High-performance low-cost antibody microarrays using enzyme-mediated silver amplification

Antibody microarrays can detect multiple proteins simultaneously, but the need for bulky and expensive fluorescence scanners limits their adaptation in clinical settings. Here we introduce a 15-plex enzyme-mediated silver enhanced sandwich immunoassay (SENSIA) on a microarray as an economic alternative to conventional fluorescence microarray assays. We compared several gold and silver amplification schemes, optimized HRP-mediated silver amplification, and evaluated the use of flatbed scanners for microarray quantification. Using the optimized assay condition, we established binding curves for 15 proteins using both SENSIA and conventional fluorescence microarray assays and compared their limits of detection (LODs) and dynamic ranges (DRs). We found that the LODs for all proteins are in the pg/mL range, with LODs for 12 proteins below 10 pg/mL. All but two proteins (ENDO and IL4) have similar LODs (less than 10-fold difference) and all but two proteins (IL1b and MCP1) are similar in DR (less than 1.5-log difference). Furthermore, we spiked six proteins in diluted serum and measured them by both silver enhancement and fluorescence detection and found a good agreement (R(2) > 0.9) between the two methods, suggesting that a complex matrix such as serum has a minimal effect on the measurement. By combining enzyme-mediated silver enhancement and consumer electronics for optical detection, SENSIA presents a new opportunity for low-cost high-sensitivity multiplex immunoassays for clinical applications.