Ultrashort separation length homogeneous electrophoretic immunoassays using on-chip discontinuous polyacrylamide gels

To realize efficient homogeneous electrophoretic immunoassays, we introduce discontinuous polyacrylamide gels that enable quantitative assay completion in separation lengths as short as 350 mum in <10 s. The discontinuous cross-linked gels reduce the required electrophoretic separation lengths and thereby significantly reduce the required applied electrical potentials needed to achieve 100's V/cm electric field strengths for rapid electrophoresis. To optimize the discontinuous polyacrylamide gel assay format, we demonstrate development of a two-color homogeneous electrophoretic immunoassay for concurrent quantitation of C reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) for monitoring inflammatory response. To achieve necessary pore-size control at the gel discontinuity, an optimized mask-based fabrication protocol is introduced. The fabrication approach improves electrophoretic separations using the discontinuous separation gels by eliminating two confounding phenomena: (1) smaller than desired pores at the discontinuity which result in undesired physical exclusion of large-species and (2) an associated transition from small to large pores aft of the interface which acts to "destack" analyte bands during the separation. With the use of the optimized discontinuous separation gels, both assays were linear and quantitative over a two-log detection range, with a lower limit of detection of 11 ng/mL for CRP and 40 ng/mL for TNF-alpha. An optimal single-point detector location was identified by balancing the separation resolution and assay duration constraints. The ultrashort separation distance electrophoretic assays developed here provide flexibility in chip and instrument design by relaxing electrical potential requirements and expanding the possibilities for assay multiplexing, therefore addressing important design considerations when developing field-portable diagnostic assays for near-patient environments.