Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device

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 multiconditional and multiparametric assays, hindering in-depth 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 digital bioassay platform with easy solution exchanges, called multidimensional (MD) digital bioassay. 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. With multiconditional assays based on our system, we could quantitatively determine inhibitor sensitivities of single influenza A virus particles and single alkaline phosphatase (ALP) molecules, which has never been achieved with conventional digital bioassays. Further, we demonstrated that ALPs from two origins can be precisely distinguished by a single-molecule multiparametric assay with our system, which was also difficult with conventional digital bioassays. Thus, MD digital bioassay is a versatile platform to gain in-depth insight into biological entities in unprecedented resolution.