To realize efficient homogeneous electrophoretic immunoassays, we optimize photopatterned polyacrylamide gels that enable quantitative assay completion in separation lengths as short as 350 um in < 10 s. The demonstrated separation length is an order of magnitude shorter than the separation length previously reported for on-chip gel electrophoresis and two orders of magnitude shorter than that achieved using capillary electrophoresis. The required separation lengths translate to less than 3.5 V for assay operation as compared to hundreds and thousands of volts currently in use for homogeneous immunoassays. A discontinuous gel sieving matrix architecture introduced in our work forms a key step towards realizing battery-operated electrophoresis systems for quantitation of protein biomarkers in near-patient environments.
In our implementation of microfluidic western blotting assay, we developed a novel strategy for on-chip protein renaturation which utilizes electrophoretic separations to isolate denaturing detergents from proteins. Furthermore, photopatterning of polyacrylamide gels within a 2D chamber enables seamless integration of protein renaturation with upstream sizing and downstream immunoaffinity recognition. The entire assay is completed within 3 minutes in a single device as compared to conventional western blotting assays which require hours to a day, multiple pieces of instruments, and frequent human intervention. Given the appreciable assay speed, this assay may comprise part of a tool set necessary to accelerate biomarker discoveries for personalized medicine.