Direct single-molecule imaging for diagnostic and blood screening assays.
Proc Natl Acad Sci U S A
; 118(14)2021 04 06.
Article
em En
| MEDLINE
| ID: mdl-33790018
ABSTRACT
Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10-18 M) for model assay systems and to the low femtomolar (10-16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10-18 to 10-5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Infecções por HIV
/
Proteína do Núcleo p24 do HIV
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Imagem Individual de Molécula
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Teste de HIV
Tipo de estudo:
Diagnostic_studies
/
Guideline
/
Prognostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article