Immunoassay on a power-free microchip with laminar flow-assisted dendritic amplification

We demonstrate a rapid (<30 min) and ultrasensitive (sub-picomolar) immunoassay on a microchip which needs no external power sources for fluid transport. We previously reported a rapid immunoassay of human C-reactive protein (CRP) on the power-free microchip with moderate sensitivity, i.e., a limit of detection (LOD) in sub-nanomolar range, due to the lack of signal amplification. In the current work, we have improved the LOD by 3 orders of magnitude by employing dendritic amplification (DA) methods. Specifically, a sandwich immunocomplex with a biotinylated secondary antibody was constructed on the inner surface of the microchannel as described in the previous report. Onto the immunocomplex, solutions of FITC-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) were supplied to grow a dendritic structure. First, we alternately supplied the two solutions for layer-by-layer growth up to three layers. As a result, we obtained an LOD of 0.21 pM with a CRP sample volume of 1.0 microL and assay time of approximately 30 min under an ordinary fluorescence microscope. Second, to reduce the number of incubation steps, we have devised a new DA method: laminar flow-assisted dendritic amplification (LFDA). In this method, F-SA and B-anti-SA were simultaneously and continuously supplied from two laminar streams formed by a Y-shaped microchannel. The immunoassay with the LFDA for 10 min (total assay time of approximately 23 min) with a CRP sample volume of 0.5 microL yielded an LOD of 0.15 pM, which is equivalent to 75 zmol. The combination of the power-free microchip and the LFDA will provide a new opportunity for ultrasensitive point-of-care testing.