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We developed a novel method to increase the sensitivity of standard enzyme-linked immunosorbent assay (ELISA) using a multiplexed electrokinetic concentration chip. The poly(dimethylsiloxane) (PDMS) molecular concentrator (1) was used to trap and collect charged fluorescent product of target-bound enzyme turnover reaction of ELISA that occurred in a standard 96 well plate. Detection sensitivities of both prostate specific antigen (PSA) and CA 19-9 (a human pancreatic and gastrointestinal cancer marker) ELISAs in serum are enhanced approximately 100 fold with a low CV of &lt;17%. We also integrated this method with an on-chip bead-based ELISA that lends itself toward a fully automated on-chip diagnostic device. Detection sensitivity of microfluidic bead-based CA 19-9 ELISA in serum is enhanced approximately 65 fold compared to the results without the electrokinetic accumulation step. This chip can be directly applied to enhance the readout sensitivity of a wide range of existing ELISA kits at concentrations below the current detection limit.

Increasing the Sensitivity of Enzyme-Linked Immunosorbent Assay Using Multiplexed Electrokinetic Concentrator