RT Journal Article SR Electronic T1 Multi-dimensional digital bioassay platform based on an air-sealed femtoliter reactor array device JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.10.25.354381 DO 10.1101/2020.10.25.354381 A1 Shingo Honda A1 Yoshihiro Minagawa A1 Hiroyuki Noji A1 Kazuhito V. Tabata YR 2020 UL http://biorxiv.org/content/early/2020/10/26/2020.10.25.354381.abstract AB Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multi-conditional or multi-parametric assays, hindering understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a new digital bioassay platform with easy solution exchanges, called multi-dimensional (MD) digital bioassay, and tested its quantitativity and utility. We immobilized single analytes in arrayed femtoliter (10−15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. To show the utility of our system, we investigated neuraminidase inhibitor (NAI) sensitivity on single influenza A virus (IAV) particles in a multi-conditional assay. We proved that IAV particles show a heterogeneous response to the NAI. Further, to demonstrate multi-parametric assays, we examined the sensitivity of individual IAV particles or model enzyme molecules to two different inhibitors. Our results support that MD digital bioassay is a versatile platform to unveil heterogeneities of biological entities in unprecedented resolution.Competing Interest StatementThe authors have declared no competing interest.