Human Tear Protein Analysis Enabled by an Alkaline Microfluidic Homogeneous Immunoassay

Abstract

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sjögren's syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.