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A fluorescence quenching based gene assay for Escherichia coli O157:H7 using graphene quantum dots and gold nanoparticles
摘要： A fluorometric assay is described for highly sensitive quantification of Escherichia coli O157:H7. Reporter oligos were immobilized on graphene quantum dots (GQDs), and quencher oligos were immobilized on gold nanoparticles (AuNPs). Target DNA was co-hybridized with reporter oligos on the GQDs and quencher oligos on AuNPs. This triggers quenching of fluorescence (with excitation/emission peaks at 400 nm/530 nm). On introducing target into the system, fluorescence is quenched by up to 95% by 100 nM concentrations of target oligos having 20 bp. The response to the fliC gene of E. coli O157:H7 increases with the logarithm of the concentration in the range from 0.1 nM to 150 nM. The limit of detection is 1.1 ± 0.6 nM for n = 3. The selectivity and specificity of the assay was confirmed by evaluating the various oligos sequences and PCR product (fliC gene) amplified from genomic DNA of the food samples spiked with E. coli O157:H7.
关键词： Food pathogens,Static quenching,fliC gene,Selectivity,Fluorophore,Quencher,Real samples
更新于2025-09-11 14:15:04
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Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore
摘要： Purpose: Imaging techniques for highly specific detection of cancer cells in vivo can have applications ranging from preclinical drug discovery studies to clinical cancer diagnosis and surgical therapy. Although fluorescence imaging using cancer-targeted antibodies has shown promise, nonspecific probe accumulation in tissue results in significant background fluorescence, reducing detection sensitivity using traditional intensity–based continuous-wave (CW) fluorescence imaging. Here we demonstrate that fluorescence lifetime (FLT) imaging can provide significant tumor contrast enhancement over CW intensity in preclinical models of human breast cancer. Experimental Design: Mice bearing MDA-MB-231 tumors were injected with anti-EGFR antibody conjugated to the fluorescent dye IRDye 800CW (anti-EGFR-800). Time domain fluorescence imaging was performed in vivo and in situ up to 48 hours after dye injection. Results: Mice injected with anti-EGFR-800 showed a significantly longer FLT (0.7 ± 0.03 ns) compared with the FLT of nonspecific probe uptake in liver (0.63 ± 0.05 ns), providing a dramatic improvement in sensitivity and specificity compared with CW intensity. IgG antibody–conjugated IRDye 800CW did not show an increased FLT compared with normal tissue, suggesting that the FLT increase of anti-EGFR-800 in tumors was associated with receptor expression. Using serial surgery, we show that FLT allows the detection of smaller residual tumors in the surgical bed than possible using CW intensity. Conclusions: Our data suggest that FLT can significantly enhance tumor contrast using fluorescently labeled antibodies, thereby accelerating the efficient clinical application of these probes for margin assessment in image-guided surgery and for highly specific detection of tumor receptors in vivo.
关键词： near-infrared fluorophore,EGFR,fluorescence lifetime imaging,image-guided surgery,tumor contrast
更新于2025-09-11 14:15:04
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Highly fluorescent materials derived from ortho-vanillin: Structural, photophysical electrochemical and theoretical studies
摘要： Small-molecule organic fluorophores are highly in demand attributed to their extensive prospective in material and biomedical applications. Particularly, luminescent π-conjugated organic molecules that possess an efficient solid-state emission are excellent candidates for optoelectronic devices. Focusing on high demand of organic fluorophores, we herein report the synthesis of three organic fluorescent materials derived from o-vanillin, viz. an ester (F1), an azine (F2) and an azo dye (F3). Interestingly, F2 exhibited very intense luminescence in its aggregate phase due to the restriction in intra-molecular rotation (RIR), as demonstrated by solution thickening studies. Further, its Single Crystal X-ray Crystallography (SCXRD) study suggested bathochromic shift in fluorescence from solution to solid phase was confirmed by its thin-film emission spectrum, which evidences the formation of J-aggregates. The observed RIR, development of J-aggregates and high conjugation in F2 impart an excellent fluorescence in its aggregated state. Thin films of both F2 and F3 on ITO plates exhibited a bathochromic shift with a deep orange to red photoluminescence on UV excitation. Furthermore, the morphological characterization revealed the presence of clear dense grains in case of F2 and F3, while the DSC analysis indicated phase transitions of all the derivatives. As seen from dielectric measurement studies, the azo dye F3 exhibited the highest dielectric constant among the three derivatives. The electronic and photophysical data based on Density Functional Theory (DFT) and Time Dependent-DFT (TD-DFT) calculations are in agreement with the experimental results. All the above data clearly advocate that, the synthesized fluorophoric o-vanillin derivatives are excellent candidates for electro-optical devices.
关键词： Fluorophore,Electrochemical,X-ray Crystallography,Aggregation,Dielectric,DFT,Azine
更新于2025-09-10 09:29:36
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Dynamic imaging of small molecule-induced protein-protein interactions in living cells with a fluorophore phase transition-based approach
摘要： Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule-induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule-induced protein-protein association and dissociation in living cells. Here we have applied fluorophore phase transition-based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule-induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drugs (IMiDs)-induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiDs analog (e.g. CC-885)-induced PPI between cereblon and GSPT1. SPPIER can be modified to image small molecule-induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intensive brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.
关键词： GFP,Small molecules,SPPIER,Living cells,Fluorophore phase transition,Protein-protein interactions
更新于2025-09-10 09:29:36
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A Single Fluorophore Ratiometric Nanosensor Based on Dual-Emission DNA-templated Silver Nanoclusters for Ultrasensitive and Selective Pb2+ Detection
摘要： Lead ion (Pb2+) is known as a major non-degradable environmental pollutant and is quite harmful to human health. In our investigation, a novel ratiometric fluorescent nanosensor with DNA-AgNCs as single fluorophore has been successfully constructed for ultrasensitive and specific detection of Pb2+. The single-strand DNA templated silver nanoclusters present green emission, which can be converted into red emission after approaching a specific DNA segment by forming duplex with its complementary DNA strands (ds-DNA-AgNCs). The ds-DNA-AgNCs contains rA cleavage site of Pb2+-dependent DNAzyme configuration. In the presence of Pb2+, the specific DNA segment would be released from the ds-DNA-AgNCs, resulting in discoloration of DNA-AgNCs from red to green. Since Pb2+ can successively cleave the rA site, the signal change from red to green emission is amplified. Thus, benefiting from the Pb2+-dependent DNAzyme and the charming properties of DNA-AgNCs, the proposed platform exhibits a good linear relationship from 0.001 nM to 10 nM with a detection limit of 1.0 pM for Pb2+ determination, which is lower than most of the reported Pb2+ biosensors. Moreover, this sensitive ratiometric fluorescent probe exhibits excellent selectivity toward Pb2+ detection in real samples, such as lake water, tap water, and human serum samples, illustrating the huge potential applications for complicated samples in the future.
关键词： Pb2+ detection,Ratiometric fluorescent nanosensor,DNA-templated silver nanoclusters,Single fluorophore
更新于2025-09-10 09:29:36
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Fabrication of a new peroxyoxalate chemiluminescence system containing the Cy3 fluorophore and its application to carvedilol detection
摘要： Cy3 is a dye that has been widely applied in fluorescence microscopy and single molecule detection due to its low hydrophobicity and intense fluorescence emission. As a new application, this study proposes using Cy3 as the fluorophore of a peroxyoxalate chemiluminescence system. The system relies on the oxidative reaction of bis-(2,4,6-trichlorophenyl) oxalate (TCPO) with H2O2 in the presence of imidazole, as the catalyst, to produce dioxetane via a chemically initiated electron exchange luminescence (CIEEL) process, excite Cy3 by the excess energy of the dioxetane and permit Cy3 to emit yellow light. The chemiluminescence behavior of the system is investigated using different concentrations of TCPO, imidazole, the oxidant and Cy3 and by considering non-linear least squares curve fitting of the obtained intensity–time profiles into the pooled intermediate model. The evaluated kinetic parameters include experimental and theoretical maximum intensity, the rise and fall rate constants of the chemiluminescence process, experimental and theoretical time required to reach the maximum intensity and total emission yield. Finally, the developed system is adopted to detect a drug, i.e., carvedilol, in different pharmaceuticals. Under the optimal conditions, the system can provide a linear response for determination of 3 × 10? 7 to 3 × 10? 5 M carvedilol (r > 0.9869, n = 6) and a detection limit of 1.21 × 10? 7 M. The relative standard deviation of the detection results for six repetitive determinations is below 3.47% and the obtained recoveries are 99.6% and 101.9%.
关键词： Cy3,Fluorophore,Carvedilol,Peroxyoxalate chemiluminescence
更新于2025-09-10 09:29:36
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SkImager: a concept device for <i>in-vivo</i> skin assessment by multimodal imaging
摘要： A compact prototype device for diagnostic imaging of skin has been developed and tested. Polarized LED light at several spectral regions is used for illumination, and round skin spot of diameter 34 mm or 11 mm is imaged by a CMOS sensor via cross-oriented polarizing filter. Four consecutive imaging series are performed: (1) RGB image at white LED illumination for revealing subcutaneous structures; (2) four spectral images at narrowband LED illumination (450, 540, 660, and 940 nm) for mapping of the main skin chromophores and diagnostic indices; (3) video-imaging under green LED illumination for mapping of skin blood perfusion; (4) autofluorescence video-imaging under UV (365 nm) LED irradiation for mapping of the skin fluorophores. Design details of the device and its software as well as preliminary results of clinical tests are presented.
关键词： optical skin diagnostics,multimodal imaging,skin chromophore and fluorophore mapping
更新于2025-09-09 09:28:46
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Fluorescence analysis of urine and its potential for ovarian cancer screening
摘要： Early diagnosis of ovarian cancer could lead to decreased mortality. We assessed the possible use of urine autofluorescence analysis in its diagnostics and screening. We analysed urine from 42 healthy volunteers, 35 patients with benign, and 36 patients with malignant ovarian tumors. Synchronous fluorescence spectra with a 70 nm wavelength difference were recorded for (1:1 – 1:1024) urine dilutions. Concentration matrices of synchronous spectra (CMSS) were used to classify samples into tested groups. CMSS analysis allowed us to distinguish patients with malignant tumors from healthy ones with a high sensitivity (91.67 %) and specificity (100 %), a positive predictive value (PPV) 100 % and a negative predictive value (NPV) 93.33 %. However, discrimination between benign and malignant ovarian tumors was weaker, with sensitivity 86.11 %, specificity 77.14 %, PPV 79.49 % and NPV 84.38 %. Fluorescence intensity and the position of peaks at 330 and 360 nm were found to be associated with the grade and stage, suggesting that different fluorescent metabolites may prevail at different stages of the disease. CMSS analysis of urine provides an alternative for ovarian cancer screening method development and could be used as a diagnostic test to detect the recurrence of the disease after therapy.
关键词： native fluorophore,ovarian cancer,urine,adnexal tumor,metabolite,synchronous fluorescence spectrum
更新于2025-09-09 09:28:46
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Multichromophoric sugar for fluorescence photoswitching
摘要： A multichromophoric glucopyranoside 2 bearing three dicyanomethylenepyran (DCM) fluorophores and one diarylethene (DAE) photochrome has been prepared by Cu(I)-catalyzed alkyne–azide cycloaddition reaction. The fluorescence of 2 was switched off upon UV irradiation, in proportion with the open to closed form (OF to CF) conversion extent of the DAE moiety. A nearly 100% F?rster-type resonance energy transfer (FRET) from all three DCM moieties to a single DAE (in its CF) moiety was achieved. Upon visible irradiation, the initial fluorescence intensity was recovered. The observed photoswiching is reversible, with excellent photo resistance.
关键词： energy transfer,monosaccharide,photochromism,click chemistry,fluorophore
更新于2025-09-04 15:30:14
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Modulating the cellular uptake of fluorescently tagged substrates of prostate-specific antigen before and after enzymatic activation
摘要： A series of peptides based on the prostate-specific antigen (PSA) specific sequence histidine-serine-serine-lysine-leucine-glutamine were functionalised with an anthraquinone fluorophore at the C-terminal residue side chain using the copper(I) catalyzed azide-alkyne cycloaddition reaction. The effect of incorporating a negatively charged N-terminal tetra-glutamic acid group to the substrate and the effect of masking the negatively charged C-terminal carboxylic acid functionality of the substrate was investigated using confocal fluorescence microscopy in two cell lines (DLD-1 and LnCaP). The addition of a tetra-glutamic acid group to the N-terminus of the intact sequence was shown to reduce cellular uptake of the intact substrate prior to activation by PSA. In contrast, masking the C-terminal carboxylic acid group of the substrate as a methyl ester was shown to improve cellular uptake of the peptide fragment after activation by PSA. The synthesized C-terminal methyl ester substrates with the anthraquinone attached to the side chain were confirmed to be cleaved by PSA in LC-MS analysis, and the cytotoxicity of the substrates was shown to increase in the presence of PSA, consistent with cleavage and uptake of the C-terminal fragment. The results indicate that C- and N- terminal functionalisation of peptide substrates targeting PSA can be used to modulate the cellular uptake of peptides before and after enzymatic activation, and may thus be an important consideration in the design of tumour activated prodrugs.
关键词： peptide substrates,cellular uptake,tumour activated prodrugs,prostate-specific antigen,anthraquinone fluorophore
更新于2025-09-04 15:30:14