a b s t r a c tThis paper reports the development of an easy to use antibody microarray-based immunoassay platform for sensitive and selective duplex detection of PSA (prostate specific antigen) and hK2 (human kallikrein 2). Using PDMS wells in a 3 Â 9 array on epoxy-coated glass slides 27 duplex immunoassays can be performed in parallel. Automated microarraying provided fast and reproducible antibody arraying in each assay well. To achieve highly sensitive and selective detection of each biomarker, we evaluated and optimized the density of each of the immobilized capture antibodies. The assay platform showed a limit of detection (LOD) of each biomarker (PSA and hK2) of less than 10 pg/mL and a dynamic range of 10 4 -10 5 orders of magnitude. Neither the PSA nor the hK2 antibody array showed any cross-reaction against each others target proteins or other plasma proteins. These results emphasize the importance of density optimization of capture antibody on the surface in order to achieve a sensitive and selective multiplex immunoassay.

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