Absolute Quantification of Amyloid Propagons by Digital Microfluidics.
Anal Chem
; 89(22): 12306-12313, 2017 11 21.
Article
em En
| MEDLINE
| ID: mdl-28972786
ABSTRACT
The self-replicating properties of proteins into amyloid fibrils is a common phenomenon and underlies a variety of neurodegenerative diseases. Because propagation-active fibrils are chemically indistinguishable from innocuous aggregates and monomeric precursors, their detection requires measurements of their replicative capacity. Here we present a digital amyloid quantitative assay (d-AQuA) with insulin as model protein for the absolute quantification of single replicative units, propagons. D-AQuA is a microfluidics-based technology that performs miniaturized simultaneous propagon-induced amplification chain reactions within hundreds to thousands of picoliter-sized droplets. At limiting dilutions, the d-AQuA reactions follow a stochastic regime indicative of the detection of single propagons. D-AQuA thus enables absolute quantification of single propagons present in a given sample at very low concentrations. The number of propagons quantified by d-AQuA was similar to that of fibrillar insulin aggregates detected by atomic-force microscopy and to an equivalent microplate-based assay, providing independent evidence for the identity of insulin propagons with a subset of morphologically defined protein aggregates. The sensitivity, precision, and accuracy of d-AQuA enable it to be suitable for multiple biotechnological and medical applications.
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Base de dados:
MEDLINE
Assunto principal:
Peptídeos beta-Amiloides
/
Técnicas Analíticas Microfluídicas
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article