Single-microchannel, multistep assay reports protein size and immunoaffinity

We introduce a fully integrated multistep protein assay that reports both protein identity and size. To report these two properties, a microfluidic design strategy integrates pore limit electrophoresis (PLE) with a heterogeneous immunoassay in a single microchannel (PLE-IA). PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation. In a single-channel multistage assay approach, an antibody to follistatin was first immobilized in a polyacrylamide PLE gradient gel, near the origin of the separation axis. Immobilization relies on pore-limit exclusion of the antibody and not on chemical functionalization of either the sieving matrix or the antibody, making assay customization by an end-user straightforward. Subsequently, target and ladder protein species were electrophoretically introduced into the antibody-patterned PLE channel. Species having an affinity for the immobilized antibody were detected via heterogeneous immunoassay. Noninteracting and, thus, unbound species electromigrated past the patterned antibodies, along the separation axis, and finally separated according to the pore-size limit of each, yielding a log-linear dependence of molecular weight on migration distance. Separations of 10 min yielded an average peak capacity of 18 ± 1.3 (separation resolution (SR) = 1) in a 10 mm separation distance. Comparison of the separated peaks in two parallel PLE channels in the presence or absence of capture antibody with a protein size ladder revealed good agreement of the target molecular weight with reported values. In addition, a more than 50-fold decrease in the detection limit (0.078 vs 5 nM) was achieved using an electrophoretic "continuous injection" technique in which sample material was continuously loaded for 40 min. On the basis of this proof-of-principle demonstration with follistatin, PLE-IA should find application in study of cell signaling, including questions related to aging and regeneration.