Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Collaborative Innovation Center of Detection Technology for Haixi Food Safety and Products (Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350116, PR China.
Institute of Environmental and Analytical Science, School of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, Henan, PR China.
Anal Chim Acta. 2017 Apr 29;964:67-73. doi: 10.1016/j.aca.2017.02.004. Epub 2017 Feb 20.
Methods based on nanostructures have been developed for potentiometric immunosensors, but most involve low sensitivity or weak signal output and are unsuitable for routine use in diagnosis. Herein, we devise an in-situ signal-amplification strategy for enhanced electrical readout of potentiometric immunosensor toward target prostate-specific antigen (PSA, one kind of cancer biomarkers), based on polyion oligonucleotide-labeled gold nanoparticles (AuNPs). To decrease the background signal, monoclonal anti-human PSA capture antibody was covalently conjugated onto an activated glassy carbon electrode via typical carbodiimide coupling. AuNPs heavily functionalized with the polyion oligonucleotides and polyclonal anti-PSA detection antibodies (pAb-AuNP-DNA) were utilized as the signal-generation nanotags. In the presence of target PSA, a sandwich-type immunoreaction was executed between capture antibody and detection antibody on the electrode. The detectable signal derived from the shift in the electric potential as a result of the change in the surface charge before and after the antigen-antibody reaction. With target PSA increased, the captured pAb-AuNP-DNA to the electrode accompanying detection antibody increased, thereby resulting in the change of the electrode potential. Due to numerous polyion oligonucleotides with the negative charge, the signal readout amplified. Under the optimal conditions, the shift in the output potential was proportional to the logarithm of target PSA concentration and displayed a dynamic linear range from 0.05 to 20 ng mL with a detection limit of 13.6 pg mL. An intermediate precision of ≤13.2% was accomplished with the batch-to-batch identification. The selectivity was acceptable. The method accuracy was evaluated for human serum specimens, and gave the consistent results between the potentiometric immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA).
基于纳米结构的方法已被开发用于电势免疫传感器，但大多数方法涉及低灵敏度或弱信号输出，不适合常规用于诊断。在此，我们设计了一种基于聚离子寡核苷酸标记的金纳米粒子（AuNPs）的原位信号放大策略，用于增强对目标前列腺特异性抗原（PSA，一种癌症生物标志物）的电势免疫传感器的电读出。为了降低背景信号，通过典型的碳二亚胺偶联将单克隆抗人 PSA 捕获抗体共价连接到活化的玻碳电极上。AuNPs 被聚离子寡核苷酸和多克隆抗 PSA 检测抗体（pAb-AuNP-DNA）高度功能化，用作信号产生的纳米标签。在存在目标 PSA 的情况下，在电极上执行捕获抗体和检测抗体之间的三明治型免疫反应。检测信号源自抗原-抗体反应前后表面电荷变化引起的电位变化。随着目标 PSA 的增加，与电极结合的捕获 pAb-AuNP-DNA 随检测抗体增加，从而导致电极电位变化。由于带负电荷的聚离子寡核苷酸众多，信号读出被放大。在最佳条件下，输出电位的变化与目标 PSA 浓度的对数成正比，呈现出 0.05 至 20 ng mL 的动态线性范围，检测限为 13.6 pg mL。批间识别的中间精密度≤13.2%。选择性可以接受。该方法用于人血清标本的准确性进行了评估，电势免疫传感器和参考酶联免疫吸附测定（ELISA）之间给出了一致的结果。
