SURFACE PLASMON RESONANCE AS A CENTRAL TECHNIQUE TO INVESTIGATE THE DIFFUSION COEFFICIENT OF ORGANIC/INORGANIC MOLECULES AND THE VISCOSITY OF BODY/EYE FLUIDS

Abstract

Purpose : We developed a novel easy user-interface Surface Plasmon Resonance (SPR) based dispersion analysis that precisely measures the diffusion coefficients (DC) and interactions of several biomolecules of eye fluids under health and disease status. The purpose of this study was to specifically tune the SPR microfluidic components to assay:
1) the DC of organic molecules and K_d (training of method)
2) biomolecular targets of interest in neurodegeneration (tau proteoforms conjugated with ubiquitin [Ub])
3) the presence of Ub-proteins in the aqueous humor (AH)
4) the viscosity of AH isolated from glaucoma and control subjects (under-way)

Methods : Microfluidic components size (tubing, loops, valves among the others) of the SPR were controlled while exploiting the repertoire of functionalization of the gold sensor, which constitutes the central part of the sensing system, to test data points (1-4). The DC of Tau-proteoforms conjugated to mono-Ub at different positions by disulfide coupling chemistry (e.g., Ub-Tau 254, 311, 353) have been calculated. Gold-sensor has been functionalized with anti-Ub antibodies (polyclonal) to address the aim #2 (n=5).

Results : For all small biomolecules (glycine, ATP, carnosine, etc.) tested, the calculated DC is in accordance with the values reported by canonical methods and the data are superimposable with those obtained by an in silico simulation (i.e. DC_glycine = 0.997 ± 0.208 * 10^-9 m²/s, DC_ATP = 0.717 ± 0.214 * 10^-9 m²/s DC_carnosine= 0.543 ± 0.048 * 10^-9 m²/s). Immobilization of anti-Ub antibodies has allowed to uncover the presence of Ub-species in AH further coupled with immunostaining by Western blotting.

Conclusions : This novel SPR application allows to measure the DC of different organic molecules as well as of macromolecular substrates of pathogenic relevance (Ub-Tau species). The method may be applied to the investigation of Ub-proteins in the AH. Tuning of the SPR method is expected to allow the determination of the DC of biological fluids in cross-sectional studies (e.g. glaucoma vs cataract) providing a novel chemical-physical approach to the study of these fluids.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.