Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications.
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.
Charles
Paul T
PT
The US Naval Research Laboratory, Center for Bio/Molecular Science and Engineering (Code 6900), 4555 Overlook Avenue, SW, Washington, DC 20375, USA. ptc@cbmse.nrl.navy.mil
Goldman
Ellen R
ER
Rangasammy
Jermain G
JG
Schauer
Caroline L
CL
Chen
Mu-San
MS
Taitt
Chris R
CR
D000936:Antigen-Antibody Complex / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation* / Q000379:methods; D020100:Hydrogels / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000936:Antigen-Antibody Complex; D020100:Hydrogels