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Kelly Karns, Nancy A. McNamara, Amy E. Herr; Development of a Microfluidic Immunoassay for Highly Alkaline Human Tear Protein Biomarkers of Sjögren’s Syndrome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3748. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A biomarker-based assay for the definitive diagnosis of Sjögren’s syndrome (SS) would have real clinical benefit by eliminating the need for labial salivary gland biopsy as is currently required. However, low throughput, large sample volume requirements and the high alkalinity of many putative SS biomarkers limit the analytical techniques available to make routine clinical assessments of tear proteins. Homogeneous microfluidic immunoassays electrophoretically separate protein species based on differences in their charge-to-mass ratios and are uniquely suited to rapidly quantify endogenous proteins in volume-limited tear samples. Here we detail a homogeneous microfluidic immunoassay capable of multiplexed quantification of highly alkaline tear film biomarkers for SS.
Tear film fluid was collected from the ocular surface of healthy and SS patients using Schirmer strips. Glass microfluidic channels were fabricated using standard wet etch processes (Caliper Life Sciences, Hopkinton, MA). A small pore-size, 40% total acrylamide (%T), polyacrylamide (PA) gel membrane and 3-6%T pore-size PA gel discontinuity were fabricated within the channels using UV photo-patterning and photo-masks. A pH 10.8 N-cyclohexyl-3-aminopropanesulfonic acid/NaOH buffer was used. Fluorescently-labeled anti-human lactoferrin (Lf) antibody and tear fluid were electrophoretically loaded, mixed at the membrane and separated in the discontinuous pore-size separation channel. Endogenous tear Lf was quantified through the measurement of bound immune complex peak area.
The assay shows a significant down-regulation of Lf in SS vs. healthy tears and is consistent with gold-standard enzyme-linked immunosorbent assay measurements. Demonstrated integrated functionality includes 1) automated sample handling, 2) on-chip mixing of tear fluid with antibody probe and 3) on-the-fly enrichment and dilution of tear fluid, enabling detection of a 10-fold Lf down-regulation between SS and healthy tears. Critical to the analysis of tear fluid in dry eye disease, the assay completes in <30 sec and uses <5 µL of sample.
This microfluidic immunoassay enables the rapid quantification of Lf in tear fluid and may be useful towards the development of a biochemical diagnostic test for SS. The alkaline buffer and optimized antibody probe allow for expansion of the assay to the multiplexed quantification of other alkaline biomarkers for SS including lysozyme and small proline-rich protein 1B.
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