Towards Next-Generation Proteomic Assays: Functional Materials as Sieving Matrices and Binding Scaffolds
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Towards Next-Generation Proteomic Assays: Functional Materials as Sieving Matrices and Binding Scaffolds Samuel Q. Tia1, Alex J. Hughes1, Kelly Karns1, M. Kursad Araz2, Mei He2, Dohyun Kim2 and Amy E. Herr1,2 1 University of California, Berkeley, Bioengineering, 308B Stanley Hall MC # 1762, Berkeley, CA 94720, U.S.A. 2 University of California, Berkeley/University of California San Francisco, Graduate Program in Bioengineering, Berkeley, CA 94720, U.S.A. ABSTRACT Next-generation bioanalytical approaches for protein-level measurements are advanced by the integration capacity of microfluidic design strategies, as well as the fine fluid and material control possible. Photopatterning of polymers within fluidic volumes is a key tool in the suite of technologies available for seamless integration of assay measurement modalities, as well as rapid target detection. Here, we overview recent advances in heterogeneous and homogeneous immunoassays using functional polymers, electrophoretic transport, and microdevices. INTRODUCTION PA gel methods at the macro-scale are well established and extensively used in electrophoretic protein and DNA separation studies. The hydrophilic nature and tunable pore size of PA gels make this material indispensable for electrophoretic assays run in aqueous solutions. Here we detail two major classes of protein assays that are advanced through the combination of tunable polymer materials and electrophoretic analyte transport. Chiefly, we detail homogeneous electrophoretic immunoassays (mobility shift immunoassays) and heterogeneous immunoassays in which one binding partner is immobilized on a PA gel support. Homogeneous electrophoretic immunoassays implemented in microfluidic formats are powerful techniques for the rapid quantitation of proteins in low volumes of complex biological fluids. In this technique, an antibody probe is used to specifically capture the analyte of interest and the free unbound antibody is electrophoretically separated from the bound immune complex allowing for analyte quantification. A successful electrophoretic immunoassay requires all analytes to be of the same net charge (either both positive or both negative). This limits the analytes that can be separated with this technique and most literature to date reports on a narrow range of weakly acidic proteins [1,2,3]. The development of a highly alkaline microfluidic immunoassay capable of separating analytes irrespective of their isoelectric (pI) point would eliminate the need to predict complex and antibody pI points and would expand the range of proteins detectable with this powerful technique. One route to developing PA gels bearing custom functionalization is via incorporation of a streptavidin linkage on acrylamide monomers. When streptavidin-acrylamide (SA) conjugates are included with acrylamide monomer in a precursor solution, they become incorporated to the backbone of the PA chain during polymerization. This SA can then be linked to any biotinylated antibody. By controlling the relative concentration of SA, monom
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