ABSTRACT
Cerebrospinal fluid (CSF) rhinorrhea is a potentially dangerous condition identified by CSF leakage into the nasal cavity. This malady stands to benefit from rapid and noninvasive screening diagnostics to complement low-throughput imaging based methods currently in use. To address this gap, we demonstrate on-chip immunosubtraction to accelerate biomarker validation and immunoassay development for a putative CSF rhinorrhea diagnostic marker, transthyretin, by combining high-specificity immunoaffinity capture with subsequent polyacrylamide gel electrophoresis (PAGE). We demonstrate the on-chip assay using photopatterned polyacrylamide immunofilters. The filter consists of polymer with controlled pore-sizes to size-exclude (i.e., "subtract") large antibody-target immune complexes from downstream PAGE separation. A control PAGE separation is also performed for comparison without immunoaffinity capture (i.e., no antibody present). We compare on-chip immunosubtraction to Western blotting and ELISA to validate CSF rhinorrhea biomarkers from nasal surgery samples. For samples representative of spontaneous rhinorrhea, the 5 min on-chip assay achieved clinical specificity of 100%, compared to 50% for ELISA which required 6 h. On-chip immunosubtraction also generated results for clinical samples not assayable via ELISA due to matrix protein spurious signals. The pilot study suggests the capability of a rapid on-chip validation tool to expedite scrutiny of putative protein markers for new clinical assays.
