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Green Fluorescent Protein-Based Glucose Indicators Report Glucose Dynamics in Living Cells
摘要： Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as "Green Glifons", to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50 μM, 600 μM and 4,000 μM), producing a ~7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 β cells. We propose that these indicators will facilitate and contribute to in vivo and multi-color imaging of energy metabolism.
关键词： biosensors,artificial sweeteners,dual-color imaging,C. elegans,live cell imaging,glucose,blood glucose level,fluorescent protein
更新于2025-11-21 11:24:58
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Discovery of Turn-On Fluorescent Probes for Detecting Bcl-2 Protein
摘要： Bcl-2 (B cell lymphoma-2 gene) family proteins play a central role in regulating programmed cell death. In cancer, anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Mcl-1, are overexpressed. However, there are few developed labeling techniques for tracing the dynamic processes of Bcl-2. To study the physiological process of Bcl-2 protein, a novel series of small molecule fluorescent probes (1-3) were designed and evaluated for their labeling properties. It’s interesting that our probes can be applied to identify tumor tissue slices and differentiate the tumor and normal tissues effectively, a feature that renders these probes compatible for future cancer diagnosis in clinical practice.
关键词： fluorescent probes,cell imaging,Bcl-2 protein
更新于2025-11-21 11:24:58
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Aggregation-Induced Emission: Lighting Up hERG Potassium Channel
摘要： Based on the scaffold of astemizole and E-4031, four AIE light-up probes (L1–L4) for Human Ether-a-go-go-Related Gene (hERG) potassium channel were developed herein using AIE fluorogen(TPE). These probes showing advantages such as low background interference, superior photostability, acceptable cell toxicity, and potent inhibitory activity, which could be used to image hERG channels at the nanomolar level. These AIE light-up probes hoped to provide guidelines for the design of more advanced AIE sensing and imaging hERG channels to a broad range of applications.
关键词： hERG channel,pharmacophore,fluorophore,cell imaging,AIE light-up probes
更新于2025-11-21 11:24:58
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MnO2 Nanosheet-mediated Ratiometric Fluorescence Biosensor for MicroRNA Detection and Imaging in Living Cells
摘要： MicroRNA (miRNA) plays significant roles in cell proliferation, differentiation and apoptosis, and has been considered to be valuable biomarker for cancer. Accurate and sensitive detection of miRNA is crucially significant for cancer diagnosis and treatment. Here, a MnO2 nanosheet-mediated ratiometric fluorescence biosensor was designed for miRNA detection and imaging in living cells. It contained MnO2 nanosheets acting as DNA carrier, and fluorescent donor (FAM)-labeled hairpin H1 (recognition probe) and fluorescent acceptor (TAMRA)-labeled hairpin H2 (amplification probe). When the biosensor entered cell by endocytosis, MnO2 nanosheets were degraded to Mn2+ via intracellular glutathione (GSH) and the adsorbed hairpins H1 and H2 were released. The intracellular target miRNA-21 hybridized with the recognition unit of H1 to initiate catalyzed hairpin assembly (CHA) and a large amount of H1-H2 duplexes were produced. This brought fluorescent donor FAM and fluorescent acceptor TAMRA into close proximity to produce fluorescence resonance energy transfer (FRET), inducing a ratiometric fluorescent response (donor signal decreased and acceptor signal enhanced) for miRNA-21 detection. Furthermore, this method could be applied to differentiate the expression levels of miRNA-21 in HeLa, HepG-2 and L02 cells. These results indicated that the proposed method possessed great potential in the early diagnosis of miRNA-related diseases.
关键词： MicroRNA detection,MnO2 nanosheets,Ratiometric,Cell imaging
更新于2025-11-21 11:24:58
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Perylenequinone-based “turn on” fluorescent probe for hydrogen sulfide with a high sensitivity in living cells
摘要： Hydrogen sulfide (H2S) is a kind of gaseous signal molecule in many physiological processes. In order to detect H2S, a novel “turn on” fluorescent probe 6,12-dihydroxyperylene-1,7-dione (DPD) was designed and synthesized. The probe DPD is fluorescence silence, while the addition of H2S induces an obvious green fluorescence with an obvious color change from dark blue to yellow-green. The probe shows excellent selectivity, fast response (2.5 minutes) and linear curve (0-90 μM) in wide effective pH range (4-10). Competition experiments are also revealed in corresponding studies and the detection limit is 3.6 μM. The response mechanism is proved to be the reduction of the probe by H2S, which is confirmed by 1H NMR. Furthermore, through the fluorescence turn-on signal toward H2S in Hela cells, probe DPD was successfully applied to monitor H2S in living Hela cells.
关键词： hydrogen sulfide,probe,fluorescence imaging,cell imaging,perylenequinone
更新于2025-11-21 11:08:12
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A fluorescent probe based on tetrahydro[5]helicene derivative with large Stokes shift for rapid and highly selective recognition of hydrogen sulfide
摘要： In this work, we have designed and synthesized a dinitrobenzene-sulfonate tetrahydro[5]helicene (H-DNP) as an effective fluorescent probe for detection of hydrogen sulfide (H2S). Upon the addition of H2S, a significant fluorescence enhancement (75-fold) at 495 nm can be observed with a distinct color change from colorless to yellow. Additionally, H-DNP shows low background spectroscopic signal, large Stokes Shift up to ~140 nm, good sensitivity, rapid response time less than 2 min, low detection limit (48 nM) and high selectivity towards common bio-thiols (Cysteine, Homocysteine and Glutathione). Compared with the previous dinitrophenoxy tetrahydro[5]helicene, this probe has shorter response time and lower detection limit. Most importantly, this probe H-DNP has low toxicity to cells and excellent cell permeability, which can be applied to visualize H2S in living cells.
关键词： Fluorescence,Cell imaging,Probe,4-dinitrobenzene,Helicene,2,Hydrogen sulfide
更新于2025-11-21 11:08:12
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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 novel peptide-based fluorescent chemosensor for Cd(II) ions and its applications in bioimaging
摘要： Nowadays, it is of great significance to develop a novel fluorescent chemosensor for Cd(II) ions detection with cost-effective, rapid, facile and applicable to environment and biological milieus. Herein, we report a new peptide-based fluorescent chemosensor DSC (Dan-Ser-Cys-NH2) based on dipeptide (Ser-Cys-NH2) conjugated with dansyl group, which was synthesized using solid phase peptide synthesis (SPPS) technology. As designed, DSC exhibited fluorescent “turn-on” response for Cd2+ in 100% aqueous solution over a wide range of pH values based on photoinduced electron transfer (PET). The stoichiometry binding of DSC and Cd2+ was determined to be 2:1 by Job’s plot and ESI-MS analysis. Furthermore, DSC showed highly sensitive for Cd2+ and a low detection limit of 13.8 nM. What's more, DSC has cell permeability and low cytotoxicity, and fluorescence imaging experiments demonstrated that DSC was capable of monitoring Cd2+ in living HK2 cells by confocal microscopy.
关键词： Fluorescent chemosensor,Cell imaging,Cd(II) ions,Dansyl group,Dipeptide
更新于2025-11-21 11:08:12
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A sequential and reversibility fluorescent pentapeptide probe for Cu(II) ions and hydrogen sulfide detections and its application in two different living cells imaging
摘要： In this study, we report a sequential and reversibility fluorescent probe (DP5) based on pentapeptide conjugated with dansyl groups using the solid phase peptide synthesis (SPPS) technology. DP5 showed immediate “turn off” response toward Cu2+ ions at an excitation wavelength of 330 nm with detection limits of 23.5 nM. The 2:1 binding ratio between DP5 and Cu2+ were confirmed using Job's plot method and fluorescence titration study, and DP5-Cu complex was observed with an association constant of 6.76 × 108 M?2. As designed, DP5-Cu complex as a promising analytical probe exhibited highly selective for H2S detection in aqueous solutions. The detection limit for H2S was obtained to be 17.2 nM, and lower than EPA and WHO guidelines. In addition, the reversibility and cyclicity were imparted to the DP5 during the detection of Cu2+ and H2S, and cycle effect is very good. Furthermore, DP5 displayed better biocompatibility and low biotoxicity, and sequential fluorescence “on-off-on” responses of DP5 to Cu2+ and H2S were successfully applied in two different living cells.
关键词： Cu2+ ions,Pentapeptide,Fluorescent probe,Cell imaging,Hydrogen sulfide,Aqueous solutions
更新于2025-11-21 11:08:12
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AIE active fluorescent organic nanoaggregates for selective detection of phenolic-nitroaromatic explosives and cell imaging
摘要： Development of organic nanoparticles with high fluorescence, good biocompatibility along with strong resistance to photobleaching through simple synthetic routes is important for diverse applications such as sensing and bioimaging. Herein, we present the development of a pyrene excimer nanoaggregate which shows aggregation induced emission (AIE) effect in a solvent mixture of THF and water. The pyrene based fluorescent probe, dimethyl-5-(pyren-1-ylmethyleneamino)isophthalate (5-DP) was synthesized through a simple single step condensation reaction from inexpensive reagents. The photophysical studies of nanoaggregated system further corroborates the AIE active behavior of 5-DP probe at different water fractions (?w = 0% to 90%), where the hydrogen bonding interaction between imine and water molecules led to suppression of photoinduced electron transfer (PET) inducing significant enhancement in fluorescence. The highly photostable nanoaggregates were explored as a selective fluorescence “turn off” sensor for phenolic nitroaromatics and the chemo-selectivity was highly pronounced for 2,4,6-trinitrophenol (picric acid), that showed efficient quenching in aqueous as well as solid phase, with a detection limit of 10 nM in aqueous medium. The quenching efficiency of the nanoaggregates can be ascribed to a combination of factors including efficient fluorescence resonance energy transfer, inner filter effect and coulombic interaction between picric acid and the aggregated probe molecules. Further, random aggregation of the pyrene derivative could be controlled for the formation of fluorescent spherical nanoparticles using Pluoronics P-123 block copolymers as encapsulating agents. The resulting composite could be used as a neoteric cell imaging probe with significantly less cytotoxicity, thus showing their potential biological applications.
关键词： aggregation induced emission,electron transfer,explosive detection,cell imaging,Fluorescent organic nanoaggregates
更新于2025-11-21 11:03:13