Using bimetallic Au@Pt nanozymes as a visual tag and as an enzyme mimic in enhanced sensitive lateral-flow immunoassays: Application for the detection of streptomycin

Highlights

• Au@Pt nanozyme with higher peroxidase-like activity was synthesized by a simple one-step method.

• Lateral-flow immunoassays based on Au@Pt as tag and enzyme mimic showed higher sensitivity.

• Lateral-flow immunoassay based on catalytic activity of Au@Pt was used to detect STR in milk.

Abstract

Because of the advantages of simplicity, cost-effectiveness and visibility, lateral-flow immunoassays (LFAs) have been widely used in the food safety field. However, the low sensitivity of LFAs needs to be solved. Nanozymes have amazing potential for application in biosensors due to their excellent and abundant enzyme-like characteristics. In this study, an Au@Pt nanozyme synthesized by a one-step method showed the higher affinity with TMB/H₂O₂ and higher catalytic efficiency than that of horseradish peroxidase (HRP). For the detection of streptomycin (STR), a typical aminoglycoside antibiotic, a novel LFA based on Au@Pt as a visual tag and an enhanced LFA based on the enzyme-like activity of Au@Pt by addition of the chromogenic substrate 3-amino-9-ethyl-carbazole (AEC) were established and compared with conventional LFA based on AuNPs. The qualitative limit of detection (LOD) was 1 ng mL⁻¹ for the LFA based on Au@Pt as the visual tag and 0.1 ng mL⁻¹ for the enhanced LFA based on the activity of Au@Pt, in comparison to 8 ng mL⁻¹ for LFA based on AuNPs. Furthermore, the level of streptomycin in milk samples from Zhenjiang City was successfully evaluated by the novel LFA based on Au@Pt nanozyme. These results suggest that LFAs based on nanozymes are a promising and effective tool for food safety.

Introduction

Lateral-flow immunoassays (LFAs) provide many advantages, such as simplicity, high efficiency, cost-effectiveness and visibility, and are widely used in the detection of various targets, such as bacteria, antibiotics, toxins and heavy metals, in the food safety field [[1], [2], [3], [4]]. For traditional LFAs, colloidal golds (AuNPs) serve as labels because of their good optical properties, chemical stability and strong biocompatibility [5,6]. However, traditional LFAs suffer from relatively low sensitivity, which limits their effectiveness in the detection of trace substances. Therefore, researchers are currently exploring new strategies to improve the performance of LFAs [[7], [8], [9]]. One possible route is to make more modifications on the surface of AuNPs to enhance the output signal, including loading enzymes. Qin et al. [10] developed double-enhanced strip biosensors for protein biomarkers based on AuNP aggregation and horseradish peroxidase (HRP)-assisted dual-class signal amplification. Another common strategy is to replace the traditional AuNPs with novel nanoparticles, such as CNTs [11], quantum dots [12], and magnetic particles [13]. However, complex material and probe preparation, expensive analytical instruments and unstable output signals limit their wide application of such novel nanoparticles in LFAs.

Nanozymes were first defined as “nanomaterials with enzyme-like activity” in a comprehensive review [14], and have been confirmed to have many kinds of enzymatic activities, including peroxidase [15], catalase [16], superoxide dismutase [17], and oxidase [18]. Furthermore, nanozymes present several advantages, such as robust catalytic activities, low production cost, and properties that can be adjusted by size- and shape-controlled synthesis and surface modification [14,19], which have attracted attention for use in the area of diagnosis, tissue imaging, and drug delivery. In biosensing fields, the use of nanozymes as direct substitutes for natural enzymes, has improved the sensitivity of analysis methods and enriched assay formats [20]. In LFA formats, some studies have been reported using nanozymes as replacements of conventional AuNPs. For example, Ouyang and coworkers developed a novel immunochromatographic test strip with integrated manganese dioxide nanoflowers (MnO₂ NFs) for detecting chlorpyrifos with a detection limit of 0.033 ng mL⁻¹ [21]. Furthermore, some previous researchers exploited original LFAs with platinum-gold nanoparticles, such as platinum-gold nanocatalysts and gold-platinum nanoflowers, for detecting Escherichia coli [22], human prostate-specific antigen (PSA) [23], p24 [24], rabbit IgG [25] and so on. It is necessary to explore more applications of nanozyme in LFAs for the more sensitive and more reproducible performance of this convenient method.

Streptomycin (STR), a typical aminoglycoside antibiotic, remains in the food of animal origin and in the environment and can affect the ecological balance and human health [26,27]. STRs are monitored in milk at a maximum residue limit (MRL) of 200 ng mL⁻¹ by the European Union [28]. Chromatography methods [29,30], including gas chromatography and high-performance liquid chromatography (HPLC), and immunoassays have been used to detect STR in animal-origin food. However, reports on rapid detection method for this target, especially LFAs, are still limited. In this study, we proposed to develop a novel LFA based on Au@Pt nanozymes to analyze STR in milk. First, a simple synthetic process for Au@Pt was described, and the obtained nanoparticles were characterized. Second, three forms of LFAs were established and compared systematically, including conventional LFAs based on AuNPs, LFAs based on Au@Pt as visual tags, and enhanced LFAs based on the enzyme-like activity of Au@Pt. Finally, bovine milk samples were monitored by the newly developed method.