A flow enzyme-linked immunosorbent assay (ELISA) method based on light absorption by enzymatically generated aniline oligomer in the presence of horseradish peroxidase (HRP), H2O2, and aniline is proposed. Aniline oligomer is rapidly formed through the polymerization reaction via the enzymatic reaction, and its fast reaction rate is beneficial for flow ELISA. An anti-3-phenoxybenzoic acid monoclonal antibody (mAb) was produced by mice, and was used for the flow competitive ELISA for the determination of 3-phenoxybenzoic acid (3PBA), which was performed on an acrylic plate having a Y-shaped channel. ABS resin beads (d = 1 mm) were filled in the channel to increase the surface area for the adsorption of the mAb. A clank-type detection chamber (optical length: 1 cm) made of polydimethylsiloxane (PDMS) containing carbon black, which can significantly decrease light scattering, was fabricated with a 3D printer. The PDMS detection chamber was connected to the outlet of the acrylic flow chip with a tube. A blue LED was used as a light source for the flow ELISA. The inhabitation concentration at 50% and the detection range (absorbance change from 90 to 10%) for the proposed flow competitive ELISA were 0.5 ppm and 0.05–5 ppm, respectively. We also performed the flow competitive ELISA in an artificial and real urine, and no significant matrix effect of the urine samples on the ELISA was found.
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