Fully automated microfluidic system for multiplexed immunoassay

Abstract

We present an automated microfluidic immunoassay system for multiplex detection of cancer biomarker. Magnetic bead was used as solid support to immobilize a capture antibody. Oligonucleotide-linked immunosorbent assay (OLISA) based on a detection antibody tethered with an oligonucleotides and RNase H-mediated signal amplification was used for biomarker detection. To prevent diffusion of fluorescent molecules, microwell structure using a pneumatic valve system was actively formed. Our microfluidic device is composed of multiple polydimethylsiloxane (PDMS) layers; top layer as pneumatic control channel, bottom layer as fluid flow channel, and 25 um PDMS membrane. Computerized numerical control (CNC) machine with poly(methyl methacrylate) (PMMA) plate to fabricate master mold (top and bottom layer). To demonstrate our microfluidic system performance, two cancer biomarker; alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were simultaneously detected in our microfluidic device. Whole assay process was automatically performed within 2h through virtual instrument software architecture (VISA) that pre-programed. Fluorescent intensity was linearly proportional to biomarker concentration, and the limit of detection (LOD) of AFP and PSA were 36.22 pg/ml and 17.35 pg/ml, respectively. Our microfluidic device showed not only multiplex and highly sensitive detection ability, but reusable capability through bead trapping and releasing with an electromagnet.