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.
In a series of recently published works, we demonstrated that the plasmon-assisted microscopy of nano-objects (PAMONO) technique can be successfully employed for the sizing and quantification of single viruses, virus-like particles, microvesicles and charged non-biological particles. This approach enables label-free, but specific detection of biological nano-vesicles. Hence, the sensor, which was built up utilizing plasmon-assisted microscopy, possesses relative versatility and it can be used as a platform for cell-based assays. However, one of the challenging tasks for such a sensor was the ability to reach a homogeneous illumination of the whole surface of the gold sensor slide. Moreover, in order to enable the detection of even relatively low concentrations of nano-particles, the focused image area had to be expanded. Both tasks were solved via modifications of previously described PAMONO-sensor set ups. Taken together, our latest findings can help to develop a research and diagnostic platform based on the principles of the surface plasmon resonance (SPR)-assisted microscopy of nano-objects.
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