Since I proved rotary catalysis of F1 ATPase motor protein (Nature 1997), I have been pursuing the elucidation of chemomechanical coupling mechanism of F1-ATPase (Cell 1998, Nature 2001, 2004, 2005, PNAS 2020a, 2020b, Science 2011, Nat. Comm. 2021). I have also established digital bioassay by use of femtoliter reactor array device (Nature biotechnology 2005, Lab on a chip 2012, Analytical chemistry 2017). This technology has been applied for artificial cell reactor project (Scientific Advances 2019, ACS Nano 2020) with aim to create autonomous artificial cell systems in bottom-up approach (ACS Syn. Bio. 2021).
We developed femto-liter reactor array device (FRAD) that display over million water-in-oil droplets with size of femto-liter range. One can readily encapsulate bio- or non-biological molecules into the FRAD reactors, by spreading assay mixture on the device and sealing the reactors with oil or air. The extremely small volume features of the reactors on FRAD allows very sensitive bioassay at single-molecule level referred as to ‘digital bioassay’. The massive number of the reactor enables to identify highly active but very rare functional molecules among a large numbers of library. Several examples will be introduced including Digital ELISA, Digital Influenza Virus Counting, and single-transporter analysis. The high biocompatibility of FRAD reactors also allows the reconstitution of molecular systems such as cell-free gene expression and replication. In the last part of the presentation, I will introduce the concept of FRAD in the context of synthetic biology, and show digital gene expression with an application for the engineering of alkaline phosphatase to enhance catalytic rate constant.