Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO) , 4-1-8 Honcho , Kawaguchi , Saitama 332-0012 , Japan.
Institute of Materials Chemistry and Engineering , Kyushu University , 6-1 Kasuga-Koen , Kasuga, Fukuoka 816-8580 , Japan.
ACS Nano. 2019 Feb 26;13(2):2262-2273. doi: 10.1021/acsnano.8b08959. Epub 2019 Feb 13.
Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far ( i. e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination.
研究人员已经展示出无机纳米线在分析细胞或细胞内成分方面的巨大应用潜力。尽管纳米线对细胞表面的隐身效应允许在分析细胞时保持完整活细胞，但作为一种完全相反的方法，通过破坏细胞来分析细胞内成分的适用性也是理解细胞信息的核心。然而，由于细胞的坚固性和迄今为止采取的错误方法（例如，纳米线穿透细胞膜），报道的裂解策略对于微生物细胞的裂解还不够充分。在这里，我们提出了一种通过引入由细胞膜拉伸引发的破裂方法来实现微生物细胞的纳米线介导的裂解方法；换句话说，纳米线不会穿透细胞膜，而是在纳米线之间破坏细胞膜。用细菌相容且灵活的纳米线缠绕细胞，并通过剪切力诱导缠绕细胞的细胞膜拉伸，对革兰氏阳性和革兰氏阴性细菌以及酵母细胞的纳米线介导的裂解起着重要作用。此外，纳米线介导的裂解很容易与环介导等温扩增（LAMP）方法兼容，因为只需引入微生物细胞即可触发裂解。我们表明，将纳米线介导的裂解与 LAMP 集成提供了一种简单、快速、一步鉴定检测方法（只需将预混合溶液引入设备中），从而实现微生物细胞的可视化显色鉴定。这种方法使研究人员能够开发一种微流控分析平台，不仅可以用于包括耐药和耐热细胞在内的微生物细胞鉴定，还可以用于现场检测而不会造成任何污染。
