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 Fe 3 O 4 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. Graphical
Surface Enhanced Raman Scattering (SERS), with good reproducibility and stability, is an analytical testing technique that uses metals or metal nanoparticles as detection substrates, and it can also be used to characterize the information from molecular vibration. In this paper, we first report a facile one-step approach that allows the fabrication of assembled Ag NPs induced by polyaniline (PANI). Ag@PANI nanocomposites have the dual functions of saccharide-oxidase-like and peroxidase-like activities. Therefore, they can be used as a tandem enzyme to produce hydrogen peroxide from saccharides and catalyze the oxidation of TMB. At the same time, they can be considered as a SERS substrate to obtain the characteristic peaks of TMBox. In addition, on the basis of SERS, the effects of the Ag+ concentration, temperature and pH on the activity of Ag@PANI nanocomposites as the tandem enzyme are further explored. Under the optimal conditions, the common saccharides (sucrose, fructose, glucose and lactose) were detected with linear correlation (R2) in different concentration ranges, and ultra-sensitive detection of various saccharides under label-free conditions was achieved.
Yangyang Zhao,Jian Yang,Guiye Shan,Zhaoyang Liu,Anni Cui,Ailin Wang,Yanwei Chen,Yichun Liu
It is greatly significant and difficult to realize the purpose of natural enzyme free and “one-step” for traditional colorimetric urine glucose detection. Here, ultra-small Ag 2-x Cu x S nanoparticles (NPs) with tandem enzyme-like activity, i.e. they simultaneously exhibit glucose oxidase (GOx)- and peroxidase-like activities, are developed for tandem glucose oxidation and colorimetric H 2 O 2 detection. Cu doping content has a great influence on the enzyme-like activity. Ag 1.8 Cu 0.2 S NPs (i.e. Ag 2-x Cu x S NPs with 10 % Cu) exhibit the best catalytic performance. The tandem enzyme-like activity can be further enhanced to ∼10 times under laser irradiation due to photothermal effect. Furthermore, accurate glucose detection has been realized in unprocessed human urine based on the photothermal-enhanced tandem enzyme-like activity of Ag 1.8 Cu 0.2 S NPs. This natural enzyme free and “one-step” colorimetric glucose detection strategy exhibits the potential application of the new generation of tandem enzyme-like nanomaterials.
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