Ultrashort Separation Length Homogeneous Electrophoretic Immunoassays Using On-Chip Discontinuous Polyacrylamide Gels

摘要

To realize efficient homogeneous electrophoretic immu-nnoassays, we introduce discontinuous polyacrylamide gelsnthat enable quantitative assay completion in separationnlengths as short as 350 µmin 10 s. The discontinuousncross-linked gels reduce the required electrophoreticnseparation lengths and thereby significantly reduce thenrequired applied electrical potentials needed to achieven100’s V/cm electric field strengths for rapid electrophore-nsis. To optimize the discontinuous polyacrylamide gelnassay format, we demonstrate development of a two-colornhomogeneous electrophoretic immunoassay for concur-nrent quantitation of C reactive protein (CRP) and tumornnecrosis factor- (TNF- ) for monitoring inflammatorynresponse. To achieve necessary pore-size control at thengel discontinuity, an optimized mask-based fabricationnprotocol is introduced. The fabrication approach improvesnelectrophoretic separations using the discontinuous sepa-nration gels by eliminating two confounding phenomena:n(1) smaller than desired pores at the discontinuity whichnresult in undesired physical exclusion of large-species andn(2) an associated transition from small to large pores aftnof the interface which acts to “destack” analyte bandsnduring the separation. With the use of the optimizedndiscontinuous separation gels, both assays were linearnand quantitative over a two-log detection range, with anlower limit of detection of 11 ng/mL for CRP and 40 ngmL for TNF- . An optimal single-point detector locationnwas identified by balancing the separation resolution andnassay duration constraints. The ultrashort separationndistance electrophoretic assays developed here providenflexibility in chip and instrument design by relaxingnelectrical potential requirements and expanding the pos-nsibilities for assay multiplexing, therefore addressingnimportant design considerations when developing field-nportable diagnostic assays for near-patient environments.