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Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration
摘要： Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are F?rster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.
关键词： FRET-based sensors,potassium ion imaging,single fluorescent protein sensors,intracellular K+ dynamics,genetically encoded indicators
更新于2025-11-21 11:24:58
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Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells
摘要： Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.
关键词： Multiple Gaussian-functions analysis,FRET imaging,Single living cell,Stoichiometry,Hetero-oligomeric complex
更新于2025-11-21 11:24:58
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Measuring the interaction of transcription factor Nrf2 with its negative regulator Keap1 in single live cells by an improved FRET/FLIM analysis
摘要： Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. Detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualise and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured Instrument Response Function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
关键词： FRET,live cell imaging,fluorescence lifetime,FLIM,sfGFP,protein-protein interaction,global binning,Keap1,Instrument Response Function,Nrf2
更新于2025-11-21 11:08:12
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A new FRET-based ratiometric fluorescence probe for hypochlorous acid and its imaging in living cells
摘要： A novel ratiometric fluorescence probe for hypochlorous acid was constructed by coumarin and pyridinium fluorophore based on the Forster resonance energy transfer (FRET) and intramolecular charge transfer (ICT) platform. In this ICT/FRET system, the energy transfer efficiency is high to 94.3%. Moreover, the probe could respond to hypochlorous acid with high selectivity and sensitivity, and exhibited a large Stokes shift. It was interesting to find that the probe could recognize hypochlorous acid via a new mechanism, in which the a -position of carbonyl group was oxidized to form a diketone derivative. More importantly, the probe was successfully applied to the ratiometric imaging of both exogenous and endogenous hypochlorous acid in living RAW 264.7 cells, with low toxicity and high photo-stability.
关键词： New reaction mechanism,FRET,Hypochlorous acid,Fluorescence probe,Cell image,Ratiometric
更新于2025-11-21 11:08:12
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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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Broad-Spectrum Tunable Photoluminescent Material Based on Cascade Fluorescence Resonance Energy Transfer between Three Fluorophores Encapsulated within the Self-Assembled Surfactant Systems
摘要： A broad spectrum tunable photoluminescent material with dual encryption based on a two-step Fluorescence Resonance Energy Transfer (FRET) between Pyrene (Py), Coumarin480 (Cou480) and Rhodamine6G (R6G) in micelles of SDS and bmimDS is presented. The phenomenon is achievable due to the encapsulation of the fluorophores within these micelles. The transfer of energy as FRET between the pair Py and Cou480 showed ON at 336 nm and OFF at 402 nm in contrast to the FRET observed between the pair Cou480 and R6G that showed ON at 402nm and OFF at 336 nm. However, the transfer of energy as FRET occurs from Py to R6G in the presence of Cou480 when excited at 336 nm, thereby making it a chain of three fluorophores with Cou480 acting as a relay fluorophore receiving energy from Py and transferring it to R6G. The different FRET scenarios between the three fluorophores in micelles provide a window for the generation of a matrix of colors, which occupies a significant 2D area in the chromaticity diagram, having potential applications in security printing. The different fluorophoric ratios generate different colors based on their individual photonic emissions and the FRET processes taking place between them. Writing tests were carried out using varied ratios of the fluorophores in the micellar systems producing different colored outputs under the UV light with insignificant visibility under the white light. We envision that this as-discovered three fluorophoric FRET system could form the basis for the future development of multi-FRET light-harvesting devices and anti-counterfeiting security inks based on much simpler non-covalent interaction aided encapsulation of the fluorophores within the self-assembled soft systems.
关键词： micelles,security printing,Rhodamine6G (R6G),SDS,Pyrene (Py),Coumarin480 (Cou480),bmimDS,Fluorescence Resonance Energy Transfer (FRET),photoluminescent material
更新于2025-11-19 16:46:39
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Excimer based FRET between non-FRET pair flourophores aided by the aromatic moiety of anionic surfactants: An experimental observation
摘要： A new FRET pair has been reported based on the apperance of excimeric emission and hence significant spectral overlap between an otherwise non-FRET pair fluorophores viz pyrene and rhodamine 6G in the anionic sodiumdodecylbenzenesulfonate (SDBS) micellar system. The occurence of FRET based on such modulation of spectral overlap integral has not been dealt with in detail in comparison to the other FRET parameters like distance between the fluorophores and orientation. Due to the presence of benzene ring in the SDBS micellar system the formation of excimers is augemented due to the π?π interactions between the pyrene molecules and the benzene units of the surfactants. The excimeric pyrene has a bathochromically shifted peak which overlaps with the absorption peak of rhodamine 6G thereby giving rise to energy transfer as FRET between these otherwise non-FRET pair molecules. The efficiency of FRET between the pyrene and rhodamine 6G can be modulated by the degree of spectral overlap between the excimeric peak of pyrene and the absorption peak of rhodamime6g. All the experiments were carried in the Sodiumdodecylsulfate (SDS) micellar system also, which unlike SDBS does not possess any benzene unit, in order to compare the results of excimer formation as well the FRET process.
关键词： rhodamine 6G,Pyrene,overlap integral,Excimer,FRET
更新于2025-11-19 16:46:39
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FRET-based dual channel fluorescent probe for detecting endogenous/exogenous H2O2/H2S formation through multicolor images
摘要： We have developed a FRET-based fluorescent probe (PHS1) as a combination of two different fluorophores (coumarin and naphthalimide); which can detect both exogenous and endogenous H2S and H2O2 in live cells through multicolor images. The precise overlap between UV-absorption of naphthalimide and the emission band of coumarin in probe PHS1 allows the acquisition of the self-calibrated information of dual analytes through FRET-based imaging. The UV–Vis absorption (λabs 390 nm) and fluorescence emission (λem 460 nm) of probe PHS1 in the presence of H2O2 are increased ∽35- fold and ∽15-fold respectively. It also allows the estimation of the levels of H2S through enhancement of emission intensity at 550 nm. The probe PHS1 exhibits high stability against various analytes, including various pH (4–9.5). The cell viability assay data indicate that the probe is not harmful to the cancer cells. The nontoxic nature of the probe PHS1 encourages application for cancer cell labeling. The probe PHS1 can detect the level of endogenous H2O2, H2S, and H2O2/H2S in cancer cells through blue, green and FRET-based green channel imaging. PHS1 is a unique probe, has potential application for diagnosing cancer by providing information on the level of dual analytes (H2S, H2O2) in cancer cells.
关键词： FRET-based fluorescent probe,Naphthalimide,Endogenous H2O2,Endogenous H2S,Coumarin
更新于2025-11-19 16:46:39
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Graphene quantum dots and carbon nano dots for the FRET based detection of heavy metal ions
摘要： We demonstrate the development of a FRET based novel optical sensing system for the efficient detection of heavy metal pollutants. The studied sensing system is comprised of graphene quantum dots (GQDs) as donor and carbon nano dots (C-Dots) as an acceptor component. When these fluorescent nano-dots are within the FRET distance, fluorescence of the donor GQDs is quenched by the non-radiative energy transfer to acceptor C-Dots. Fluorescence lifetime is measured by time resolved photo-luminescence spectroscopic study to validate the FRET efficacy of the mix dot based sensor system. Upon gradual addition of heavy metals like arsenic (As5+) and mercury (Hg2+) into this sensor system, a significant amount of reduction in the investigated FRET signal is experienced. The detailed mechanisms of the molecular interactions between GQDs and C-Dots are thoroughly studied by UV–Visible absorption, infrared, steady state and time resolved spectroscopy.
关键词： FRET,Carbon nano dot,Metal ions,Sensor,Graphene quantum dot
更新于2025-11-19 16:46:39
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Glutaraldehyde non-conjugated chitosan polymer fluorophores for selective determination of picric acid via fluorescence resonance energy transfer strategy
摘要： Water-dispersed glutaraldehyde (GA) non-conjugated chitosan polymer fluorophores (GCPF) with quantum yield of 16 % is synthesized by stirring chitosan and GA for 6 h at room temperature in the present work. It is a facile and mild method and fluorescent GCPF can be stabled for two months. Owing to the spectral overlap of fluorescent spectrum of GCPF and absorption spectrum of picric acid (PA), a novel sensitive fluorescent method using fluorescent GCPF for PA detection from 10 nM to 50 μM via fluorescence resonance energy transfer (FRET) strategy is established. The distance between donor of GCPF and acceptor of PA (R0 value) is calculated to be 3.5 nm. FRET method using fluorescent GCPF possesses high sensitivity (LOD of 2.8 nM), and selectivity and fast response within 2 min. Moreover, fluorescent GCPF is also utilized in visual analysis of PA using cotton swabs. Fluorescence quenching effect can be observed by eyes irradiated with 365 nm ultraviolet light at cotton swabs and using GCPF solid on quartz glasses, which paves an effect and wide way for the application of fluorescent GCPF in our daily life.
关键词： Glutaraldehyde non-conjugated chitosan polymer fluorophores (GCPF),picric acid (PA),fluorescence resonance energy transfer (FRET),glutaraldehyde (GA),chitosan
更新于2025-11-14 15:23:50