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基于三维刚性DNA桥式纳米探针的DNA甲基化光电化学传感器的制备和效果评价
Preparation and effectiveness evaluation of DNA methylation photoelectrochemical sensor based on 3-D rigid DNA bridge nanoprobes
- 陆军军医大学学报 2022年44卷第7期 页码：665-672
- 作者机构：
1.510515 广州, 南方医科大学检验与生物技术学院
2.400038 重庆, 陆军军医大学(第三军医大学)药学与检验医学系临床检验与野战检验教研室
- 作者简介：
[曾涛, E-mail: zengt@smu.edu.cnZENG Tao, E-mail: zengt@smu.edu.cn
[郑峻松, E-mail: zhengalpha@sina.comZHENG Junsong, E-mail: zhengalpha@sina.com
- 基金信息：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)
DOI：10.16016/j.2097-0927.202112211
中图分类号： R197.39;R312;R446收稿：2021-12-23，
修回：2022-01-24，
纸质出版：2022-04-15
- 稿件说明：
移动端阅览
朱垂雨, 黄辉, 李艳, 等. 基于三维刚性DNA桥式纳米探针的DNA甲基化光电化学传感器的制备和效果评价[J]. 陆军军医大学学报, 2022,44(7):665-672.
Chuiyu ZHU, Hui HUANG, Yan LI, et al. Preparation and effectiveness evaluation of DNA methylation photoelectrochemical sensor based on 3-D rigid DNA bridge nanoprobes[J]. Journal of Army Medical University, 2022, 44(7): 665-672.
朱垂雨, 黄辉, 李艳, 等. 基于三维刚性DNA桥式纳米探针的DNA甲基化光电化学传感器的制备和效果评价[J]. 陆军军医大学学报, 2022,44(7):665-672. DOI： 10.16016/j.2097-0927.202112211.
Chuiyu ZHU, Hui HUANG, Yan LI, et al. Preparation and effectiveness evaluation of DNA methylation photoelectrochemical sensor based on 3-D rigid DNA bridge nanoprobes[J]. Journal of Army Medical University, 2022, 44(7): 665-672. DOI： 10.16016/j.2097-0927.202112211.
摘要
目的
2
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
2
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
2
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10 μg/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=-0.004 7+4.011 4×N，决定系数
R
2
为0.999 7。
结论
2
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。
Abstract
Objective
2
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
2
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
2
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 10 μg/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 was ΔI=-0.004 7+4.011 4×N
and the coefficient of determination
R
2
was 0.999 7.
Conclusion
2
The developed photoelectrochemical sensor involves excellent stability and specificity
and can be used for quantitative detection of multi-site DNA methylation.
关键词
Keywords
references
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