Fang Xian, Bai Lijuan, Han Xiaowei, Wang Jiao, Shi Anqi, Zhang Yuzhong
College of Chemistry and Materials Science, Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Laboratory of Molecule-Based Materials, and Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China.
School of Chemistry and Ecological Engineering, Guangxi University for Nationalities, Nanning 530006, People's Republic of China.
Anal Biochem. 2014 Sep 1;460:47-53. doi: 10.1016/j.ab.2014.05.019. Epub 2014 Jun 2.
In this study, an ultra-sensitive hairpin DNA-based electrochemical DNA biosensor for K-ras gene detection is described. Gold nanoparticles (Au-NPs) and horseradish peroxidase (HRP)-streptavidin capped Au-NPs (HAS) conjugates are used for signal amplification. Initially, hairpin DNA dually labeled with thiol at its 5' end and with biotin at its 3' end is immobilized on the surface of Au-NPs modified electrode, and the hairpin DNA is in a "closed" state; hence, the HAS conjugates are shielded from being approached by the biotin due to steric hindrance. However, in the presence of target DNA, the target DNA hybridizes with the loop structure of hairpin DNA and causes conformational change, resulting in biotin forced away from the electrode surface, thereby becoming accessible for the HAS conjugates. Thus, the HAS conjugates are linked to the electrode surface via the specific interaction between biotin and streptavidin. Electrochemical detection was performed in phosphate-buffered saline (PBS) containing tetramethylbenzidine (TMB) and H2O2. Under optimal conditions, the peak current differences (ΔI) are linear with the target DNA in the range from 0.1 fM to 1 nM with a detection limit of 0.035 fM. Furthermore, this biosensor also demonstrates its excellent specificity for single-base mismatched DNA.
在本研究中，描述了一种用于检测K-ras基因的基于超灵敏发夹DNA的电化学DNA生物传感器。金纳米颗粒（Au-NPs）和辣根过氧化物酶（HRP）-链霉亲和素包覆的Au-NPs（HAS）共轭物用于信号放大。最初，在其5'端用硫醇和3'端用生物素双重标记的发夹DNA固定在Au-NPs修饰电极的表面，且发夹DNA处于“封闭”状态；因此，由于空间位阻，HAS共轭物被屏蔽而无法接近生物素。然而，在存在靶DNA的情况下，靶DNA与发夹DNA的环结构杂交并引起构象变化，导致生物素被迫远离电极表面，从而使得HAS共轭物能够接近。因此，HAS共轭物通过生物素与链霉亲和素之间的特异性相互作用连接到电极表面。在含有四甲基联苯胺（TMB）和H2O2的磷酸盐缓冲盐水（PBS）中进行电化学检测。在最佳条件下，峰电流差（ΔI）与0.1 fM至1 nM范围内的靶DNA呈线性关系，检测限为0.035 fM。此外，该生物传感器对单碱基错配DNA也表现出优异的特异性。
