Ge Xiaoxiao, Zhang Aidong, Lin Yuehe, Du Dan
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China; School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164, United States.
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China.
Biosens Bioelectron. 2016 Jun 15;80:201-207. doi: 10.1016/j.bios.2016.01.058. Epub 2016 Jan 24.
A novel electrochemical immunosensor has been developed to detect phosphorylated proteins, phospho-p53(15) and phospho-p53(392), simultaneously. Different apoferritin templated metal phosphates were used as distinguishable signal reporters (apoferritin templated cadmium phosphates (ATCP) and apoferritin templated lead phosphates (ATLP)) to enhance the detection sensitivity. Here, magnetic Fe3O4 nanoparticles functionalized phospho-p53(15) capture antibody (MP-p53(15)c-Ab) and phospho-p53(392) capture antibody (MP-p53(392)c-Ab), respectively, were used to specifically capture phospho-p53(15) and phospho-p53(392) antigens, followed by immunorecognition with p53(15) detection antibody (p53(15)d-Ab) and p53(392) detection antibody (p53(392)d-Ab) to form sandwich-like immunocomplexes. SiO2@Au nanocomposites served as nanocarriers for co-immobilization of both d-Ab and signal reporters (ATCP/SiO2@Au/p53(15)d-Ab, ATLP/SiO2@Au/p53(392)d-Ab), which greatly amplified the detection signal. The distinguished current responses were achieved by electrochemical detection of cadmium ions and lead ions with square wave voltammetry (SWV) after dissolution with acid. The proposed immunoassay exhibited high sensitivity and selectivity for the detection of phospho-p53(15) and phospho-p53(392) simultaneously. The linear relationships between electrochemical responses and the concentrations of phospho-p53(15) and phospho-p53(392) were obtained in the range of 0.1-20 ng/mL and 0.05-20 ng/mL, respectively. The detection limits were 0.05 and 0.02 ng/mL (S/N=3), respectively. This strategy provides a new platform for simultaneous immunoassay of multiple protein biomarkers.
一种新型电化学免疫传感器已被开发出来，用于同时检测磷酸化蛋白、磷酸化p53(15)和磷酸化p53(392)。不同的脱铁铁蛋白模板化金属磷酸盐被用作可区分的信号报告物（脱铁铁蛋白模板化镉磷酸盐（ATCP）和脱铁铁蛋白模板化铅磷酸盐（ATLP）），以提高检测灵敏度。在此，分别用功能化的磷酸化p53(15)捕获抗体（MP-p53(15)c-Ab）和磷酸化p53(392)捕获抗体（MP-p53(392)c-Ab）的磁性Fe3O4纳米颗粒特异性捕获磷酸化p53(15)和磷酸化p53(392)抗原，随后用p53(15)检测抗体（p53(15)d-Ab）和p53(392)检测抗体（p53(392)d-Ab）进行免疫识别，形成夹心样免疫复合物。SiO2@Au纳米复合材料用作纳米载体，用于共固定检测抗体和信号报告物（ATCP/SiO2@Au/p53(15)d-Ab，ATLP/SiO2@Au/p53(392)d-Ab），极大地放大了检测信号。在用酸溶解后，通过方波伏安法（SWV）对镉离子和铅离子进行电化学检测，实现了明显的电流响应。所提出的免疫分析方法对同时检测磷酸化p53(15)和磷酸化p53(392)具有高灵敏度和选择性。在0.1 - 20 ng/mL和0.05 - 20 ng/mL范围内分别获得了电化学响应与磷酸化p53(15)和磷酸化p53(392)浓度之间的线性关系。检测限分别为0.05和0.02 ng/mL（S/N = 3）。该策略为多种蛋白质生物标志物的同时免疫分析提供了一个新平台。
