Laboratory of Process Engineering and Environment, Faculty of Sciences and Techniques, Hassan II University of Casablanca, P.A. 146, Mohammedia, Morocco.
Mikrochim Acta. 2022 May 3;189(5):209. doi: 10.1007/s00604-022-05302-9.
In contrast to reported enzyme-based immunoassays, an enzyme-free immunoassay (optical and electrochemical) is presented here for the first time that can be used as point-of-need detection bioplatforms of bovine IgG as goat milk adulterant. In the first format, Prussian blue nanoparticles (PBNPs) were used as antibody catalytic labels in a competitive colorimetric microplate immunoassay. Absorbance measurement was performed photometrically at 450 nm. After in-depth optimization, excellent sensitivity was achieved (0.01% cow/goat volume ratio), which is 100 times lower than the limit allowed by the European legislation (EL) (1% v/v), thanks to the high catalytic activity of PBNPs compared with natural peroxidase. Moreover, the antibody-PBNPs bioconjugates showed excellent stability over 4 weeks (> 94% of the initial response) confirming the successful anchoring of the antibodies to the surface of the PBNPs. On the other hand, a label-free voltammetric immunoassay for the detection of bovine IgG was developed. The sensing principle was based on the hindrance of charge transfer between ferri-ferrocyanide redox couple and the screen-printed gold electrodes modified with bovine IgG antibody. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step modification of the electrode surface. Under optimal conditions, this single-step electrochemical analysis achieved a high sensitivity of 0.1% (cow/goat) when monitoring the ferrocyanide oxidation at + 0.092 V (vs. Ag/AgCl) using differential pulse voltammetry (DPV). The selectivity of the developed immunoassays was evaluated for different species of milk of similar composition, and both immunoassays exhibited a selective response only to bovine IgG. Unlike conventional immunoassays, the developed enzyme-free immunoassays have many attractive features for the detection of milk adulteration, whether they are used in quality control laboratories for routine milk analysis (optical immunoassay) or at on-site checkpoints (electrochemical immunoassay) using wireless electrochemical detectors. The sensors provide high sensitivity (≤ 0.1%), excellent precision (RSD < 6%), low cost (no enzyme is required) and ease of operation, including handling of milk samples.
与报道的基于酶的免疫分析相反，本文首次提出了一种无酶免疫分析（光学和电化学），可作为牛 IgG 作为羊奶掺杂物的即时检测生物平台。在第一种格式中，普鲁士蓝纳米粒子 (PBNP) 被用作竞争性比色微板免疫测定中的抗体催化标记。在 450nm 处进行吸光度光度法测量。经过深入优化，实现了优异的灵敏度（牛/羊体积比 0.01%），比欧洲法规 (EL) 允许的最低限（1%v/v）低 100 倍，这要归功于 PBNP 与天然过氧化物酶相比的高催化活性。此外，抗体-PBNP 生物缀合物在 4 周以上表现出优异的稳定性（初始响应的 >94%），证实了抗体成功地固定在 PBNP 的表面上。另一方面，开发了一种用于检测牛 IgG 的无标记伏安免疫测定。传感原理基于在金电极表面上修饰有牛 IgG 抗体的情况下，阻碍铁氰化物/亚铁氰化物氧化还原对之间的电荷转移。循环伏安法 (CV) 和电化学阻抗谱 (EIS) 用于表征电极表面的逐步修饰。在最佳条件下，使用差分脉冲伏安法 (DPV) 在监测+0.092V（相对于 Ag/AgCl）处的亚铁氰化物氧化时，这种单步电化学分析实现了 0.1%（牛/羊）的高灵敏度。评估了开发的免疫分析对具有相似组成的不同种类的牛奶的选择性，并且两种免疫分析仅对牛 IgG 表现出选择性响应。与传统免疫分析不同，开发的无酶免疫分析具有许多用于检测牛奶掺假的吸引人的特点，无论是在用于常规牛奶分析的质量控制实验室中（光学免疫分析）还是在使用无线电化学探测器的现场检查站（电化学免疫分析）中。传感器提供高灵敏度（≤0.1%）、优异的精度（RSD<6%）、低成本（无需酶）和易于操作，包括处理牛奶样品。
