Honda Shingo, Minagawa Yoshihiro, Noji Hiroyuki, Tabata Kazuhito V
Anal Chem. 2021 Apr 6;93(13):5494-5502. doi: 10.1021/acs.analchem.0c05360. Epub 2021 Mar 11.
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 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.
单分子实验通过揭示单个分子的实际工作方式，帮助我们更深入地了解生物现象。数字生物测定法是单分子生物学中的一项新兴技术，它将分析物分子单独限制在小隔室中进行分析，并适用于各种生物实体（如细胞和病毒颗粒）。然而，数字生物测定法与多条件和多参数测定不兼容，阻碍了对分析物的深入理解。这是因为当前的数字生物测定法缺乏一个可重复的溶液交换系统来将分析物保持在隔室内。为了应对这一挑战，我们开发了一种具有简便溶液交换功能的数字生物测定平台，称为多维（MD）数字生物测定法。我们将单个分析物固定在排列好的飞升（10⁻¹⁵升）反应器中，并用气流密封。每个反应器中的溶液稳定，在超过2小时的时间内未因溶液泄漏而出现串扰，并且成功进行了30多轮完美的溶液交换。基于我们的系统进行多条件测定时，我们能够定量测定单个甲型流感病毒颗粒和单个碱性磷酸酶（ALP）分子的抑制剂敏感性，这是传统数字生物测定法从未实现过的。此外，我们证明，利用我们的系统通过单分子多参数测定法可以精确区分来自两个来源的碱性磷酸酶，这也是传统数字生物测定法难以做到的。因此，MD数字生物测定法是一个通用平台，能够以前所未有的分辨率深入洞察生物实体。
