Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, College of Chemistry and Materials, Nanning Normal University, Nanning 530001, P. R. China.
Analyst. 2022 May 30;147(11):2412-2424. doi: 10.1039/d2an00367h.
A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 (ORAOV1) gene was constructed exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. Capture DNA with a sulfhydryl group at its 3' terminus was modified onto the surface of a bare gold electrode an Au-S bond. Assisted DNA hybridized with basal DNA to form hybrid DNA in advance, and ORAOV1 gene hybridized continuously with such a hybrid DNA from the other terminus to construct intact double-stranded DNA. Exonuclease III digested basal DNA in such intact double-stranded DNA specifically, and both ORAOV1 gene and assisted DNA were released into solution. ORAOV1 gene induced another intact double-stranded DNA digestion for target recycling, while assisted DNA hybridized with the capture DNA to form double-stranded DNA on the modified electrode surface. Unhybridized capture DNA on the modified electrode surface was hydrolyzed by RecJf exonuclease to reduce the background electrochemical signal. The 3' terminus of double-stranded DNA on the modified electrode surface was prolongated to be guanine-rich oligonucleotides under the catalysis of terminal deoxynucleotidyl transferase. In the presence of K ions, hemin adsorbed onto guanine-rich oligonucleotides to construct a G-quadruplex/hemin complex with a large steric hindrance effect to efficiently avoid the charge transfer of the [Fe(CN)] probe toward the electrode surface. The electrochemical impedance value was increased significantly after the addition of ORAOV1 gene exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. The electrochemical impedance value was linearly related to the logarithmic concentration of ORAOV1 gene in the range from 0.05 fM to 20 pM, and the detection limit of ORAOV1 gene was low to 0.019 fM. This biosensor was used to detect ORAOV1 gene in complicated human saliva samples with satisfactory results.
构建了一种无标记和超灵敏的电化学生物传感器，用于检测口腔癌过表达 1（ORAOV1）基因，该传感器基于外切酶 III 辅助的目标循环和双酶辅助信号放大策略。巯基修饰的捕获 DNA 修饰在裸金电极表面形成 Au-S 键。辅助 DNA 与基底 DNA 预先杂交形成杂交 DNA，然后 ORAOV1 基因从另一个末端连续与该杂交 DNA 杂交，构建完整的双链 DNA。外切酶 III 特异性地消化完整双链 DNA 中的基底 DNA，同时将 ORAOV1 基因和辅助 DNA 释放到溶液中。ORAOV1 基因诱导另一个完整双链 DNA 进行目标循环，而辅助 DNA 则与捕获 DNA 杂交，在修饰电极表面形成双链 DNA。修饰电极表面未杂交的捕获 DNA 被 RecJf 外切酶水解，以降低背景电化学信号。修饰电极表面双链 DNA 的 3'末端在末端脱氧核苷酸转移酶的催化下延伸为富含鸟嘌呤的寡核苷酸。在 K 离子存在下，血红素吸附在富含鸟嘌呤的寡核苷酸上，形成具有大位阻效应的 G-四链体/血红素复合物，有效地阻止 [Fe(CN)]探针向电极表面的电荷转移。加入 ORAOV1 基因后，电化学阻抗值显著增加，这是在外切酶 III 辅助的目标循环和双酶辅助信号放大策略的作用下。电化学阻抗值与 ORAOV1 基因的对数浓度在 0.05 fM 至 20 pM 范围内呈线性关系，检测 ORAOV1 基因的检测限低至 0.019 fM。该生物传感器用于检测复杂的人唾液样本中的 ORAOV1 基因，结果令人满意。
