Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Abstract

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.