This paper presents a simple fluid handling technique for microchip immunoassay. Necessary solutions were sequentially injected into a microchannel by air-evacuated poly(dimethylsiloxane), and were passively regulated by capillary force at the inlet opening. For heterogeneous immunoassay, microchips are potentially useful for reduction of sample consumption and assay time. However, most of the previously reported microchips have limitations in their use because of the needs for external power sources for fluid handling. In this paper, an on-chip heterogeneous immunofluorescence assay without such an external power source is demonstrated. The microchip consisting of poly(dimethylsiloxane) (PDMS) and glass has a simple structure, and therefore is suitable for single-use applications. Necessary solutions were sequentially injected into a microchannel in an autonomous fashion with the power-free pumping technique, which exploits the high solubility and the rapid diffusion of air in PDMS. For deionized water, this method yielded flow rates of 3-5 nL s-1 with reproducibility of 4-10%. The inlet opening of the microchannel functioned as a passive valve to hold the solution when the flow was finished. Rabbit immunoglobulin G (rIgG) and human C-reactive protein (CRP) were detected using the microchannel walls as reaction sites. With the sample consumption of 1 microL and the assay time of approximately 20 min including the antibody immobilization step, the sandwich immunoassay methods for rIgG and CRP exhibited the limits of detection of 0.21 nM (0.21 fmol) and 0.42 nM (0.42 fmol), respectively.

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.