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Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications
Journal article   Peer reviewed

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

Paul T Charles, Ellen R Goldman, Jermain G Rangasammy, Caroline L Schauer, Mu-San Chen and Chris R Taitt
Biosensors & bioelectronics, v 20(4), pp 753-764
01 Nov 2004
DOI: https://doi.org/10.1016/j.bios.2004.04.007
PMID: 15522590
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Reproducibility of Results Antigen-Antibody Complex - analysis Sensitivity and Specificity Biosensing Techniques - instrumentation Biosensing Techniques - methods Protein Array Analysis - instrumentation Fluorescence Polarization Immunoassay - instrumentation Fluorescence Polarization Immunoassay - methods Equipment Design Equipment Failure Analysis Hydrogels - chemistry Protein Array Analysis - methods
We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.

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Web of Science research areas
Biophysics
Biotechnology & Applied Microbiology
Chemistry, Analytical
Electrochemistry
Nanoscience & Nanotechnology
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