基于微纳米技术的免疫检测新方法  

Construction of a new immunodetection method based on micro-nanometer technology

作　　者：吴玉娟 李姝亭 翟荣云 张玮莹 郭向东 贲亚琍[1] WU Yu-juan;LI Shu-ting;ZHAI Rong-yun;ZHANG Wei-ying;GUO Xiang-dong;BEN Ya-li(School of Medicine,Jianghan University,Wuhan 430056,China;College of Life Sciences,Jianghan University,Wuhan 430056,China)

机构地区：[1]江汉大学医学部,武汉430056 [2]江汉大学生命科学学院,武汉430056

出　　处：《兰州大学学报(自然科学版)》2025年第2期231-237,245,共8页Journal of Lanzhou University(Natural Sciences)

基　　金：国家自然科学基金项目(11804121);中央引领地方科技发展专项资金项目(2016ZYYD049)。

摘　　要：通过构建一种基于微纳米技术的免疫检测新方法,对结直肠癌患者外周血中循环肿瘤DNA(ctDNA)的PIK3CA基因E542k甲基化的表达量进行高灵敏度、高特异性检测.制备线性阵列结构的微芯片,在其表面孵育功能化的肽核酸金纳米探针(PNA-AuNPs)复合物、ctDNA及5'-甲基胞嘧啶单克隆抗体-羧基化多壁碳纳米管复合物制备出ctDNA纳米生物传感器,采用电化学工作站检测并通过回收实验验证其对临床血浆中ctDNA浓度检测的可行性.结果表明,该生物传感器能够特异性检测PIK3CA基因E542k甲基化水平,检测范围为50~10~4 fmol/L,最低检测限为10 fmol/L,回收率为89.11%~102.64%;分别对正常人与结直肠癌患者血浆构建的传感器进行电化学检测,发现电流峰值具有显著性差异(P<0.01),通过线性方程可以计算出患者血浆中的PIK3CA基因E542k甲基化水平;采用共聚焦显微镜验证,发现正常人与结直肠癌患者构建的传感器中ctDNA荧光染色表达量具有明显不同,证明其可以用于临床血样检测ctDNA浓度.To construct a new immunodetection method based on micro-nanometer technology,for the catalytic subunit of phosphatidylinositol 4,5-diphosphate 3-kinase of circulating tumor DNA (ctDNA) in the peripheral blood of colorectal cancer patients,with a high sensitivity and specificity detection of the expression level of E542k methylation in PIK3CA gene.Linear array microchips were fabricated,and a ctDNA nanobiosensor using peptide nucleic acids,probe-gold nanoparticle complex,ctDNA and anti-5-methylcytosine monoclonal antibody-multi-walled carbon nanotubes-COOH complex was demonstrated.The electrochemical workstation was used to detect the current and feasibility of the biosensor for detecting ctDNA concentration in clinical plasma verified through recycling experiments.The results showed that the biosensor could specifically detect E542k methylation of the PIK3CA gene,in the detection range of 50 to 10~4 fmol/L with a low detection limit of 10 fmol/L.The recovery rate of the biosensor ranged from 89.11%to 102.64%,the peak current detection of the biosensor constructed from the plasma of normal individuals and colorectal cancer patients showed significant differences (P<0.01),and the PIK3CA gene E542k methylation concentration in the plasma of colorectal cancer patients was calculated by a linear equation.It could be seen that the expression levels of ctDNA fluorescence staining in the biosensor constructed from normal individuals and colorectal cancer patients were significantly different by the confocal microscope,proving the feasibility of the biosensor for clinical blood sample detection of ctDNA.

关 键 词：生物传感器 羧基化多壁碳纳米管 循环肿瘤DNA 结直肠癌 液态活检 

分 类 号：R73[医药卫生—肿瘤]