Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device
Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device
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DOI：
10.1021/acs.analchem.0c05360
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发表时间：
2021-03-12
影响因子：
7.4
通讯作者：
Tabata, Kazuhito, V
中科院分区：
文献类型：
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作者：
Honda, Shingo;Minagawa, Yoshihiro;Tabata, Kazuhito, V
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（其中分析物分子被单独地限制在要分析的小隔室中）是单分子生物学中的新兴技术，并且适用于各种生物实体（例如，细胞和病毒颗粒）。然而，数字生物测定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，我们开发了一个数字生物测定平台，可以轻松交换解决方案，称为多维（MD）数字生物测定。我们将单个分析物固定在阵列式的毫微微升（10（-15）L）反应器中，并用气流密封它们。每个反应器中的溶液是稳定的，并且在超过2小时的时间内没有显示出通过溶液泄漏的串扰，并且成功地进行了超过30轮的完美溶液交换。基于我们的系统的多条件检测，我们可以定量地确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，这是传统的数字生物检测从未实现过的。此外，我们证明了来自两个来源的ALP可以通过使用我们的系统的单分子多参数测定来精确区分，这也是传统数字生物测定难以实现的。因此，MD数字生物测定是一个多功能的平台，以前所未有的分辨率深入了解生物实体。
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.