Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, Aarhus C DK-8000, Denmark.
Department of Environmental Science, Aarhus University, Frederiksborgvej 399, Roskilde DK-4000, Denmark.
Anal Chem. 2020 Sep 15;92(18):12451-12459. doi: 10.1021/acs.analchem.0c02262. Epub 2020 Aug 28.
Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, , was performed in a sandwich reaction with -specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single cell detection and from 1 to 4 × 10 CFU mL quantification. No interference/cross-reactivity from , , and was observed when the assay was performed on Ab-modified MBs, and could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.
病原体相关感染是对人类健康的主要威胁之一，因此需要可靠的方法来立即、准确地鉴定致病微生物。在这里，我们开发了一种廉价的纤维素酶联免疫磁珠方法，可在 3 小时内实现对单个细菌细胞水平的细菌进行特异性和超灵敏分析。通过使用 -特异性适体或抗体（Ab）修饰的磁珠（MBs）和与纤维素酶相连的 Ab/适体报告分子进行夹心反应，对模型细菌 进行了检测。将标记有纤维素酶的免疫适体夹心应用于修饰有硝化纤维素膜的电极上，可消化膜并改变其电导率。在没有任何氧化还原指示剂的情况下，电极的计时库仑响应在 0.3 V 下允许单个细胞的检测和从 1 到 4 × 10 CFU mL 的定量。当在 Ab 修饰的 MBs 上进行测定时，未观察到来自 、 、 和 的干扰/交叉反应，并且可以在自来水中和牛奶中定量检测到 。这种电化学无标记方法足够快速、高度特异和灵敏，可用于直接现场应用。该测定方法可适应于相同或不同物种的其他细菌菌株的特异性检测，并为临床、食品和环境中细菌的快速、特异性和可靠分析提供了新的分析工具。
