State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. China; Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China.
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Jiangxi Normal University, Nanchang 330022, P. R. China.
J Dairy Sci. 2019 Dec;102(12):10877-10886. doi: 10.3168/jds.2019-17067. Epub 2019 Sep 11.
The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-HO) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of HO through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 10 cfu/mL. This method also revealed a good linear range (3 × 10 to 3 × 10 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 10 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 10 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.
传统的基于金纳米粒子（AuNP）生长的等离子体酶联免疫吸附测定（pELISA）严格且直接受还原试剂控制，可以实现高灵敏度，但对周围环境仍然很脆弱。本工作通过 DNA 介导 AuNP 生长，开发了一种用于粉状婴儿配方样品中克罗诺杆菌检测的夹心 pELISA。在该测定中，吸附在金纳米种子表面的 DNA 引导具有羟胺作为还原剂的各向异性晶体生长，并且引入了过氧化氢酶-过氧化氢（Cat-HO）系统将 DNA 导向的 AuNP 生长与 pELISA 桥接，因为 DNA 可以通过芬顿试剂氧化产生的羟基自由基断裂成片段。在优化条件下，该 pELISA 可以通过肉眼定性检测克罗诺杆菌属（Cronobacter muytjensii ATCC 51329），截止值为 3×10 cfu/mL。该方法还揭示了用于定量检测 C. muytjensii ATCC 51329 的良好线性范围（3×10 至 3×10 cfu/mL），检测限为 1.6×10 cfu/mL，约比辣根过氧化物酶基于常规 ELISA（2.6×10 cfu/mL）低 162.5 倍。通过利用高度稳定的 DNA 导向的 AuNP 生长，该方法在添加不同浓度的 C. muytjensii ATCC 51329 的粉状婴儿配方样品中表现出良好的性能，平均回收率范围为 90.79%至 119.09%，变异系数范围为 4.24%至 9.55%。这些值对应于该方法的可接受的准确性和精密度。总之，这项工作为食品安全、临床诊断和环境监测中的其他分析物筛选展示了潜力。
