130 Measuring molecular binding kinetics with SPRM Impedance SPR Microscopy (SPRM) is a powerful tool for measuring the binding kinetics of ligands or drug compounds with membrane proteins, including GPCRs. It has many other unique features that lead to new applications. A recent example is the integration of SPRM with electrochemical methods for molecular binding kinetics quantification. Researchers at Arizona State University and Johns Hopkins University developed a virion oscillator technology using SPRM Impedance 1. The technology displayed (VirD) GPCRs in the herpesvirus envelope to detect ligand binding kinetics with the GPCRs with a self-assembled virion-oscillator microarray detection (Fig. 1). To measure ligand binding to the GPCRs in the virial envelopes, each oscillator consists of a VirD tethered to a SPR sensor surface with a flexible molecular linker. When applying an alternating electric field normal to the sensor surface with a three-electrode electrochemical setup, where WE, RE and CE are the working (the Au chip), quasi-reference (Ag wire) and counter electrodes (Pt coil), respectively. The virion particle is forced to oscillate with the electric field, and the oscillation amplitude is measured with a SPRM with sub-nm resolution. Upon ligand binding to the GPCRs, the oscillation amplitude changes. Therefore, by measuring the oscillation amplitude in real time with SPRM, the kinetics of ligand binding to the GPCRs is obtained. Fig. 1 Measuring ligand binding to GPCRs in their native membrane environment with SPRM. a) Herpesvirus (HSV-1) particles are transfected to display human GPCRS (VirD); b) the particles (virions) are tethered to a gold sensing surface via flexible PEG linkers to form virion oscillators; c) VirD-Oscillator microarray are driven by an alternating electrical field into oscillate, and the oscillation amplitudes are detected by a plasmonic imaging technique with sub-nm precision; d) molecular binding events change the oscillation amplitudes, allowing determination of the binding kinetics, described by association (ka ) and dissociation ( kd ) rate constants, and affinity ( KD ) with SPRM Impedance. Each virion appears as parabolic pattern formed by the scattering a propagating plasmonic wave by the virion. ka kd KD AC electric field Ligand association Ligand dissociation Oscillation amplitude Time Evanescent field Spacers Displayed GPCR proteins Small molecule ligands PEG linker HSV-1 virion Virions express human GPCRs Transfection VirD: Virion displayed membrane proteins HSV-1 vector Vectors for GPCR expression Structure of a VirD-Oscillator Binding induced oscillation amplitude change Gold coated sensor chip Light Prism camera Plasmonic detection Base line VirD-Oscillator Microarray Using SPRM Impedance, the ASU-JHU team measured the oscillation amplitude of the virions on the SPR sensor surface from the average plasmonic imaging intensity and determined the binding kinetic constants. Fig. 2 shows an example of the binding kinetics measurement between peptide ligands and different GPCRs. The measured binding kinetics are distinctively different between intended and off-target ligands. The work demonstrated that the SPRM Impedance system can provide binding kinetics constants via monitoring the plasmonic imaging intensity. Fig. 2. (a) and (b) VirD oscillators spotted on a SPR sensor chip and imaged with SPRM. Although the individual VirD oscilltors cannot be resolved, the image intensity of each spot oscillates with the applied AC electrical field (c), providing the average oscillation amplitude of the VirD oscillators. The red line in (c) is the image intensity from the background regions, which was subtracted out in data analysis. (d) and (e) show the kinetics of a peptide ligand (SPRIF14 MW=1560) binding to two different types of GPCRs (GABBR1 and NTSR1), where the solid lines are global fitting of the data to the first order kinetics. The extracted kinetic constants are shown on the graphs. Applied voltage: 0.3 V amplitude at 5 Hz. Buffer: PBS. 1 G.Z. Ma, G-D Syu, X.N. Shan, B. Henson, S.P. Wang, P. Desai, H. Zhu and N.J. Tao, “Measuring ligand binding kinetics to membrane proteins using virion nano-oscillators”, Journal of the American Chemical Society, 2018 Sep 12;140(36):11495-11501
The SPRm 200 from Biosensing Instrument Inc. is a system that seamlessly combines both optical bright-field and SPR microscopies thus enabling the unique in vitro binding kinetic measurement of individual cells in their native environment. The architecture of the system consists of using the SPR phenomenon to measure binding kinetics while also simultaneously permitting use of bright-field microscopy to study topographical and phenotypical changes in the sample (Fig. 1). Typically, the cells are cultured and fixed on a poly-L-lysine coated SPR sensor chip. The integrated optical microscope allows for visualization of the sample with x10 magnification and 1 um resolution. The SPRm 200 exceeds the performance of homemade systems by demonstrating a much larger field of view, simultaneous bright-field and SPR microscopies, and greater stability. SPRm 200 has the resolution to observe sub-cellular binding activities (Fig. 3). In conclusion, SRPm 200 is a novel instrument that seamlessly integrates bright-field and SPR microscopies into one system. This instrument enables the kinetic binding study of multiple individual cells as well as the localized responses on a single cell.
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The Surface Plasmon Resonance (SPR) Microscopy system, which combines optical microscopy with SPR, is a powerful and label-free technology for measuring the binding affinity and kinetics of membrane proteins directly on whole cells in vitro. This integrated approach enables the simultaneous analysis of phenotypic changes via brightfield imaging and molecular interactions through SPR, offering a comprehensive view of cell-surface binding events. SPR Microscopy has been replacing radioligand binding assays and has become a go-to platform in both pharmaceutical and academic research for studying the interactions of small molecules, peptides, antibodies, and proteins with membrane targets such as GPCRs, ion channels, and other difficult-to-purify membrane proteins. It supports measurements on both fixed and live cells, whether adherent or in suspension. Thanks to its high spatial resolution (~1 μm), the technology also enables real-time monitoring of nanoparticles, virus and bacterial binding activity, expanding its applications in infectious disease and microbiology research.
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SPR microscopy integrates optical imaging and SPR technology into one system. It provides high-quality data on binding kinetics and affinity of cell membrane proteins and spatial visualization of cellular structures. Real-time interactions of the drug and membrane protein can be measured in its native state without needing to extract proteins from the cell. Because of its unique measurement of cell membrane proteins on the cell, SPR microscopy has become a powerful tool that provides binding information about various drug compounds and drug efficacy. Antibody-interaction studies are also used to investigate disease pathways and targets for potential drug therapies, and much more. In addition, the system measures nanometer scale binding activities needed for the study of bacteria and virus interactions and the development of new methods for nanoparticle drug delivery.
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The 5-channel SPR instrument, when paired with the BI Autosampler, offers an ideal solution for high-throughput assay development, combining efficiency with low operating and maintenance costs. Featuring advanced BI-DirectFlow™ technology—integrated into the BI-4500 system—it ensures precise sample delivery for exceptional binding kinetic measurements. This innovative flow system, together with temperature-controlled SPR detection, provides highly sensitive and reproducible binding responses, even for low immobilization levels and small molecule targets (<100 Daltons). The system also supports compatibility with electrochemistry and chemical vapor analysis modules, enabling integrated EC-SPR and gas-SPR measurements. These capabilities make it especially well-suited for a wide range of biosensing applications.
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