Absolute Quantification of Amyloid Propagons by Digital Microfluidics

作者：Pfammatter Manuela; Andreasen Maria; Meisl Georg; Taylor Christopher G; Adamcik Jozef; Bolisetty Sreenath; Sanchez Ferrer Antoni; Klenerman David; Dobson Christopher M; Mezzenga Raffaele; Knowles Tuomas P J; Aguzzi Adriano; Hornemann Simone^*

来源：Analytical Chemistry, 2017, 89(22): 12306-12313.

DOI：10.1021/acs.analchem.7b03279

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.

• 出版日期2017-11-21