ArticleJournal of the American Chemical Society2018
Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration.
Article in Journal of the American Chemical Society, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 37 citations in OpenAlex.
- Image analysis optimization for nanowire-based optical detection of molecules.Nanophotonics (Berlin, Germany) · 2025Article
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- Digital and Absolute Assays for Low Abundance Molecular Biomarkers.Accounts of chemical research · 2023Article
- Evaluation of probe-based ultra-sensitive detection of miRNA using a single-molecule fluorescence imaging method: miR-126 used as the model.Frontiers in bioengineering and biotechnology · 2023Article
- CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging.Chemical science · 2022Article
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- Photonic crystal enhanced fluorescence emission and blinking suppression for single quantum dot digital resolution biosensing.Nature communications · 2022Article
- Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification.The Analyst · 2022Article
- Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers.Bioengineering (Basel, Switzerland) · 2021Article
- Multilabel hybridization probes for sequence-specific detection of sepsis-related drug resistance genes in plasmids.Talanta open · 2021Article
- Direct single-molecule imaging for diagnostic and blood screening assays.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Critical Review: digital resolution biomolecular sensing for diagnostics and life science research.Lab on a chip · 2020Review
- Article
- Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays.Angewandte Chemie (International ed. in English) · 2020Review
- Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging.Chemical science · 2020Article
- High-Fidelity Single Molecule Quantification in a Flow Cytometer Using Multiparametric Optical Analysis.ACS nano · 2020Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.