Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, People's Republic of China.
Ji'an Vocational Polytechnic College, Ji'an, 343000, People's Republic of China.
Mikrochim Acta. 2018 Jan 10;185(2):92. doi: 10.1007/s00604-017-2651-z.
The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B (AFB). It is based on the just-in-time generation of an MnO 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 (KMnO) is converted into manganese dioxide (MnO) 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), KMnO is reduced to Mn(II) ions. This results in a decrease in the amount of MnO nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB and magnetic beads carrying bovine serum albumin conjugated to AFB are used for the determination of AFB. In presence of AFB, it will compete with the BSA-conjugated AFB (on the magnetic beads) for the labeled antibody against AFB on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 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 AFB concentrations in the range from 0.1 to 100 ng mL, with a 0.1 ng mL detection limit (at the 3S 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 abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B (AFB) by coupling with hydrolytic reaction of AOx toward AA and the KMnO-Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].
作者描述了一种用于模型分析物黄曲霉毒素 B (AFB) 的比色免疫分析方法。它基于 MnO 纳米催化剂的即时生成。与以前开发的免疫分析不同，比色反应依赖于没有底物辅助的氧化酶模拟物的即时形成。高锰酸钾 (KMnO) 转化为二氧化锰 (MnO)，后者作为氧化酶模拟物，通过氧气催化 3,3',5,5'-四甲基联苯胺 (TMB) 的氧化，生成蓝色产物。在抗坏血酸 (AA) 的存在下，KMnO 被还原为 Mn(II) 离子。这导致 MnO 纳米催化剂的量减少。因此，TMB 的氧化不会发生。通过添加抗坏血酸氧化酶，AA 转化为脱氢抗坏血酸，无法还原 KMnO。基于这些观察结果，开发了一种比色竞争酶免疫分析法，其中使用抗坏血酸氧化酶和金纳米颗粒标记的针对 AFB 的抗体以及携带与 AFB 结合的牛血清白蛋白的磁性珠用于测定 AFB。在存在 AFB 的情况下，它将与磁性珠上的 BSA 结合的 AFB（与磁性珠结合）竞争针对金纳米颗粒上的 AFB 的标记抗体。这使得与磁性珠结合的抗坏血酸氧化酶/抗-AFB 抗体标记的金纳米颗粒的量减少，并且抗坏血酸增加。在最佳条件下，吸光度（在 652nm 处测量）随 AFB 浓度在 0.1 至 100ngmL 的范围内增加而降低，检测限为 0.1ngmL（在 3S 水平）。通过分析添加花生样品验证了该测定方法的准确性。结果与使用商业 ELISA 试剂盒获得的结果吻合良好。可以想象，该方法不仅限于黄曲霉毒素，而且具有广泛的应用范围，因为它可应用于许多其他具有相应抗体的分析物。
