Electrokinetic concentration on a microfluidic chip using polyelectrolytic gel plugs for small molecule immunoassay

We have devised a new method to enhance the sensitivity of competitive immunoassay using electrokinetic concentration near a pair of highly charge-selective polymer [poly-2-acrylamido-2-methyl-1-propanesulfonic acid (pAMPSA)] plugs on a microfluidic chip. The polyelectrolytic gel, which was photopolymerized in a microfluidic channel network, served as the effective charge-selective extractor to sophisticatedly control the ion distribution. In this system, the fluorescent indicators on magnetic microbeads dispersed in the sample were spontaneously displaced by unlabeled target molecules and then electrokinetically preconcentrated in a single spot on the microfluidic chip. The locally preconcentrated fluorescent indicators were detected by laser-induced fluorescence. As a proof-of-concept, the competitive displacement assay of unlabeled 1 nM biotin was conducted to observe ca. 2000-fold enrichment within 3 min. In addition to the sensitive assessment of the unlabeled small target molecules, the proposed immunoassay system also showed good selectivity for biotin analogs such as biocytin, 2-iminobiotin, desthiobiotin.