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.

中文翻译：

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基于气密飞升反应器阵列装置的多维数字生物测定平台

单分子实验通过阐明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。数字生物测定法是将分析物分子分别限制在小隔间中进行分析的数字生物测定法，是单分子生物学中的一种新兴技术，适用于各种生物实体（例如细胞和病毒颗粒）。但是，数字生物测定法与多条件或多参数测定法不兼容，这妨碍了对分析物的理解。这是因为当前的数字生物测定法缺乏将分析物保留在隔室内的可重复溶液交换系统。为了应对这一挑战，我们开发了一种易于交换解决方案的新型数字生物测定平台，称为多维（MD）数字生物测定，并测试了其定量性和实用性。我们将单个分析物固定在阵列的飞升（10-15 L）反应器中，并用气流密封。每个反应器中的溶液均稳定，并且在超过2小时内没有因溶液泄漏引起的串扰，并且成功进行了30多次完美的溶液交换。为了显示我们系统的实用性，我们在多条件分析中研究了神经氨酸酶抑制剂（NAI）对单个甲型流感病毒（IAV）颗粒的敏感性。我们证明IAV粒子显示出对NAI的异质反应。此外，为了证明多参数分析，我们检查了单个IAV颗粒或模型酶分子对两种不同抑制剂的敏感性。我们的结果支持MD数字生物测定法是一个以前所未有的分辨率揭示生物实体异质性的多功能平台。