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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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Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-induced Hybridization Chain Reaction
摘要： On cell membrane surfaces, receptor protein dimers play a fundamental role in many signaling pathways, which are crucial for normal biological processes and cancer development. Efficient and sensitive analysis of receptor dimers in the native environment is highly desirable. Herein, we present a strategy for amplified imaging of receptor dimers in zebrafish and living cells, which relies on aptamer recognition and proximity-induced hybridization chain reaction. Taking advantages of specific aptamer recognition and enzyme-free signal amplification, this strategy is successfully applied to amplified visualize c-Met receptor dimers in the HGF-independent or -dependent manner. Therefore, the developed imaging strategy paves the way for further investigation of protein dimerization or oligomerization state of cell surface receptors and corresponding activation processes in zebrafish and living cells.
关键词： aptamer recognition,zebrafish,living cells,receptor dimers,hybridization chain reaction
更新于2025-09-10 09:29:36
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A dual-responsive fluorescent probe for detection of fluoride ion and hydrazine based on test strips
摘要： Hydrazine (N2H4) and fluoride ion (F-) are regarded as environmental pollutants and potential carcinogens. A dual-functional fluorescent probe (probe 1) was developed for both F- and N2H4 with high selectivity and sensitivity. 1 was based on nucleophilic aromatic substitution reaction for N2H4 detection and selective cleavage of 4-nitrobenzenesulphonyl group for the determination of F-. The limits of detection of probe for F- and N2H4 were 77.82 nM and 29.34 nM, respectively, which are far below the threshold limit value (TLV) of United States Environmental Protection Agency (EPA). The home-made test strips of 1 provided the positive tool for F- and gaseous N2H4 in different system. And the confocal fluorescence images indicated that 1 can quantitatively detect N2H4 in living PC12 cells. Promisingly, 1 has great prospects for N2H4 imaging and determining in living system.
关键词： Living cells,Fluoride ion,Dual-functional fluorescent probe,Hydrazine,Home-made test strips
更新于2025-09-10 09:29:36
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In Nano, Volume 12, Issue 11
摘要： Nanoprobes have become critical components of the many near-field imaging techniques developed over the past decade. These tools are typically constructed by integrating nanostructures on the tip of an optical fiber, for example, by coating them with an ultrathin layer of metal; etching with plasmonic nanoantennas; or attaching them with a single gold nanorod, semiconductor nanowire, metal nanoparticle, or photonic crystal nanocavity. The optical resonances of these materials to be concentrated into a confined region, enable light illuminating samples with nanometer resolution. However, these nanostructures are usually made with noble metals or semiconductors that lack biocompatibility and can easily rupture cells when interfacing with them. In addition, their preparation often requires sophisticated nanofabrication processes and electrochemical reactions. To avoid these issues, Li et al. (DOI: 10.1021/acsnano.8b05235) created nanoprobes made of living cells. The researchers made these tools by inserting a tapered fiber light source into a mixture of yeast and Lactobacillus acidophilus cells. Using light the researchers captured a single yeast cell onto its tip. Light conducted through that cell acted as a subsequent trapping laser beam to secure a string of connected L. acidophilus cells on top of the yeast cell. Tests showed that these nanoprobes could be used for near-field scanning imaging with a subwavelength spatial resolution to illuminate leukemia cells stained with green fluorescent protein in blood. The nanoprobes also demonstrated flexibility and deformability, bending when they contacted cells instead of rupturing them. The authors suggest that these living nanoprobes could find relevant applications in biosensing and imaging.
关键词： nanoprobes,imaging,living cells,near-field imaging,biosensing
更新于2025-09-10 09:29:36
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Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra
摘要： Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra. The SPEES-FRET method we developed here is very robust against cellular autofluoresence. SPEES-FRET method can obtain stable and accurate E and RC values for FRET tandem constructs with high or low FRET efficiency in HEK293 cells with strong autofluorescence, and can also obtain consistent results for the bright and dim cells expressing FRET constructs or co-expressing Cerulean and Venus or co-expressing CFP-Bax and YFP-Bax. Therefore, the SPEES-FRET is applicable to the quantitative FRET measurements for the live cells with strong autofluorescence, and must become a powerful and robust tool for monitoring the weak protein-protein interaction in living cells.
关键词： spontaneous excitation-emission spectra,FRET,living cells,quantitative measurement,spectral unmixing
更新于2025-09-09 09:28:46
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Visualization of Endogenous Hydrogen Sulfide in Living Cells based on Au Nanorods@Silica Enhanced Fluorescence
摘要： Hydrogen sulfide as a gas indicator molecule plays an important role in various human physiological processes. However, due to the high volatility and diffusivity of H2S in biological systems, it is very difficult to implement a precise assay for H2S detection. Compared with the destructive instrumental methods, assays based on fluorescence probes provide noninvasive and real-time detections of H2S in living cells. In this work, we presented a fluorescent nanoprobe based on dye-functionalized Au nanorods (NRs)@silica for sensitive and selective detection of H2S in vitro and living cells. With the metal enhanced fluorescence effect, the fluorescence turn-on and turn-off were controlled by the formation and disassembly of coordination compound between dyes and copper ions. Silica matrix was used to coat the Au NRs to prevent them from the biological cytotoxicity. The effects of the different distances between Au NRs and fluorophores on fluorescent enhancement were explored and approximately 5-fold fluorescence enhancement was obtained with a distance of 22 nm. A detection of limit of 17 nM was achieved. In addition, visualization of exogenous and endogenous H2S in living cells was validated.
关键词： nanoprobes,Au nanorods@silica,metal enhanced fluorescence,living cells,endogenous H2S
更新于2025-09-04 15:30:14
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Iminocoumarin-based red to near-infrared fluorescent turn-on probe with a large Stokes shift for imaging H2S in living cells and animals
摘要： The development of organic dye-based fluorescent probes for detection of H2S in living systems has attracted considerable attention in recent years. In this work, a novel tetrahydroquinoxaline iminocoumarin-based red to near-infrared (red-to-NIR) fluorescent probe was developed for detection of H2S. This probe shows a rapid and distinct red-to-NIR fluorescent turn-on detection process for H2S with high selectivity and sensitivity (the detection limit was determined to be as low as 10 nM at physiological pH). In addition, this probe exhibits a remarkable large Stokes shift (128 nm) and low cytotoxicity, and can be applied for imaging H2S in living cells and animals. All these results demonstrated that this new probe is promising for detection of H2S both in vitro and in vivo.
关键词： large Stokes shift,hydrogen sulfide,living cells and animals,near-infrared fluorescent probe,iminocoumarin
更新于2025-09-04 15:30:14
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Direct visualization of single-molecule membrane protein interactions in living cells
摘要： Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors’ dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.
关键词： co-immunoimmobilization assay,β2-AR,single-molecule visualization,living cells,membrane protein interactions,EGFR
更新于2025-09-04 15:30:14
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Fast response two-photon fluorogenic probe based on Schiff base derivatives for monitoring nitric oxide levels in living cells and zebrafish
摘要： Numerous excellent fluorogenic probes for NO detection based on NO-mediated reactions have been developed. However, some of them still suffer from limitations such as low selectivity, slow response, and a short excitation wavelength (o500 nm). Herein, a novel two-photon fluorogenic probe (XNO1) based on a Schiff base derivative has been reported for the first time. This new mechanism with a Schiff base structure as the specific response moiety towards NO endows the probe with fast responsibility, high selectivity and pH-independent properties. Furthermore, XNO1 has been demonstrated to be lysosome-targeted and to successfully monitor exogenous/endogenous NO in living cells and zebrafishes with one- and two-photon fluorescence imaging.
关键词： living cells,nitric oxide,two-photon fluorogenic probe,zebrafish,Schiff base derivatives
更新于2025-09-04 15:30:14