Xiang Tianxin, Wen Jin, Fang Jianhua, Nie Chuang, Cheng Na, Ying Ying, Tan Hongliang
Jiangxi Provincial Key Laboratory of Prevention and Treatment of Infectious Diseases, Jiangxi Medical Center for Critical Public Health Events, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, 330052, P.R. China.
Jiang Xi Hospital of China-Japan Friendship Hospital, Nanchang, 330052, P.R. China.
Mikrochim Acta. 2025 Aug 9;192(9):573. doi: 10.1007/s00604-025-07449-7.
Enzymatic assays represent a promising alternative to conventional multiplex optical methods for multi-target detection due to their exceptional specificity and catalytic versatility. Nevertheless, most existing enzymatic systems are limited to single-target detection. In this work, we developed a dual-signal multiplex assay based on non-tandem enzymes for the simultaneous detection of multiple pathogenic bacteria. This assay utilizes the distinct catalytic activities of horseradish peroxidase (HRP) and invertase (INV) to produce two orthogonal signal outputs. Specifically, HRP catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine to produce a blue chromogenic signal measurable by absorbance, while INV hydrolyzes sucrose to yield glucose, which is quantified with a personal glucose meter. To implement this assay, HRP and INV are individually encapsulated within zeolitic imidazolate framework-90, and FeO nanoparticles functionalized with phenylboronic acid are employed for dual-recognition of target pathogens. By integrating spatiotemporal control of enzymatic reactions with distinct signal separation, the assay effectively minimizes crosstalk and enhances detection accuracy and reliability. This assay demonstrates excellent sensitivity and selectivity in milk samples, highlighting its potential for practical applications. We believe this work establishes a versatile and robust platform for multiplex pathogen detection, with significant implications for food safety, clinical diagnostics, and environmental monitoring.
由于其卓越的特异性和催化多功能性，酶法检测是传统多目标检测的多重光学方法的一种有前途的替代方法。然而，大多数现有的酶系统仅限于单目标检测。在这项工作中，我们开发了一种基于非串联酶的双信号多重检测方法，用于同时检测多种病原菌。该检测方法利用辣根过氧化物酶（HRP）和转化酶（INV）的不同催化活性产生两种正交信号输出。具体而言，HRP催化3,3',5,5'-四甲基联苯胺氧化，产生可通过吸光度测量的蓝色显色信号，而INV水解蔗糖生成葡萄糖，用个人血糖仪进行定量。为了实现该检测，将HRP和INV分别封装在沸石咪唑酯骨架-90中，并使用用苯硼酸功能化的FeO纳米颗粒对目标病原体进行双重识别。通过将酶促反应的时空控制与不同的信号分离相结合，该检测方法有效地减少了串扰，提高了检测的准确性和可靠性。该检测方法在牛奶样品中表现出优异的灵敏度和选择性，突出了其实际应用潜力。我们相信这项工作建立了一个通用且强大的多重病原体检测平台，对食品安全、临床诊断和环境监测具有重要意义。
