High-throughput single-molecule bioassay using micro-reactor arrays with a concentration gradient of target molecules

Micro-reactor arrays enable highly sensitive and quantitative bioassays at a single-molecule level. Accordingly, they are widely used for sensitive "digital" bioassays, e.g., digital PCR and digital ELISA. Despite high integration, individual reactors in digital bioassays are filled with a uniform reaction solution, thus limiting the ability to simultaneously conduct multiple bioassays under different conditions using integrated reactors in parallel, resulting in the loss of potential throughput. We developed micro-reactor arrays with a concentration gradient of target molecules, in which individual reactors sealed with a lipid-bilayer membrane contained a precise amount of target molecules. Using the arrays, we successfully demonstrated multiple single-molecule bioassays in parallel using alkaline phosphatase or α-hemolysin, key components in various biomedical sensors. This new platform extends the versatility of micro-reactor arrays and could enable further analytical and pharmacological applications.