Enzyme-linked immunosorbent assay utilizing thin-layered microfluidics

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) is required for on-site clinical diagnosis. Previously, a microfluidic ELISA in which antibody-immobilized beads are packed in a microchannel for a high surface-to-volume (S/V) ratio was developed, but utilizing beads led to complicated fluidic operation. Recently, we have reported nanofluidic ELISA that utilizes antibody-immobilized glass nanochannels (10²-10³ nm) to achieve a high S/V ratio without beads, enabling even single-molecule detection, but it is not applicable to clinical diagnosis owing to its fL sample volume, much smaller than the nL-μL sample volume in clinical diagnosis. Here, we propose an antibody-immobilized, thin-layered microfluidic channel as a novel platform. Based on the method of nanofluidic ELISA, the channel width was expanded from 10³ nm to 10⁰ mm to expand the volume of the reaction field to 10² nL, while the channel depth (10³ nm) was maintained to retain the high S/V ratio. A device design which incorporates a taper-shaped interface between the thin-layered channel and the microchannel for sample injection was proposed, and the uniform introduction of the sample into the high-aspect-ratio (width/depth ∼ 200) channel was experimentally confirmed. For the proof of concept, a thin-layered ELISA device with the same S/V ratio as the bead-based ELISA format was designed and fabricated. By measuring a standard C-reactive protein solution, the working principle was verified. The limit of detection was 34 ng mL^-1, which was comparable to that of bead-based ELISA. We believe that the thin-layered ELISA can contribute to medicine and biology as a novel platform for sensitive and rapid ELISA.