A-Sense Lab, University of Antwerp, 2020 Antwerp, Belgium.
Department of Biomedical Sciences, Institute of Tropical Medicine, 2000 Antwerp, Belgium.
Anal Chem. 2021 Oct 12;93(40):13606-13614. doi: 10.1021/acs.analchem.1c02876. Epub 2021 Sep 29.
Detection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (O) that, in turn, readily reacts with -nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens.
检测痕量存在的抗原生物标志物对于医学诊断至关重要。尽管寄生虫病——人弓蛔虫病在世界范围内发生，但目前还缺乏一种足够的诊断方法。目前使用的血清学检测方法即使在可能未被注意到的感染多年后仍可能呈阳性，而由于循环寄生虫抗原浓度低，直接检测再感染或仍处于活动状态的感染仍然具有挑战性。我们报告了一种使用源自骆驼重链抗体的小重组单域抗体（纳米抗体）的时间高效夹心免疫传感器，这些纳米抗体针对特定抗原。通过使用由光催化氧化和电化学还原步骤组成的氧化还原循环，可以实现对 pg/mL 水平的增强灵敏度。光催化氧化是通过光催化剂产生单线态氧（O）来实现的，单线态氧反过来很容易与碱性条件下酶促产生的 -硝基苯酚反应。光氧化产生苯醌，苯醌被电化学还原为对苯二酚，产生安培响应。光驱动过程可以很容易地与背景分离，从而使安培检测更可靠。与使用相同纳米抗体的其他检测方案相比，用于检测弓蛔虫病抗原标志物的方法表现出优越的性能，并且至少比基于常规抗体的检测方法高出两个数量级，这为具有挑战性的低抗原水平的电化学免疫分析提供了新的机会。
