Department of Molecular Sciences and Nanosystems, University Ca'Foscari of Venice, via Torino 155, 30172 Venezia Mestre, Italy.
Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", 34100 Trieste, Italy.
Sensors (Basel). 2019 Mar 11;19(5):1233. doi: 10.3390/s19051233.
Celiac disease (CD) is a gluten-dependent autoimmune disorder affecting a significant percentage of the general population, with increasing incidence particularly for children. Reliable analytical methods suitable for the serological diagnosis of the disorder are urgently required for performing both the early diagnosis and the follow-up of a patient adhering to a gluten-free diet. Herein we report on the preparation and application of a novel electrochemical immunosensor based on the use of ensembles of gold nanoelectrodes (NEEs) for the detection of anti-tissue transglutaminase (anti-tTG), which is considered one reliable serological marker for CD. To this end, we take advantage of the composite nature of the nanostructured surface of membrane-templated NEEs by functionalizing the polycarbonate surface of the track-etched membrane with tissue transglutaminase. Incubation of the functionalized NEE in anti-tTG samples results in the capture of the anti-tTG antibody. Confirmation of the recognition event is achieved by incubating the NEE with a secondary antibody labelled with horseradish peroxidase (HRP): in the presence of H₂O₂ as substrate and hydroquinone as redox mediator, an electrocatalytic current is indeed generated whose increment is proportional to the amount of anti-tTG captured from the sample. The optimized sensor allows a detection limit of 1.8 ng mL, with satisfactory selectivity and reproducibility. Analysis of serum samples from 28 individuals, some healthy and some affected by CD, furnished analytical results comparable with those achieved by classical fluoroenzyme immunoassay (FEIA). We note that the NEE-based immunosensor developed here detects the IgG isotype of anti-tTG, while FEIA detects the IgA isotype, which is not a suitable diagnostic marker for IgA-deficient patients.
乳糜泻（CD）是一种依赖于麸质的自身免疫性疾病，影响着相当大比例的普通人群，尤其是儿童的发病率呈上升趋势。为了进行疾病的早期诊断和对坚持无麸质饮食的患者的随访，迫切需要可靠的分析方法来进行血清学诊断。在此，我们报告了一种新型电化学免疫传感器的制备和应用，该传感器基于使用金纳米电极（NEE）的集合来检测抗组织转谷氨酰胺酶（anti-tTG），anti-tTG 被认为是 CD 的一种可靠的血清学标志物。为此，我们利用了膜模板化 NEE 的纳米结构表面的复合性质，通过用组织转谷氨酰胺酶对聚碳酸酯膜的多孔表面进行功能化。将功能化的 NEE 孵育在抗-tTG 样品中，可捕获抗-tTG 抗体。通过将 NEE 与标记有辣根过氧化物酶（HRP）的二级抗体孵育来确认识别事件：在 H₂O₂作为底物和对苯二酚作为氧化还原介体的存在下，确实会产生电催化电流，其增量与从样品中捕获的抗-tTG 的量成正比。优化后的传感器允许检测限为 1.8 ng mL，具有令人满意的选择性和重现性。对来自 28 个人的血清样本进行分析，其中一些人健康，一些人患有 CD，得出的分析结果与经典荧光酶免疫分析（FEIA）相当。我们注意到，这里开发的基于 NEE 的免疫传感器检测抗-tTG 的 IgG 同种型，而 FEIA 检测 IgA 同种型，对于 IgA 缺乏的患者，IgA 同种型不是合适的诊断标志物。
