Chip-based immunoassays.

Microfluidic immunoassay techniques offer advantages in speed, automation, and portability over -bench-top gold standard counterparts. In particular, on-chip immunosubtraction is a rapid homogeneous immunoassay used for reporting both protein native mobility and binding specificity. Immunosubtraction is performed by removing antibody-bound target proteins from electrophoretic detection via a size-based exclusion filter, while unbound nontarget proteins are able to pass through the filter for downstream detection. Immunosubtraction is achieved on-chip by fabrication of discrete patterned polyacrylamide (PA) gel regions. Additionally, PA gel regions are used to define on-chip sample preparation regions for protein enrichment, fluorescent labeling, and antibody-target binding prior to immunosubtraction. Here we describe the immunosubtraction device fabrication technique as well as the electrophoretic assay protocol for determining target protein mobility and binding specificity within complex biological samples including cerebrospinal fluid.