A galactose polyacrylate-based hydrogel scaffold for the detection of cholera toxin and staphylococcal enterotoxin B in a sandwich immunoassay format

A galactoside-based polyacrylate hydrogel was used as a scaffold to immobilize antibodies for the development of a sandwich immunoassay to detect cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The hydrogel possesses large pores and simulates a solution-like environment allowing easy penetration of large biomolecules. Highly crosslinked hydrogels containing pendant amine or carboxyl functionalities were polymerized through a free-radical polymerization process. Covalent crosslinking of the antibodies on hydrogel films was accomplished using a homobifunctional crosslinker or carbodiimide chemistry. Utilizing the two different crosslinking methodologies, our results demonstrated the effectiveness of repetitive additions of crosslinker reactant into a single location on the gel surface. This approach in fact increased the amount of immobilized antibody. Patterned arrays of the immobilized antibodies for sandwich immunoassay development were achieved using a PDMS template containing micro-channels. This template provided a suitable means for applying reagents in multiple cycles. Fluorescence and three-dimensional (3D) imaging by confocal microscopy and laser scanning confocal microscopy of Cy3-labeled anti-CT and/or Cy3-anti-SEB tracer molecules provided qualitative and quantitative measurements on the efficiency of protein immobilization, detection sensitivity and signal-to-noise ratios. As a result of using the galactose polyacrylate-base hydrogel as a platform for immunoassay development, we have successfully been able to achieve low limits of detection for SEB and cholera toxins (1.0 ng mL −1). Repetitive additions (>3 cycles) of the crosslinker and antibody have also shown a dramatic increase in the immobilization of antibody resulting in improved immunoassay sensitivity. Fluorescence signal-to-noise ratios using the hydrogel-based immunoassays have been observed as high a 40:1.