A Temperature-Controlled Stopped-Flow Droplet-Based Microfluidic Reactor for Fast Biomolecular Kinetics

Abstract

Several important biomolecular reactions, including folding/unfolding, aggregation, and binding, occur on a time scale of sub-seconds or seconds. One relevant example is represented by microscopic reactions underlying, the formation of amyloid fibrils from normal soluble proteins and peptides, which is associated with the onset and progression of Alzheimer's and Parkinson's diseases. The protein aggregation process consists of a network of several microscopic steps of nucleation and growth. Each individual step has different contributions to the formation of the final fibrils and of the soluble intermediates generated during the reactions, which are currently thought to represent the most toxic species. The characterization of individual reactions in a sub-second time scale is challenging with the current available biophysical techniques. In this work, we leverage the power of droplet-based microfluidics to quantitatively explore aggregation kinetics within a time window of milliseconds to seconds. Herein we present a universal droplet-based microfluidic platform for rapid and high-precision kinetics using stroboscopic illumination and fluorescence detection within a single experiment. The microfluidic platform is also integrated with a heating element at the reactor zone, which is isolated from the droplet generation and mixing part. The droplets can be heated up rapidly to a set temperature (within a range of 24 to 90°C) once they enter the heating zone enabling fast biomolecular kinetics to be extracted at different temperatures. We prove the potential of this platform with two systems: the denaturation kinetics of hen egg white lysozyme under various temperatures, and the elongation rate of fibrils composed of the peptide Abeta42, the peptide associated with Alzheimer's disease.