College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of MoleCular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, P. R. China.
Analyst. 2020 Mar 16;145(6):2389-2397. doi: 10.1039/c9an02470k.
In this work, we report a simple ratiometric electrochemiluminescence (ECL) method for ultra-sensitive immunoanalysis. A glassy carbon electrode (GCE) was modified by a mixture of porous g-C3N4 nanosheets and carbon nanotubes (CNTs). Secondary antibodies were labeled using CuS nanoparticles as the tags. After immune recognition, CuS nanoparticles in the immunocomplex were dissolved as Cu2+, which can quench the ECL of g-C3N4. The amount of Cu2+ was determined to quantify the concentration of the target antigen. To enhance the sensitivity, Cu2+ ions were firstly enriched and reduced to Cu on the surface of GCE/CNTs-g-C3N4, and the cathodic ECL of g-C3N4 was measured as the reference signal in the ratiometric ECL measurements. After applying a potential of 0.6 V (vs. Ag/AgCl) for 6 s, Cu was dissolved as Cu2+, which can quench the ECL of g-C3N4 with much higher efficiency because the freshly dissolved Cu2+ ions were distributed mainly within the Helmholtz layer of GCE/CNTs-g-C3N4. By using the ECL intensity ratio of GCE/CNTs-g-C3N4 (Cu2+) to GCE/CNTs-g-C3N4 (Cu) measured under the potentiostatic model as the signal indictor, the ratiometric ECL method was used to detect a biomarker of alpha fetoprotein with the limit of detection of 0.1 fg mL-1. It was shown that the influence of the difference in electrode modification and ECL measurement conditions on the determination of Cu2+ is suppressed greatly in the ratiometric ECL method. The combination of ratiometric ECL with electrochemical enrichment and biometallization is a useful strategy to enhance the sensitivity and reproducibility in immunoanalysis.
在这项工作中，我们报告了一种简单的比率型电化学发光（ECL）方法，用于超灵敏免疫分析。玻碳电极（GCE）通过多孔 g-C3N4 纳米片和碳纳米管（CNTs）的混合物进行修饰。使用 CuS 纳米粒子作为标记物标记二级抗体。免疫识别后，免疫复合物中的 CuS 纳米粒子溶解为 Cu2+，可以猝灭 g-C3N4 的 ECL。通过测定 Cu2+的量来定量检测目标抗原的浓度。为了提高灵敏度，首先在 GCE/CNTs-g-C3N4 表面富集和还原 Cu2+为 Cu，然后作为参考信号在比率型 ECL 测量中测量 g-C3N4 的阴极 ECL。施加 0.6 V（相对于 Ag/AgCl）的电位 6 s 后，Cu 溶解为 Cu2+，由于新鲜溶解的 Cu2+离子主要分布在 GCE/CNTs-g-C3N4 的亥姆霍兹层内，因此可以更高效率猝灭 g-C3N4 的 ECL。通过使用在恒电位模型下测量的 GCE/CNTs-g-C3N4（Cu2+）与 GCE/CNTs-g-C3N4（Cu）的 ECL 强度比作为信号指示剂，使用比率型 ECL 方法检测甲胎蛋白的生物标志物，检测限为 0.1 fg mL-1。结果表明，在比率型 ECL 方法中，大大抑制了电极修饰和 ECL 测量条件差异对 Cu2+测定的影响。比率型 ECL 与电化学富集和生物金属化相结合是提高免疫分析灵敏度和重现性的有效策略。
