Microfluidic Validation of Diagnostic Protein Markers for Spontaneous Cerebrospinal Fluid Rhinorrhea

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.