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• W2890537151 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 applied to expand the limit of detection and dynamic range of a variety of surface fluorescence assays." @default.
• W2890537151 created "2018-09-27" @default.
• W2890537151 creator A5021900842 @default.
• W2890537151 creator A5029964030 @default.
• W2890537151 creator A5072709323 @default.
• W2890537151 date "2018-09-14" @default.
• W2890537151 modified "2023-10-12" @default.
• W2890537151 title "Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration" @default.
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• W2890537151 doi "https://doi.org/10.1021/jacs.8b08879" @default.
• W2890537151 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6462413" @default.
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• W2890537151 hasPublicationYear "2018" @default.
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