Research Center of Mycotoxins, Jiangxi Agricultural University, Nanchang, 330045, China; Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables/Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits &Vegetables in Jiangxi Province, Jiangxi Agricultural University, Nanchang, 330045, China.
Key Lab for Agro-product Processing and Quality Control of Nanchang City, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045, China.
Biosens Bioelectron. 2023 Jan 15;220:114906. doi: 10.1016/j.bios.2022.114906. Epub 2022 Nov 12.
Nanobody(Nb) is too small to carry more signal reporters, which often leads to low sensitivity in immunoassay. Herein, we proposed a novel immunoprobe integration of Nb and biomimetic mineralized metal-organic frameworks(MOF), in which plenty of succinylated horseradish peroxidase(sHRP) were encapsulated within a single MOF and the Nb was assembled on the biomimetic mineralized MOF. It overcomes the dilemma that the Nb is difficult to carry more signal reporters. Meanwhile, the mineralized MOF can protect the sHRP from denaturation and facilitate the transport of substrates to the active sites of sHRP. Electrosensing of aflatoxin B(AFB) was realized with a competitive format in which the target AFB and immobilized artificial antigen were competing for binding with the immunoprobe. Additionally, the detection signal was enlarged by the catalysis of this immunoprobe to 4-chloro-1-naphthol for producing precipitations, which blocked the channels of the immunoprobe and the redox probes of Fe(CN) was difficult to reach the electrode surface through the channels. Hence, the as-prepared immunosensor exhibited good voltammetry responses towards the determined AFB in a linear range of 50.0 fg/mL - 20.0 ng/mL with a detection limit of 20.0 fg/mL. The specificity, stability, and reproducibility of this immunosensor were satisfactory. This work may provide an alternative idea for the application of Nb in immunoassay, and the idea may also be applicable to other bio-recognition elements for immunoassay.
纳米抗体（Nb）太小，无法携带更多的信号报告分子，这通常导致免疫分析的灵敏度较低。在此，我们提出了一种新型免疫探针，将 Nb 与仿生矿化金属有机骨架（MOF）集成在一起，其中大量琥珀酰化辣根过氧化物酶（sHRP）被包裹在单个 MOF 内，而 Nb 则组装在仿生矿化 MOF 上。它克服了 Nb 难以携带更多信号报告分子的困境。同时，矿化 MOF 可以保护 sHRP 免于变性，并促进底物向 sHRP 的活性位点的传输。通过竞争格式实现了黄曲霉毒素 B（AFB）的电传感，其中目标 AFB 和固定化人工抗原竞争与免疫探针结合。此外，通过该免疫探针对 4-氯-1-萘酚的催化作用放大了检测信号，从而产生沉淀，这阻止了免疫探针的通道以及 Fe(CN)的氧化还原探针到达电极表面。因此，所制备的免疫传感器在 50.0 fg/mL-20.0 ng/mL 的线性范围内对测定的 AFB 表现出良好的伏安响应，检测限为 20.0 fg/mL。该免疫传感器具有良好的特异性、稳定性和重现性。这项工作可为 Nb 在免疫分析中的应用提供另一种思路，该思路也可能适用于免疫分析中的其他生物识别元件。
