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Benjamin D Sullivan, Wendy Mendiola, Melissa Lee, Brandon Westerberg, Matthew Solomon, Christian Potzner, Roy Chung, Hans Boehringer, Steve Zmina, Erol Harvey; A Rapid Multiplexed Ultrasensitive Nanoliter Lab-on-a-Chip Immunoassay Platform for Human Tear Film Analysis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):322.
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© ARVO (1962-2015); The Authors (2016-present)
Existing methods for point of care molecular diagnostics require volumes of tear than are not readily available from dry eye disease patients. Manual sample transfers and long reaction times can compromise test integrity, reduce precision and impede clinical workflow. The purpose of this work was to categorize the dominant variables that influence nanoliter assay performance.
A three-dimensional microfluidic platform was constructed to accurately collect, meter and assay nanoliters of tear fluid. Integration of a long Stokes shifted fluorescent nanoparticle label with flow timing and homogenization structures facilitated an on-chip chromatographic platform for tear immunoassays. The geometry and chemistry of the upstream nanoparticle distribution was modulated to optimize the speed and sensitivity of the assay. Assays were run using 200 nL spiked human tear samples (BioreclamationIVT).
After a 5 second incubation time, steady state signals were observed 20 seconds after co-localization of reactants, with a total sample-to-answer time under one minute. Test line integration from 45-60 seconds showed a significant difference (p < 2.4e-11) between control (1.59 ± 1.16 AU) vs. spiked samples (9.72 ± 0.95 AU), with a signal-to-noise ratio of 6.1 and a coefficient of variation of 9.7% for a 0.5 nM concentration (n=9 each). Significant differences in assay time and signal isotropy were observed for the various initial nanoparticle distributions.
Optimization of input signal distributions substantially improved sensitivity and speed of detection for point-of-care nanoliter assays.
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