Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes, and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.
Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.
Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors.
A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.
Timely diagnosis of acute diseases improves treatment outcomes and saves lives, but it requires fast and precision quantification of biomarkers. Here, we report a time-resolved digital immunoassay based on plasmonic imaging of binding of single nanoparticles to biomarkers captured on a sensor surface. The real-time and high contrast of plasmonic imaging lead to fast and precise counting of the individual biomarkers over a wide dynamic range. We demonstrated the detection principle, evaluated the performance of the method using procalcitonin (PCT) as an example, and achieved a limit of detection of ∼2.8 pg/mL, dynamic range of 4.2−12500 pg/mL, for a total detection time of ∼25 min.
Measuring molecular binding is critical for understanding molecular-scale biological processes and screening drugs. Label-free detection technologies, such as surface plasmon resonance (SPR), have been developed for analyzing analytes in their natural forms. However, the specificity of these methods is solely relying on surface chemistry and has often nonspecific binding issues when working with samples in complex media. Herein, we show that single-molecule-based measurement can distinct specific and nonspecific binding processes by quantifying the mass and binding dynamics of individual-bound analyte molecules, thus allowing the binding kinetic analysis in complex media such as serum. In addition, this single-molecule imaging is realized in a commonly used Kretschmann prism-coupled SPR system, thus providing a convenient solution to realize high-resolution imaging on widely used prism-coupled SPR systems.
Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.
Membrane proteins play vital roles in cellular signaling processes and serve as the most popular drug targets. A key task in studying cellular functions and developing drugs is to measure the binding kinetics of ligands with the membrane proteins. However, this has been a long-standing challenge because one must perform the measurement in a membrane environment to maintain the conformations and functions of the membrane proteins. Here, we report a new method to measure ligand binding kinetics to membrane proteins using self-assembled virion oscillators. Virions of human herpesvirus were used to display human G-protein-coupled receptors (GPCRs) on their viral envelopes. Each virion was then attached to a gold-coated glass surface via a flexible polymer to form an oscillator and driven into oscillation with an alternating electric field. By tracking changes in the oscillation amplitude in real-time with subnanometer precision, the binding kinetics between ligands and GPCRs was measured. We anticipate that this new label-free detection technology can be readily applied to measure small or large ligand binding to any type of membrane proteins and thus contribute to the understanding of cellular functions and screening of drugs.
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