Analysis of Ion Channel Dynamics by Single Molecule Tracking in Live Cells

Protein dynamics play an important role in signal transduction in association with their activation mechanisms, functions, and so on. Single molecule tracking technique have been widely used for investigating diffusion behavior of protein in live cells. Especially membrane proteins have been studied since they are important to understand cell responding to the surroundings. However, not many ion channel proteins such as AMPAR, NMDAR, etc., have been monitored due to their complex system. Here, we observed ionotropic glutamate receptor in neuroblastoma SH-SY5Y cell using single-molecule imaging. The diffusion coefficient of the receptor was significantly low because the receptor has four subunits (tetramers) and each subunit possesses a four transmembrane domain. Furthermore, we also analyzed interaction between subunits using single molecule tracking, further investigation of the protein dynamics of the membrane proteins such as ionotropic receptors, their structure and protein activation mechanism will be possible.1

Observation of reactions in single molecules/nanoparticles using light microscopy

Recent techniques for direct observation of single molecules or nanoparticles provide methodologies for imaging the activation sites of heterogeneous catalysts (spatially resolved) and observing intermediates that are not visible in the ensemble average (temporally resolved). Accordingly, the primary challenge for related experiments is obtaining sufficient spatial and temporal resolutions for microscopic observation of the chemical reaction of interest. This review discusses recent advances in fluorescence-for example, total internal reflection fluorescence (TIRF)-and dark-field microscopy-for example, imaging plasmonic probes-used for observing organic, inorganic, and biological reactions. The following key factors for microscopic observation of chemical reactions are discussed: (1) design of the chemical reaction and probe, (2) selection of microscope based on reaction's temporal information, and (3) use of machine learning algorithms to analyze the sequence imaging data. This review summarizes experimental techniques and detailed examples of reactions at the single molecule and nanoparticle level. Furthermore, it discusses avenues of development. These observations can guide the development of new and systematic methodological approaches for investigating important unsolved problems in chemistry. © 2022 Korean Chemical Society, Seoul & Wiley-VCH GmbH.FALS

Poly(ethylene glycol)- and carboxylate-functionalized gold nanoparticles using polymer linkages: Single-step synthesis, high stability, and plasmonic detection of proteins

Gold nanoparticles with suitable surface functionalities have been widely used as a versatile nanobioplatform. However, functionalized gold nanoparticles using thiol-terminated ligands have a tendency to aggregate, particularly in many enzymatic reaction buffers containing biological thiols, because of ligand exchange reactions. In the present study, we developed a one-step synthesis of poly(ethylene glycol) (PEG)ylated gold nanoparticles using poly(dimethylaminoethyl methacrylate) (PDMAEMA) in PEG as a polyol solvent. Because of the chelate effect of polymeric functionalities on the gold surface, the resulting PEGylated gold nanoparticles (Au@P-PEG) are very stable under the extreme conditions at which the thiol-monolayer-protected gold nanoparticles are easily coagulated. Using the solvent mixture of PEG and ethylene glycol (EG) and subsequent hydrolysis, gold nanoparticles bearing mixed functionalities of PEG and carboxylate are generated. The resulting particles exhibit selective adsorption of positively charged chymotrypsin (ChT) without nonselective adsorption of bovine serum albumin (BSA). The present nanoparticle system has many advantages, including high stability, simple one-step synthesis, biocompatibility, and excellent binding specificity; thus, this system can be used as a versatile platform for potential bio-related applications, such as separation, sensing, imaging, and assays.112151sciescopu

Single-Molecule Imaging of Membrane Proteins on Vascular Endothelial Cells

Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis. A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes state-of-the-art single-molecule imaging techniques, such as super-resolution microscopy, single-molecule tracking, and protein–protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed. © 2023 The Korean Society of Lipid and Atherosclerosis.TRU

Plasmonic Monitoring of Catalytic Hydrogen Generation by a Single Nanoparticle Probe

Plasmonic nanostructures such as gold nanoparticles are very useful for monitoring chemical reactions because their optical properties are highly dependent upon the environment surrounding the particle surface. Here, we designed the catalytic structure composed of platinized cadmium sulfide with gold domains as a sensitive probe, and we monitored the photocatalytic decomposition of lactic acid to generate hydrogen gas in situ by single-particle dark-field spectroscopy. The plasmon band shift of the gold probe throughout the reaction exhibits significant particle-to-particle variation, and by simulating the reaction kinetics, the rate constant and structural information (including the diffusion coefficient through the shell and the relative arrangement of the active sites) can be estimated for individual catalyst particles. This approach is versatile for the monitoring of various heterogeneous reactions with distinct components at a single-particle level

Sunlight-Activatable ROS Generator for Cell Death Using TiO2/c-Si Microwires

Solar-driven reactive oxygen species (ROS) generation is an attractive disinfection technique for cell death and water purification. However, most photocatalysts require high stability in the water environment and the production of ROS with a sufficient amount and diffusion length to damage pathogens. Here, a ROS generation system was developed consisting of tapered crystalline silicon microwires coated with anatase titanium dioxide for a conformal junction. The system effectively absorbed >95% of sunlight over 300-1100 nm, resulting in effective ROS generation. The system was designed to produce various ROS species, but a logistic regression analysis with cellular survival data revealed that the diffusion length of the ROS is similar to 9 mu m, implying that the most dominant species causing cell damage is H2O2. Surprisingly, a quantitative analysis showed that only 15 min of light irradiation on the system would catalyze a local bactericidal effect comparable to the conventional germicidal level of H2O2 (similar to 3 mM)

Monitoring Rotation Dynamics of Membrane Protein in Live Cells

Dynamic behavior of membrane protein provides critical information in molecular and cellular mechanisms. To have access to the mobility of a membrane protein, single-particle tracking has been advanced for the microscopic mechanism understandings. Among various molecular motions, however, only the lateral motion of the protein has been monitored due to the lack of in situ imaging tool enabling observation for rotation and vibration. Here, we developed plasmonic nanoparticles which can monitor rotational diffusion dynamics as well as lateral motion. This nanoparticle probe allows direct evidence and quantitative analysis of rotation dynamics, and furthermore, observation of conformation changes of proteins and the protein-protein interactions in live cells. This study provides an insight into the molecular mechanism regarding the intracellular signaling process.1

In Situ Monitoring of Individual Plasmonic Nanoparticles Resolves Multistep Nanoscale Sulfidation Reactions Hidden by Ensemble Average

The generation of complex nanostructures to obtain novel characteristics and improved performance has been achieved by coupling multiple nanoscale reactions. Because reactions at the nanometer scale directly govern the morphology of nanostructures, understanding the reaction mechanism is critical to precisely control the morphology and, eventually, the physicochemical properties of the materials. However, because of the ensemble-average effect, investigating the reaction mechanism at the bulk level does not provide sufficient information. In this study, we investigated the overall sulfidation reaction mechanism that occurred on individual silver nanocubes in real time at high temperature. Using the single-particle dark-field imaging technique, three discrete steps of the sulfidation reaction were clearly resolved in the profiles of the plasmon peak shift and the intensity change of individual particles according to time progress: (I) reactant diffusion to the silver surface by passing through a ligand barrier, (II) silver sulfide formation by C-S bond cleavage of cysteine molecules, and (III) diffusion of silver atoms in the silver sulfide layer until the complete formation of silver sulfide. By a combination of simulation and control experiments, physical constants were derived for each step, which is completely hidden in the ensemble measurements. Each individual nanoparticle exhibited a large variation of physical values, such as the reaction rate constant and diffusivity, mainly resulting from the intrinsic structural heterogeneity. Dark-field microscopy image processing based on surface plasmon scattering would be helpful to analyze the reaction kinetics and understand the reaction mechanisms of the numerous multistep nanoscale reactions in real time with high spatial and temporal resolutions under actual reaction conditions. © 2019 American Chemical Society.1