3 years ago

Absolute Quantification of Amyloid Propagons by Digital Microfluidics

Absolute Quantification of Amyloid Propagons by Digital Microfluidics
Adriano Aguzzi, Christopher M. Dobson, Georg Meisl, Manuela Pfammatter, David Klenerman, Christopher G. Taylor, Simone Hornemann, Antoni Sánchez-Ferrer, Maria Andreasen, Jozef Adamcik, Tuomas P. J. Knowles, Raffaele Mezzenga, Sreenath Bolisetty
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.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b03279

DOI: 10.1021/acs.analchem.7b03279

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