Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration
| Autor: | Lucas D. Smith, Andrew M. Smith, Manish Kohli |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Brightness Analyte Fluorophore 01 natural sciences Biochemistry Fluorescence Article Catalysis 03 medical and health sciences chemistry.chemical_compound Colloid and Surface Chemistry Limit of Detection Calibration Fluorescent Dyes chemistry.chemical_classification 010405 organic chemistry Dynamic range Biomolecule Detector Phycoerythrin General Chemistry Single Molecule Imaging 0104 chemical sciences MicroRNAs Spectrometry Fluorescence 030104 developmental biology chemistry Biological system Nucleic Acid Amplification Techniques Algorithms |
| Zdroj: | Journal of the American Chemical Society. 140:13904-13912 |
| ISSN: | 1520-5126 0002-7863 |
| Popis: | Surface capture assays can measure fluorescently labeled analytes across a 1,000-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. |
| Databáze: | OpenAIRE |
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