A bead-based immunogold-silver staining assay on capillary-driven microfluidics

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in "bead lanes", which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using "classical" enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL^-1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL^-1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.