Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration

Journal of the American Chemical Society
Lucas SmithAndrew M Smith

Abstract

Surface capture assays can measure fluorescently labeled analytes across a 1000-fold concentration range and at the sub-nanomolar level, but many biological molecules exhibit 1,000,000-fold variations in abundance down to the femtomolar level. The goal of this work is to expand the dynamic range of fluorescence assays by using imaging to combine molecular counting with single-molecule calibration of ensemble intensities. We evaluate optical limits imposed by surface-captured fluorescent labels, compare performances of different fluorophore classes, and use detector acquisition parameters to span wide ranges of fluorescence irradiance. We find that the fluorescent protein phycoerythrin provides uniquely suitable properties with exceptionally intense and homogeneous single-fluorophore brightness that can overcome arbitrary spot detection threshold biases. Major limitations imposed by nonspecifically bound fluorophores were then overcome using rolling circle amplification to densely label cancer-associated miRNA biomarkers, allowing accurate single-molecule detection and calibration across nearly 5 orders of magnitude of concentration with a detection limit of 29 fM. These imaging and molecular counting strategies can be widely ap...Continue Reading

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Citations

Mar 17, 2021·Chemistry, an Asian Journal·Tracey LuuYuning Hong
Apr 2, 2021·Proceedings of the National Academy of Sciences of the United States of America·Qiaoqiao RuanSergey Y Tetin
Jun 12, 2020·ACS Nano·Phuong LeAndrew M Smith
Feb 8, 2020·Analytical Chemistry·Lucas D SmithBrian T Cunningham

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