摘要
目的构建一种基于三维刚性DNA桥式纳米探针(3D-TPN)的光电化学(photoelectro-chemistry,PEC)传感器,用于DNA甲基化位点分析研究。方法将二氧化钛(TiO_(2))悬浮液滴加到ITO电极表面,形成薄膜,通过电位溶出法在TiO_(2)纳米膜表面沉积金纳米粒子(AuNPs),进一步通过Au-S将三维刚性DNA桥式探针固载到电极表面。制备5-甲基胞嘧啶抗体和碲化镉量子点(Ab-CdTe QDs)偶联物。靶序列存在时,5-甲基胞嘧啶与抗体特异性结合,使Ab-CdTe QDs连接到电极表面,与AuNPs/TiO_(2)形成共敏化结构提高光电信号。在含L-抗坏血酸(AA)的磷酸盐缓冲液(PBS)中检测该传感器的光电化学信号响应。结果成功制备了三维刚性DNA桥式纳米探针。TiO_(2)悬浮液最佳浓度为4 mg/mL,5-甲基胞嘧啶抗体最佳浓度为10μg/mL。在最优实验条件下,平行检测同一条件制备的5支电极,其相对标准差(RSD)为1.8%;4周后光电流仍保持初始值的92.5%;且该传感器对目标序列有良好的选择性。DNA甲基化位点数与光电响应值呈线性关系,回归方程为ΔI=-0.0047+4.0114×N,决定系数R^(2)为0.9997。结论该光电化学传感器具有优异的稳定性和特异性,可用于多位点DNA甲基化的定量检测。
Objective To construct a photoelectrochemical(PEC)sensor based on a 3-dimensional(3-D)rigid DNA bridge nanoprobe for the analysis of DNA methylation sites.Methods Titanium dioxide(TiO_(2))suspension was dropped onto the surface of ITO electrode to form a thin film;gold nanoparticles(AuNPs)were deposited on the TiO_(2)nanofilm by potentiometric stripping,and then the 3-D rigid DNA bridge probe was fixed to the electrode surface by Au-S binding.Furthermore,the 5-methylcytosine antibody and cadmium telluride quantum dots(Ab-CdTe QDs)conjugate were prepared.In the presence of the target sequence,the 5-methylcytosine specifically bound to the antibody,and made Ab-CdTe QDs connect to the electrode surface,and then a co-sensitized structure with AuNPs/TiO_(2)was formed to enhance the photoelectric signal.The PEC signal response of the sensor was measured in phosphate buffer(PBS)containing L-ascorbic acid(AA).Results 3-D rigid DNA bridge nanoprobes were successfully prepared.The optimal concentration of TiO_(2)suspension was 4 mg/mL,and that of 5-methylcytosine antibody was 10μg/ml.Under the optimal experimental conditions,5 electrodes prepared under the same condition were tested parallelly,and the relative standard deviation(RSD)was only 1.8%.After 4 weeks,the photocurrent of the sensor still maintained 92.5%of the initial value.Meanwhile,the sensor presented good selectivity to the target sequence,and the photoelectric response value exhibited a linear relationship with the number of DNA methylation sites.The regression equation wasΔI=-0.0047+4.0114×N,and the coefficient of determination R^(2)was 0.9997.Conclusion The developed photoelectrochemical sensor involves excellent stability and specificity,and can be used for quantitative detection of multi-site DNA methylation.
作者
朱垂雨
黄辉
李艳
方立超
汪莉娜
刘华敏
李承红
曾涛
郑峻松
ZHU Chuiyu;HUANG Hui;LI Yan;FANG Lichao;WANG Lina;LIU Huamin;LI Chenghong;ZENG Tao;ZHENG Junsong(School of Laboratory Medicine and Biotechnology,Southern Medical University,Guangzhou,Guangdong Province,510515;Department of Clinical and Military Laboratory Medicine,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第7期665-672,共8页
Journal of Army Medical University
基金
国家自然科学基金面上项目(81873982)
重庆市基础研究与前沿探索专项面上项目(cstc2019jcyj-zdxm0037)
关键词
DNA甲基化
桥式纳米探针
光电化学
生物传感技术
DNA methylation
bridge DNA nanoprobes
photoelectrochemistry
biosensing techniques
