Akama Kenji, Noji Hiroyuki
Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Analyst. 2021 Feb 21;146(4):1303-1310. doi: 10.1039/d0an02056g. Epub 2020 Dec 23.
Digital homogeneous non-enzymatic immunosorbent assay (digital Ho-Non ELISA) is a new class of digital immunoassay that enables highly sensitive quantification of biomolecules using a simple protocol. In digital Ho-Non ELISA, nanoparticles are tethered onto the surface of femtoliter reactors via captured target molecules. The tethered particles capturing target molecules are identified as those showing a confined Brownian motion with root-mean-square displacement (RMSD) values in a defined range. The present work aims to improve the specificity to discriminate tethered particles via single-target molecules from non-specifically immobilized particles by analyzing two nanoparticle parameters. First, in order to suppress the broadening of RMSD due to the heterogeneity of bead size, we corrected the RMSD with the fluorescence intensity of the beads. Second, focusing on the shape of Brownian motion in the x-y trajectory, we classified motion patterns by aspect ratio of the trajectory. By using multiparameter single-particle motion analysis with corrected RMSD and aspect ratio, a 3.9-fold enhanced sensitivity in PSA assay was achieved compared to the conventional RMSD analysis approach. This new strategy would increase the potential of digital immunoassays.
数字均相非酶免疫吸附测定法（数字Ho-Non ELISA）是一类新型的数字免疫测定法，它能够使用简单的方案对生物分子进行高灵敏度定量。在数字Ho-Non ELISA中，纳米颗粒通过捕获的目标分子连接到飞升反应器的表面。捕获目标分子的连接颗粒被识别为那些显示出具有在定义范围内的均方根位移（RMSD）值的受限布朗运动的颗粒。目前的工作旨在通过分析两个纳米颗粒参数来提高从非特异性固定颗粒中区分通过单目标分子连接的颗粒的特异性。首先，为了抑制由于珠子尺寸的异质性导致的RMSD变宽，我们用珠子的荧光强度校正了RMSD。其次，关注x-y轨迹中布朗运动的形状，我们通过轨迹的纵横比来对运动模式进行分类。通过使用具有校正的RMSD和纵横比的多参数单颗粒运动分析，与传统的RMSD分析方法相比，在PSA测定中实现了3.9倍的灵敏度提高。这种新策略将增加数字免疫测定的潜力。
