The Employment of the Surface Plasmon Resonance (SPR) Microscopy Sensor for the Detection of Individual Extracellular Vesicles and Non-Biological Nanoparticles.

A wide-field surface plasmon resonance (SPR) microscopy sensor employs the surface plasmon resonance phenomenon to detect individual biological and non-biological nanoparticles. This sensor enables the detection, sizing, and quantification of biological nanoparticles (bioNPs), such as extracellular vesicles (EVs), viruses, and virus-like particles. The selectivity of bioNP detection does not require biological particle labeling, and it is achieved via the functionalization of the gold sensor surface by target-bioNP-specific antibodies. In the current work, we demonstrate the ability of SPR microscopy sensors to detect, simultaneously, silica NPs that differ by four times in size. Employed silica particles are close in their refractive index to bioNPs. The literature reports the ability of SPR microscopy sensors to detect the binding of lymphocytes (around 10 µm objects) to the sensor surface. Taken together, our findings and the results reported in the literature indicate the power of SPR microscopy sensors to detect bioNPs that differ by at least two orders in size. Modifications of the optical sensor scheme, such as mounting a concave lens, help to achieve homogeneous illumination of a gold sensor chip surface. In the current work, we also characterize the improved magnification factor of the modified SPR instrument. We evaluate the effectiveness of the modified and the primary version of the SPR microscopy sensors in detecting EVs isolated via different approaches. In addition, we demonstrate the possibility of employing translation and rotation stepper motors for precise adjustments of the positions of sensor optical elements-prism and objective-in the primary version of the SPR microscopy sensor instrument, and we present an algorithm to establish effective sensor-actuator coupling.

Absolute Analysis of Nanoparticle Suspension with Surface Plasmon Microscopy.

The recently developed surface plasmon microscopy of single nanoparticles offers a powerful tool for monitoring particle transfer from bulk solution to the adsorbing surface. In the present work, the possibility of absolute analysis of suspension, based on particle transfer observation, was examined. A model based on particle diffusion in a laminar flow, which allows analytical solution of mass transport equation, was considered. It was demonstrated that direct determination of particle concentration, based on counting the particles that bind to the sensor surface, is possible. The model contains only independent, easy to determine parameters: flow cell geometry, flow rate, particle size, and liquid viscosity. For applicability of the model, particles should be irreversibly bound when touching the sensor surface. It was demonstrated that the required "perfect binding" is provided in certain electrolyte concentration range because even particles oppositely charged to the surface do not bind in pure water. If the binding probability is less than 100%, "absoluteness" of the method is not violated. It will require the extension of the transport model with the sticking coefficient, which can be also measured by the surface plasmon microscopy directly. The present work shows the feasibility of the absolute analysis. It demonstrates the great potential of the method toward the comprehensive nanoparticle analytics and investigation of binding processes.