn can be used as vehicle for delivering multimodal imaging agents and therapeutic material to specific target sites in vivo. In this sense, bimodal imaging probes that simultaneously enable magnetic resonance imaging and fluorescence imaging have gained tremendous attention because disease sites can be characterized quick and precisely through synergistic multimodal imaging. But such hybrid nanocomposite materials have limitations such as low chemical stability (magnetic component) and harsh cytotoxic effects (fluorescent component) and, hence, require a biocompatible protecting agent. Silica micro/nanospheres have shown promise as protecting agent due to the high stability and low toxicity. This review will cover a full description of MRI-active and fluorescent multifunctional silica micro/nanospheres including the design of the probe, different characterization methods and their application in imaging and treatment in cancer. PMID:25821696

  18. A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes.

    PubMed

    Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji; Nakamura, Akiko; Yanagisawa, Hiroyuki; Hayashi, Yasunori; Okamoto, Akimitsu

    2012-01-01

    Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.

  19. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

    NASA Astrophysics Data System (ADS)

    Ha, Taekjip; Tinnefeld, Philip

    2012-05-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

  20. Photophysics of Fluorescence Probes for Single Molecule Biophysics and Super-Resolution Imaging

    PubMed Central

    Ha, Taekjip; Tinnefeld, Philip

    2013-01-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of certain color and brightness but instead have their own ‘personalities’ regarding spectroscopic parameters, redox properties, size and water solubility, photostability and several more. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single molecule biophysical studies which was later overcome using Trolox through reducing/oxidizing system, but a boon for super-resolution imaging due to the controllable photoswitching. Understanding photophysics is critical in design and interpreting single molecule experiments. PMID:22404588

  1. A novel flavone-based fluorescent probe for relay recognition of HSO3(-) and Al(3+).

    PubMed

    Xu, Shuai; Tang, Ruiren; Wang, Zhen; Zhou, Yin; Yan, Rui

    2015-01-01

    In this work, a new flavone-based fluorescent probe 3-hydroxy-3'-formylflavone (3HFF) was designed to achieve highly selective relay recognition of HSO3(-) and Al(3+) in DMSO-H2O (2:8, v/v) solution. 3HFF displayed a highly selective response to HSO3(-) with a green fluorescence appearing at 524 nm. Moreover, the in situ generated 3HFF+HSO3(-) system demonstrated eminent relay recognition capability for Al(3+) with a blue fluorescence appearing at 453 nm by the formation of a 1:1 complex between 3HFF and Al(3+) in DMSO-H2O (2:8, v/v) solution. However, only slight change was observed in emission intensity with addition of Al(3+) to 3HFF, and indicated HSO3(-) was essential for the sensing of Al(3+). This work achieves the detection of HSO3(-) and Al(3+) by only one probe and provides another example for this rare combination (anion/metal).

  2. Study of fluorescence quenching in aluminum-doped ceria nanoparticles: potential molecular probe for dissolved oxygen.

    PubMed

    Shehata, N; Meehan, K; Leber, D

    2013-05-01

    This work investigates a novel usage of aluminum-doped ceria nanoparticles (ADC-NPs), as the molecular probe in optical fluorescence quenching for sensing the dissolved oxygen (DO). Cerium oxide (ceria) nanoparticles can be considered one of the most unique nanomaterials that are being studied today due to the diffusion and reactivity of oxygen vacancies in ceria, which contributes to its high oxygen storage capability. Aluminum can be considered a promising dopant to increase the oxygen ionic conductivity in ceria nanoparticles which can improve the sensitivity of ceria nanoparticles to DO. The fluorescence intensity of ADC-NPs, synthesized via chemical precipitation, is found to have a strong inverse relationship with the DO concentration in aqueous solutions. Stern-Volmer constant of ADC-NPs at room temperature is determined to be 454.6 M(-1), which indicates that ADC-NPs have a promising sensitivity to dissolved oxygen, compared to many presently used fluorophores. In addition, Stern-Volmer constant is found to have a relatively small dependence on temperature between 25 °C to 50 °C, which shows excellent thermal stability of ADC-NPs sensitivity. Our work suggests that ADC-NPs, at 6 nm, are the smallest diameter DO molecular probes between the currently used optical DO sensors composed of different nanostructures. This investigation can improve the performance of fluorescence-quenching DO sensors for industrial and environmental applications.

  3. Laser fluorescence detection of subgingival calculus using the DIAGNOdent Classic versus periodontal probing.

    PubMed

    Shakibaie, Fardad; Walsh, Laurence J

    2016-11-01

    Deposits of subgingival calculus on the root surfaces of the teeth are difficult to detect with conventional methods such as tactile probing. This study compared the performance of a 655-nm wavelength laser fluorescence (LF) system (DIAGNOdent Classic with a periodontal tip) with conventional periodontal probing under defined conditions in a laboratory simulation, using an experienced examiner. Models with a total of 30 extracted teeth with varying levels of subgingival deposits scattered on their root surfaces were prepared, and silicone impression material applied to replicate periodontal soft tissues. The models were located in a phantom head, and the presence of subgingival calculus recorded at 8 points per tooth (240 sites), then the entire scoring process repeated after 1 and 2 weeks. The optimal LF threshold was determined as a fluorescence score of 7 using Receiver Operating Characteristic curves. When compared the gold standard of direct microscopic examination of roots, LF was more sensitive, specific, and accurate than tactile probing (68.3 vs. 51.7 %; 92.1 vs. 67.8; and 80.2 vs. 59.8 %, respectively), and was also more reproducible (Cohen kappa 0.60 vs. 0.39). These data support the concept of using LF as an adjunct to clinical diagnosis.

  4. Smart lanthanide coordination polymer fluorescence probe for mercury(II) determination.

    PubMed

    Liu, Baoxia; Huang, Yankai; Zhu, Xu; Hao, Yuanqiang; Ding, Yujie; Wei, Wei; Wang, Qi; Qu, Peng; Xu, Maotian

    2016-03-17

    Lanthanide coordination polymers (LCPs) have recently emerged as attractive biosensor materials due to their flexible components, high tailorable properties and unique luminescence features. In this work, we designed a smart LCP probe of Tb-CIP/AMP {(CIP, ciprofloxacin) (AMP, adenosine monophosphate)} for Hg(2+) detection by using lanthanide ions as metal nodes, CIP as ligand molecule, and AMP as bridging linker and recognition unit. Tb-CIP/AMP emits strong green luminescence due to the inclusion of AMP, which withdraws the coordinated water molecules and shields Tb(3+) from the quenching effect of O-H vibration in water molecules. The subsequent addition of Hg(2+) into Tb-CIP/AMP can strongly quench the fluorescence because of the specific coordination interaction between AMP and Hg(2+). As a kind of Hg(2+) nanosensor, the probe exhibited excellent selectivity for Hg(2+) and high sensitivity with detection limit of 0.16 nM. In addition, the probe has long fluorescence lifetime up to millisecond and has been applied to detect Hg(2+) in drinking water and human urine samples with satisfactory results. We envision that our strategy, in the future, could be extended to the designation of other LCP-based hypersensitive time-gated luminescence assays in biological media and biomedical imaging.

  5. Characterization of human OCT1-mediated transport of DAPI as a fluorescent probe substrate.

    PubMed

    Yasujima, Tomoya; Ohta, Kinya; Inoue, Katsuhisa; Yuasa, Hiroaki

    2011-09-01

    The present study was conducted to assess the functional characteristics of human organic cation transporter 1 (hOCT1) for the transport of 4',6-diamidino-2-phenylindol (DAPI), a fluorescent compound that may be used as a probe substrate for rapid assays of its functionality. The specific uptake of DAPI by hOCT1 heterologously introduced into Madin-Darby canine kidney II cells by stable transfection was found to be, when assessed by DAPI-derived fluorescence intensity, rapid and saturable with a Michaelis constant of 8.94 µM, indicating that DAPI is a good substrate of hOCT1. The specific uptake of DAPI was insensitive to the membrane potential and extracellular pH, indicating a mode of operation different from that for typical cationic substrates such as tetraethylammonium (TEA), for which hOCT1 has been suggested to be driven by an inside-negative membrane potential and favor higher pH for optimal operation. However, many organic cations were found to inhibit the specific DAPI uptake with extents well correlated with those of inhibition of the specific uptake of [(14) C]TEA, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of hOCT1.

  6. Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay.

    PubMed

    Wu, Zai-Sheng; Hu, Peng; Zhou, Hui; Shen, Guoli; Yu, Ruqin

    2010-03-01

    This work reported the G-quadruplex structure of pyrene-labeled G-rich DNA probe and its application in the immunoglobulin E (IgE) detection, providing plausibly an insight into the biological function of human telomere. Based on the intermolecular G-quadruplex, a terminal-single-pyrene-labeled oligonucleotide signaling probe was developed and a novel protein assay strategy was proposed via combining specific DNA cleavage by S1 nuclease with target-recognizing aptamer. This assay platform not only circumvented the optimization of specific sites for reporter attachment and the pyrene monomer fluorescence quenching by flanking nucleotide bases but also presented a signal-on mechanism. Thus, ultrasensitive homogeneous detection of IgE was successfully conducted. A linear dynamic range of 4.72 x 10(-12) to 7.56 x 10(-9)m, a regression coefficient of 0.9941 and a detection limit of 9.45 x 10(-14)m were given. Additionally, a preliminary concept of the single-pyrene-labeled excimer fluorescence probes associated with G-quadruplex for screening biological markers was described. Importantly, the unexpected structural property of G-quadruplex discovered seems to provide valuable information to allow understanding of the structure and function of human telomere and exploring of useful structure-based anticancer drug.

  7. Probing chromium(III) from chromium(VI) in cells by a fluorescent sensor

    NASA Astrophysics Data System (ADS)

    Hu, Xiangquan; Chai, Jie; Liu, Yanfei; Liu, Bin; Yang, Binsheng

    2016-01-01

    Cellular uptake of Cr(VI), followed by its reduction to Cr(III) with the formation of kinetically inert Cr(III) complexes, is a complex process. To better understand its physiological and pathological functions, efficient methods for the monitoring of Cr(VI) are desired. In this paper a selective fluorescent probe L, rhodamine hydrazide bearing a benzo[b]furan-2-carboxaldehyde group, was demonstrated as a red chemosensor for Cr(III) at about 586 nm. This probe has been used to probe Cr(III) which is reduced from Cr(VI) by reductants such as glutathione (GSH), vitamin C, cysteine (Cys), H2O2 and Dithiothreitol (DTT) by fluorescence spectra. Cr(VI) metabolism in vivo is primarily driven by Vc and GSH. Vc could reduce CrO42 - to Cr(III) in a faster rate than GSH. The indirectly detection limit for Cr(VI) by L + GSH system was determined to be 0.06 μM at pH = 6.2. Moreover, the confocal microscopy image experiments indicated that Cr(VI) can be reduced to Cr(III) inside cells rapidly and the resulted Cr(III) can be captured and imaged timely by L.

  8. Microwave assisted one-pot synthesis of graphene quantum dots as highly sensitive fluorescent probes for detection of iron ions and pH value.

    PubMed

    Zhang, Chunfang; Cui, Yanyan; Song, Li; Liu, Xiangfeng; Hu, Zhongbo

    2016-04-01

    Recently, carbon nanomaterials have received considerable attention as fluorescent probes owing to their low toxicity, water solubility and stable photochemical properties. However, the development of graphene quantum dots (GQDs) is still on its early stage. In this work, GQDs were successfully synthesized by one-step microwave assisted pyrolysis of aspartic acid (Asp) and NH4HCO3 mixture. The as-prepared GQDs exhibited strongly blue fluorescence with high quantum yield up to 14%. Strong fluorescence quenching effect of Fe(3+) on GQDs can be used for its high selectivity detection among of general metal ions. The probe exhibited a wide linear response concentration range (0-50 μM) to Fe(3+) and the limit of detection (LOD) was calculated to be 0.26 μM. In addition, GQDs are also sensitive to the pH value in the range from 2 to 12 indicating a great potential as optical pH sensors. More importantly, the GQDs possess lower cellular toxicity and high photostability and can be directly used as fluorescent probes for cell imaging.

  9. Thiophene functionalized silicon-containing aggregation-induced emission enhancement materials: applications as fluorescent probes for the detection of nitroaromatic explosives in aqueous-based solutions.

    PubMed

    Wang, Xuefeng; Bian, Jiangyan; Xu, Lichao; Wang, Hua; Feng, Shengyu

    2015-12-28

    Two novel aggregation-induced emission enhancement (AIEE) molecules, namely, 3,4-diphenyl-2,5-di(2-thienyl)phenyltrimethylsilane (DPTB-TMS) and bis[3,4-diphenyl- 2,5-di(2-thienyl)phenyl]methylphenylsilane (DPTB-TMS) were designed and synthesized. The optical properties of the two silanes were completely opposite to the traditional luminescent materials. Unlike the aggregation caused quenching, they all emit faint fluorescence in the dispersed state, while emission intensity increased sharply in aggregate states. Fluorescence spectra showed that the two compounds exhibited AIEE properties and that is due to the weak π-π stacking caused by the restriction of intramolecular rotations of dye segments, particularly the -SiMe3 and thienyl groups in the aggregate state. As fluorescent (FL) probes, the fluorescence quenching behavior was further investigated. Thanks to the richer-electron thiophene groups, both compounds showed good performance in detecting nitroaromatics, especially picric acid (PA). The two AIEE FL probes exhibited better quenching efficiency in aqueous-based than in organic-based solutions. For DPTB-MPS, the addition of 80 μM nitrobenzene, 60 μM m-nitrobenzene and 40 μM PA resulted in about 50% quenching in aqueous solutions. The quenching mechanism would be electron transfer from silanes to nitroaromatics. This work provides a basis for designing organic-silanes with "abnormal" but useful optical properties and FL probes with AIEE properties for the detection of nitroaromatics.