AbstractThe authors describe a colorimetric immunoassay for the model nalyte aflatoxin B1 (AFB1). It is based on the just-in-time generation of an MnO2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO4) is converted into manganese dioxide (MnO2) which acts as an oxidase mimic that catalyzes the oxidation 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO4. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB1 and magnetic beads carrying bovine serum albumin conjugated to AFB1 are used for the determination of AFB1. In presence of AFB1, it will compete with the BSA-conjugated AFB1 (on the magnetic beads) for the labeled antibody against AFB1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB1 concentrations in the range from 0.1 to 100 ng mL−1, with a 0.1 ng mL−1 detection limit (at the 3Sblank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstractSchematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO4)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B1 (AFB1) by coupling with hydrolytic reaction of AOx toward AA and the KMnO4-Mn(II)-TMB system [note: 3,3′,5,5′-tetramethylbenzidine: TMB].

中文翻译：

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用于非常规测定的常规化学反应：通过使用酶响应及时生成基于 MnO2 的纳米催化剂对黄曲霉毒素 B1 进行比色免疫测定

摘要作者描述了模型分析物黄曲霉毒素 B1 (AFB1) 的比色免疫分析。它基于 MnO2 纳米催化剂的即时生成。与以前开发的免疫分析不同，显色反应依赖于氧化酶模拟物的及时形成，无需底物的帮助。高锰酸钾 (KMnO4) 转化为二氧化锰 (MnO2)，二氧化锰 (MnO2) 充当氧化酶模拟物，催化氧气将 3,3',5,5'-四甲基联苯胺 (TMB) 氧化，生成蓝色产物。在抗坏血酸 (AA) 存在下，KMnO4 被还原为 Mn(II) 离子。这导致MnO2纳米催化剂的量减少。因此，TMB 的氧化不会发生。通过加入抗坏血酸氧化酶，AA转化为不能还原KMnO4的脱氢抗坏血酸。基于这些观察，开发了比色竞争性酶免疫分析法，其中抗坏血酸氧化酶和金纳米颗粒标记的抗 AFB1 抗体以及携带与 AFB1 偶联的牛血清白蛋白的磁珠用于测定 AFB1。在 AFB1 存在的情况下，它将与 BSA 偶联的 AFB1（在磁珠上）竞争金纳米颗粒上针对 AFB1 的标记抗体。这使得结合在磁珠上的抗坏血酸氧化酶/抗 AFB1 抗体标记的金纳米粒子的量减少，并导致抗坏血酸的增加。在最佳条件下，吸光度（在 652 nm 处测量）随着 AFB1 浓度的增加而降低，范围从 0.1 到 100 ng mL-1，检测限为 0.1 ng mL-1（在 3Sblank 水平）。通过分析加标花生样品来验证该测定的准确性。结果与商业 ELISA 试剂盒获得的结果非常吻合。可以想象，该方法不限于黄曲霉毒素，而是具有广泛的范围，因为它可以应用于许多其他有相应抗体的分析物。图形摘要 抗坏血酸氧化酶 (AOx) 介导的高锰酸钾 (KMnO4)-响应性抗坏血酸 (AA) 的示意图，通过与 AOx 对 AA 和 KMnO4-Mn(II) 的水解反应偶联，对黄曲霉毒素 B1 (AFB1) 进行视觉比色免疫测定-TMB 系统 [注：3,3',5,5'-四甲基联苯胺：TMB]。