Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting

Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real-time tracking of the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker–antibody binding in biofluids with biomarker–protein binding on the cellular membrane.