Accurate identification of both abundant and rare proteins hinges on the development of single protein sensing methods. Given the immense variation in protein expression levels in a cell, separating proteins by weight would improve protein classification strategies. Here, we scale a bulk analysis method for protein separation, SDS-PAGE, to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel with only ~ 600 nm depth. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. We demonstrate the separation of a set of Atto647N-covalently labeled recombinant proteins and even more show the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle tracking and advanced image analysis methods, we created detailed profiles of the separation kinetics of each labeled protein. In addition, the device is also suitable for multicolor detection and tracking. We can achieve additional information on the sample by dual-labeling with two different fluorophores, each tagging a different amino acid. The intensity of each color and the ratio between them provide additional data for distinguishing the variety of proteins and even allows the identification and quantification of proteins with the same molecular weight. By downstream coupling or on its own, this device serves as a single-protein identification sensor and may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.

On-chip protein separation with single-molecule resolution using nanochannels

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.