检测核基质蛋白22的电化学免疫传感器的构建  

Establishment of electrical immunosensor for the detection of nuclear matrix protein-22

作　　者：荣胜忠 邹立娜[3] 张泽 张颖聪 张慧 于洪伟[1] 潘洪志 常东[1] Rong Shengzhong;Zou Lina;Zhang Ze;Zhang Yingcong;Zhang Hui;Yu Hongwei;Pan Hongzhi;Chang Dong(Department of Clinical Laboratory,Pudong Hospital Affiliated to Fudan University,Shanghai 201399,China;Public Health School,Mudanjiang Medical University,Mudanjiang157011,China;Hongqi Hospital Affiliated to Mudanjiang Medical University,Mudanjiang 157011,China;Collaborative Research Center,Shanghai University of Medicine and Health Sciences,Shanghai 201318,China)

机构地区：[1]复旦大学附属浦东医院临床检验科,上海201399 [2]牡丹江医学院公共卫生学院,牡丹江157011 [3]牡丹江医学院附属红旗医院,牡丹江157011 [4]上海健康医学院协同研究中心,上海201318

出　　处：《中华检验医学杂志》2019年第10期841-847,共7页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(81703269,81773480,81973097,81673229,81872703,81573216);上海市浦东新区重要薄弱学科计划(PWZbr2017-02);黑龙江省自然科学基金(QC2018120);黑龙江省普通本科高等学校青年创新人才培养计划项目(UNPYSCT-2017173);黑龙江省省属高等学校基本科研业务费项目(2017-KYYWF-0648);牡丹江市科学技术计划项目(Z2016s0027)。

摘　　要：目的建立免标记电化学免疫传感器检测膀胱癌标志物核基质蛋白22(NMP22)。方法方法学建立与验证研究。收集2017年9月至2019年7月在牡丹江医学院附属红旗医院就诊的20名对照与20例膀胱癌患者的尿液对方法进行评价。采用还原性氧化石墨烯-四乙烯五胺固定金掺杂的金属有机框架材料ZIF8复合膜修饰丝网印刷电极,再将NMP22抗体固定在修饰电极表面,用牛血清白蛋白封闭。采用循环伏安和电化学阻抗法对传感器的组装进行表征。采用差分脉冲伏安法评估传感器的线性范围、回收率、精密度、选择性和稳定性。采用Mann-Whitney U检验分析实验数据。结果最佳检测条件下,传感器的线性范围为0.01~1000 ng/ml,检出限为3.33 pg/ml(S/N=3),加标回收率在97.65%~107.05%之间,利用其分析尿液中NMP22水平,膀胱癌组[66.03(4.34,91.74)ng/ml]显著高于对照组0.54(0.06,8.84)ng/ml(P=0.001)。结论该电化学免疫传感器可以灵敏、快速、准确检测尿液中NMP22,在监测肿瘤标志物方面具有潜在的应用前景。Objective A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22(NMP22)as a biomarker of bladder cancer.Methods The study was based on the establishment and validation of the methodology.Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method.A screen-printed electrode(SPE)was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine(rGO-TEPA)immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles(rGO-TEPA@Au-ZIF8).Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE,which then was blocked with bovine serum albumin to elimiate nonspecific binding sites.The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy.Differential pulse voltammetry was used to evaluate the linear range,recovery,precision,selectivity and stability.The data were analyzed by Mann-Whitney U test.Results Under optimal conditions,the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml(S/N=3)and a standard recovery of 97.65%-107.05%.The levels of NMP22 in urine samples from patients with bladder cancer[66.03(4.34,91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06,8.84)ng/ml(P=0.001).Conclusion The immunosensor can achieve sensitive,rapid and acucurate detection of NMP22,and has potential application prospects in monitoring tumor markers.

关 键 词：膀胱肿瘤 核蛋白质类 生物传感技术 电化学技术 免疫测定 石墨 聚胺类 生物标记 肿瘤 

分 类 号：R73[医药卫生—肿瘤]