- 标题
- 摘要
- 关键词
- 实验方案
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Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications
摘要： Protein post-translational modifications (PTMs) are typically enzyme-catalyzed events generating functional diversi?cation of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable ?uorescent “signal-on” and “signal-o?” detection scheme in the peptide-break PTM detection. Taking the advantage of time-resolved ?uorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing di?erent soluble Eu3+-signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu3+-reporter. The concept functionality was demonstrated with four Eu3+-signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-o? readout at 615 nm or time-resolved Fo?rster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional ?uorescence-based single-label detection concept not limited only to PTM detection.
关键词： time-resolved fluorescence,signal-off,high-throughput screening,peptide-break technology,protein post-translational modifications,FRET,signal-on
更新于2025-11-19 16:56:35
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Photo-physical properties of substituted 2,3-distyryl indoles: Spectroscopic, computational and biological insights
摘要： The structural dependence of the photo-physical properties of substituted 2,3-distyryl (23DSI) indoles were studied using several spectroscopic techniques including steady-state UV-VIS spectroscopy, steady-state fluorescence spectroscopy, steady-state excitation spectroscopy, time correlated single photon counting (TCSPC) spectroscopy, and time-resolved fluorescence upconversion spectroscopy (TRFLS). Each of 23DSI derivatives investigated showed distinct fluorescence emission and UV-VIS spectra, indicating strong structural dependence of the emission and the excitation. The UV-VIS spectra of the 23DSI derivatives showed three main identical absorption bands with minor deviations in the absorbance caused by substituent groups on the distyryl rings. The time-resolved fluorescence up-conversion studies indicated that the fluorescence undergoes a mono-exponential decay whereas the calculated fluorescence lifetime showed relatively short fluorescence lifetimes of approximately 1 ns. All of the 23DSI derivatives showed two-photon absorption upon direct excitation of 1.6 W laser pulses at 800 nm. These studies suggest that the substituents, attached to distyryl core, are capable of boosting or hindering fluorescence intensities by distorting the π-conjugation of the 23DSI molecule. Our studies showed that 23DSI (p-F) has the highest fluorescence emission quantum yield. Theoretical calculations for the ground state of 23DSI derivatives confirmed differences in electron densities in 23DSI derivatives in the presence of different substituent attachments. The excellent fluorescence emission, high fluorescence quantum yield and two-photon absorption properties of these 23DSI molecules make them attractive candidates for potential applications in the fields of biological imaging, biomedicine, fluorescent probes, and photodynamic inactivation (PDI). B. subtilis samples, treated with micro molar solutions of 23DSI (p-OCH3) and 23DSI (p-CH3), showed very effective photodynamic inactivation (PDI) upon irradiation with white light.
关键词： Two photon absorption,Time-resolved fluorescence upconversion laser spectroscopy,Photodynamic inactivation,Gaussian calculations,Photo-physical properties,Light-activation
更新于2025-11-14 15:26:12
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A comprehensive photophysical and NMR investigation on the interaction of a 4-methylumbelliferone derivative and cucurbit[7]uril
摘要： The host-guest interaction of a 7-hydroxycoumarin (3-[2-(diethylamino)ethyl]-7-hydroxy-4-methylcoumarin, 3ED4MU) with cucurbit[7]uril, CB7, was investigated in aqueous solution. From the steady-state fluorescence Job plot and 1H NMR spectroscopy the formation of a 1:1 inclusion complex, with a binding constant of 1.75×105 M?1 was obtained. Time-resolved fluorescence data in water, DMSO and dioxane for 3ED4MU and for umbelliferone (UM, here taken has the model compound) show that the decays are double or triple-exponentials mirroring the presence of excited neutral (N*), anionic (A*), tautomeric (T*) and zwiterionic (T*’) species (the latter only present in 3ED4MU). In the time-resolved experiments, the interaction of 3ED4MU with CB7 is mirrored by an increase of the pre-exponential factor associated to the zwiterionic species (thus giving support for the stabilization of this species when interacting with CB7) and a dramatic change in the pKa* value (from ~ 0.5 to ~8), associated to a displacement of the water environment probed by 3ED4MU.
关键词： Time-resolved fluorescence,Host-guest chemistry.,Binding constant,Fluorescence,Hydroxycoumarins,Cucurbit[7]uril
更新于2025-09-23 15:23:52
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[Methods in Enzymology] || Fluorescence Depolarization Kinetics to Study the Conformational Preference, Structural Plasticity, Binding, and Assembly of Intrinsically Disordered Proteins
摘要： Fluorescence depolarization kinetics measured by the time-resolved fluorescence anisotropy decay serves as a sensitive and powerful methodology to study the conformational dynamics of macromolecules. This methodology allows us to delineate the different modes of biomolecular motional dynamics including the local, segmental, and global rotational dynamics on the timescale ranging from picoseconds to nanoseconds. In this chapter, we describe the principles and applications of this methodology to obtain unique molecular insights into the intrinsically disordered proteins (IDPs). Fluorescence depolarization kinetics, when performed in a site-specific manner, can offer a reliable tool to monitor the intrinsic backbone torsional dynamics of expanded IDPs and is capable of discerning the conformational preference of IDPs. Additionally, the time-resolved fluorescence anisotropy measurements allow us to investigate the mechanism of binding and assembly of a wide range of IDPs that are involved in crucial function and disease.
关键词： Fluorescence depolarization kinetics,conformational dynamics,time-resolved fluorescence anisotropy decay,biomolecular interactions,intrinsically disordered proteins
更新于2025-09-23 15:21:01
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Photophysical Properties of Fluorescent Self-Assembled Peptide Nanostructures for Singlet Oxygen Generation
摘要： In this work, a drug delivery system for perillyl alcohol based on the peptide self-assembly containing 3-(2-benzothiazolyl)-7-(diethylamino)coumarin (C6) as a fluorescent additive is obtained, and its photophysical characteristics as well as its release dynamics were studied by steady-state and time-resolved fluorescence spectroscopy. Results proved the dynamics of drug release from the peptide nanostructures and showed that the system formed by the self-assembled peptide and C6, along with perillyl alcohol, presents unique photophysical properties that can be exploited to generate singlet oxygen (1O2) upon irradiation, which is not achieved by the sole components. Through epifluorescence microscopy combined with time-correlated single photon counting fluorescence spectroscopy, the release mechanism was proven to occur upon peptide structure interconversion, which is controlled by environmental changes.
关键词： photophysical properties,perillyl alcohol,drug delivery system,time-resolved fluorescence spectroscopy,fluorescent additive,peptide self-assembly,epifluorescence microscopy,singlet oxygen generation
更新于2025-09-23 15:21:01
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Digital scanned laser lighta??sheet fluorescence lifetime microscopy with widea??field timea??gated imaging
摘要： We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a sub-resolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimeters and can be utilized as a powerful and robust method for biomedical research, for example as a readout of protein-protein interactions via F?rster resonance energy transfer.
关键词： Fluorescence lifetime imaging,light-sheet fluorescence microscopy,time-resolved fluorescence imaging
更新于2025-09-23 15:19:57
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Optical properties and zeta potential of carbon quantum dots (CQDs) dispersed nematic liquid crystal 4a?2- heptyl-4-biphenylcarbonitrile (7CB)
摘要： In this work, carbon quantum dots (CQDS) of three concentrations 0.1, 0.2 and 0.5 wt% have been dispersed in nematic liquid crystal 4-Heptyl-4-biphenylcarbonitrile (7CB). The results on the optical properties and zeta potential of pure and CQDs dispersed nematic 7CB have been reported. It has been observed that the dispersion of CQDs in host nematic matrix induces quenching of photoluminescence and reduces the Ultra-Violet-Visible (UV–Vis) absorbance of pure nematic at a lower wavelength. From the result of fluorescence decay profile, the fluorescence life time of pristine and dispersed nematic systems have been obtained. No remarkable changes in nematic lifetime in the presence of CQDs have been observed, which confirm that no energy is transferred from nematic to CQDs. Further, it has been found that due to dispersion of CQDs the numerical values of zeta potential are increased for the dispersed systems with 0.1 and 0.2 wt% concentrations of CQDs as compared to pure 7 CB. This result demonstrates that the study of zeta potential provides useful information on the stability and aggregation of the CQDs dispersed nematic systems.
关键词： Carbon quantum dots,UV–Vis absorbance,Zeta potential,Nematic liquid crystal,Time resolved fluorescence
更新于2025-09-23 15:19:57
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Interaction of Zinc Oxide and Copper Oxide Nanoparticles with Chlorophyll: A Fluorescence Quenching Study
摘要： The present study aims to investigate the interactions of zinc oxide nanoparticles and copper oxide nanoparticles with the major photosynthetic pigment chlorophyll using ultraviolet-visible, steady state, and time resolved laser induced fluorescence spectroscopy. The steady state fluorescence measurements show that zinc oxide and copper oxide nanoparticles quench the fluorescence of chlorophyll in concentration-dependent manner. The Stern-Volmer plot for the chlorophyll-zinc oxide nanoparticles is linear, and the value of quenching constant has been observed to increase with temperature indicating the possibility of dynamic quenching. A decrease in the lifetime of chlorophyll with increase in the concentration of zinc oxide nanoparticles confirms the involvement of dynamic quenching in the chlorophyll–zinc oxide nanoparticle interaction. In the case of copper oxide nanoparticles, the Stern-Volmer plot deviates from linearity observed in the form of upward curvature depicting the presence of both static and dynamic quenching. In addition, the lifetime of chlorophyll decreases with increase in the concentration of copper oxide nanoparticles displaying the dominance of dynamic quenching in the chlorophyll-copper oxide nanoparticle interaction. The decrease observed in the value of binding constant with increasing temperature and negative values of change in enthalpy, entropy, and Gibb’s free energy indicates that van der Waal and hydrogen bonding are the prominent forces during the interaction of chlorophyll with both zinc oxide and copper oxide nanoparticles and that the process is spontaneous and exothermic. The interaction of zinc oxide and copper oxide nanoparticles with chlorophyll occurs through electron transfer mechanism. The obtained results are useful in understanding the sensitization processes involving chlorophyll and zinc oxide and copper oxide nanoparticles.
关键词： chlorophyll and nanoparticles,molecular interaction,ultraviolet-visible spectroscopy,laser-induced fluorescence spectroscopy,Attenuated total reflectance–Fourier transform infrared spectroscopy,steady state and time resolved fluorescence,X-ray diffraction
更新于2025-09-19 17:15:36
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Simultaneous detection of fumonisin B1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2) as labels
摘要： A rapid and highly sensitive time-resolved fluorescence (TRF)-based aptasensor for simultaneous recognition of mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) was developed using multi-color, Ln3+-doped time-resolved fluorescence nanoparticles (TRF-NPs) (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2 NPs) coupled with complementary strand DNA (cDNA) as luminescence probe and aptamers-conjugated amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) act as a capture probe. Under the optimized conditions, the time-resolved fluorescence intensities at 544 and 618 nm corresponded with Tb3+ and Eu3+, respectively, were used to measure FB1 (Y = 19,177.1 + (? 12,054.4)x, R2 = 0.9917) and OTA (Y = 4138.8 + (? 11,182.6)x, R2 = 0.9924), respectively. The limits of detection (LODs) for FB1 and OTA were 0.019 pg mL?1 and 0.015 pg mL?1, respectively, which were much lower than previously described methods for simultaneous recognition of mycotoxins OTA and FB1 while detection range varied from 0.0001–0.5 ng mL?1. This aptasensor was effectively applied to quantity FB1 and OTA in maize samples and results were compared with ELISA method. This is the first reported time-resolved fluorescence (TRF)-based aptasensor to detect two agriculturally important toxins in the maize. The developed aptasensor has potential to be used for detection of toxins in food safety fields.
关键词： Simultaneous detection,Aptamer,Time-resolved fluorescence nanoparticles,Mycotoxins
更新于2025-09-19 17:15:36
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Study of Interfacial Charge Transfer from an Electron Rich Organic Molecule to CdTe Quantum Dot by using Sterna??Volmer and Stochastic Kinetic Models
摘要： Photoinduced electron transfer from N-methylaniline (NMA) to a photoexcited CdTe quantum dot (QD*) is studied in toluene. The PET mechanism at low to moderate quencher (NMA) concentrations (< 0.08 M) remains mostly collisional with some contributions from QD-NMA complex formation. However, at high quencher concentrations (> 0.10 M), QDs form larger numbers of static complexes with NMA molecules leading to a steep positive deviation in the steady-state Stern–Volmer curves. An isothermal titration calorimetry (ITC) study confirms the formation of QD-NMA complexes (K ~ 150 M?1) at high quencher concentrations. Fitting our experimental data using a stochastic kinetic model indicates that the number of NMA molecules attached per QD at highest NMA concentration (~ 0.16 M) used in this study decreases from ~ 0.76 to ~ 0.47 with reducing the QD size from ~ 5.2 nm to ~ 3.2 nm. However, the PET rate increases with decreasing QD size, which is commensurate with the observation that the chemical driving force (ΔG) increases with decreasing the QD particle size. We have analyzed the PET kinetics mainly by using Stern-Volmer fittings. However, in some cases Tachiya’s stochastic kinetic model is used for stoichiometric analysis, which seems to be useful only at high quencher concentrations. The measured PET rate coefficients in all the cases are found to be at least an order of magnitude lower when compared to the diffusion-controlled rate of the reaction medium.
关键词： quantum dot,time-resolved fluorescence,photoinduced electron transfer,Stern–Volmer analysis,bimolecular quenching
更新于2025-09-19 17:13:59