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Ultrasensitive tantalum oxide nano-coated long-period gratings for detection of various biological targets
摘要： In this work we discussed a label-free biosensing application of long-period gratings (LPGs) optimized in refractive index (RI) sensitivity by deposition of thin tantalum oxide (TaOx) overlays. Comparing to other thin film and materials already applied for maximizing the RI sensitivity, TaOx offers good chemical and mechanical stability during its surface functionalization and other biosensing experiments. It was shown theoretically and experimentally that when RI of the overlay is as high as 2 in IR spectral range, for obtaining LPGs ultrasensitive to RI, the overlay’s thickness must be determined with subnanometer precision. In this experiment the TaOx overlays were deposited using Atomic Layer Deposition method that allowed for achieving overlays with exceptionally well-defined thickness and optical properties. The TaOx nano-coated LPGs show RI sensitivity determined for a single resonance exceeding 11,500 nm/RIU in RI range nD=1.335-1.345 RIU, as expected for label-free biosensing applications. Capability for detection of various in size biological targets, i.e., proteins (avidin) and bacteria (Escherichia coli), with TaOx-coated LPGs was verified using biotin and bacteriophage adhesin as recognition elements, respectively. It has been shown that functionalization process, as well as type of recognition elements and target analyte must be taken into consideration when the LPG sensitivity is optimized. In this work optimized approach made possible detection of small in size biological targets such as proteins with sensitivity reaching 10.21 nm/log(ng/ml).
关键词： protein detection,label-free biosensing,optical fiber sensor,tantalum oxide,bacteria detection,long-period grating,atomic layer deposition
更新于2025-11-28 14:23:57
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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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Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
摘要： Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.
关键词： Phospholipid nanodiscs,G-protein coupled receptors,Conformational dynamics,β2 adrenergic receptor,Single-molecule fluorescence
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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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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A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation
摘要： Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identi?ed changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS. Our ?ndings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for dif?cult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
关键词： ocular pharmacology,retinal degeneration,GPCR,phototransduction,rod photoreceptors,rhodopsin,misfolded protein
更新于2025-11-21 11:20:48
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Modulating Protein-Protein Interactions with Visible-Light Responsive Peptide Backbone Photoswitches
摘要： Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of these complex cross-talks has recently attracted attention, even as a novel therapeutic strategy. Here, we describe the synthesis and characterization of two visible-light responsive peptide backbone photoswitches based on azobenzene derivatives to exert optical control over protein-protein interactions (PPI). Our novel peptidomimetics undergo fast isomerization and reversibility with low photochemical fatigue under alternatively blue/green-light irradiation cycles. Both bind in the nanomolar rage to the protein of interest. Importantly, our best peptidomimetic displays a clear difference between isomers in its protein-binding capacity and, in turn, in its potential to inhibit enzymatic activity via PPI disruption. In addition, crystal structure determination, docking and MD calculations give a molecular interpretation and open new avenues in the design and synthesis of future photoswitchable PPI modulators.
关键词： protein-protein interactions,photopharmacology,visible-light irradiation,azobenzene,photoswitches
更新于2025-11-21 11:20:42
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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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[Methods in Molecular Biology] Plant Long Non-Coding RNAs Volume 1933 (Methods and Protocols) || Trimolecular Fluorescence Complementation (TriFC) Assay for Visualization of RNA-Protein Interaction in Plants
摘要： RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.
关键词： RNA-protein interaction,Long noncoding RNA,TriFC,Tobacco transient expression,In vivo visualization
更新于2025-11-21 11:08:12
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Silicon nanowire luminescent sensor for cardiovascular risk in saliva
摘要： Cardiovascular diseases are some of the today major cause of death in the world. C-reactive protein (CRP) is well known as the main biomarker related to cardiovascular risk and heart attack occurrence. The standard CRP analyses are performed in a hospital or in a biochemical laboratory with blood analysis after a long chemical and labelling preparation that require expert personnel. In this scenario, a health care analysis that can be performed by the same patient at his own home appears extremely revolutionary. In this paper, the study of an innovative sensing platform based on the luminescence at room temperature of silicon nanowires (NWs) is reported. This NWs sensor is label-free and does not require a chemical treatment of the analyte, is strongly selective to the CRP demonstrating a femtomolar limit of detection and a wide operating range. This proposed silicon sensing platform can be realized with an industrial compatible approach and permits to reveal the strategic CRP level in saliva in order to prevent a heart attack, with great advantages for the patient.
关键词： Cardiovascular risk,Saliva,C-reactive protein,Silicon nanowires,Luminescent sensor
更新于2025-11-21 11:01:37