Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China.
School of Pharmaceutical Sciences, Jiangnan University, Wuxi, 214122, China.
Anal Bioanal Chem. 2021 Nov;413(28):7081-7091. doi: 10.1007/s00216-021-03677-x. Epub 2021 Sep 29.
A colorimetric biosensor assay has been developed for Cd and Hg detection based on Cd-dependent DNAzyme cleavage and Hg-binding-induced conformational switching of the G-quadruplex fragment. Two types of multifunctional magnetic beads (Cd-MBs and Hg-MBs) were synthesized by immobilizing two functionalized DNA sequences on magnetic beads via avidin-biotin chemistry. For Cd detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd-specific DNAzyme (Cdzyme). In the presence of Cd, the PS substrate is cleaved by Cdzyme, and single-stranded DNA is released as the signal transduction sequence. After molecular assembly with the other two oligonucleotides, duplex DNA is produced, and it can be recognized and cleaved by FokI endonuclease. Thus, a signal output component consisting of a G-quadruplex fragment is released, which catalyzes the oxidation of ABTS with the addition of hemin and HO, inducing a remarkably amplified colorimetric signal. To rule out false-positive results and reduce interference signals, Hg-MBs modified with poly-T fragments were used as Hg accumulation probes during the course of Cd detection. On the other hand, Hg-MBs can perform their second function in Hg detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg, the G-quadruplex structure in Hg-MBs is disrupted upon Hg binding. In the absence of Hg, an intensified color change can be observed by the naked eye for the formation of intact G-quadruplex/hemin DNAzymes. The biosensor assay exhibits excellent selectivity and high sensitivity. The detection limits for Cd and Hg are 1.9 nM and 19.5 nM, respectively. Moreover, the constructed sensors were used to detect environmental water samples, and the results indicate that the detection system is reliable and could be further used in environmental monitoring. The design strategy reported in this study could broadly extend the application of metal ion-specific DNAzyme-based biosensors.
基于依赖于 Cd 的 DNA 酶切割和 G-四链体片段的 Hg 结合诱导构象转变，开发了用于 Cd 和 Hg 检测的比色生物传感器测定法。通过将两种功能化 DNA 序列通过亲和素-生物素化学固定在磁性珠上，合成了两种类型的多功能磁性珠（Cd-MBs 和 Hg-MBs）。用于 Cd 检测时，Cd-MBs 用作识别探针，其用单个硫代磷酸核糖核苷酸（rA）底物（PS 底物）和 Cd 特异性 DNA 酶（Cdzyme）进行修饰。在 Cd 的存在下，PS 底物被 Cdzyme 切割，单链 DNA 作为信号转导序列释放。与另外两个寡核苷酸分子组装后，产生双链 DNA，其可被 FokI 内切酶识别和切割。因此，释放出由 G-四链体片段组成的信号输出成分，其催化在血红素和 HO 的加入下 ABTS 的氧化，引起明显放大的比色信号。为了排除假阳性结果并减少干扰信号，在 Cd 检测过程中，使用修饰有聚 T 片段的 Hg-MBs 作为 Hg 积累探针。另一方面，Hg-MBs 可以通过改变 G-四链体/血红素 DNA 酶的催化活性在 Hg 检测中执行其第二个功能。在 Hg 的存在下，Hg 结合会破坏 Hg-MBs 中的 G-四链体结构。在没有 Hg 的情况下，通过肉眼可以观察到完整 G-四链体/血红素 DNA 酶形成时的颜色变化增强。该生物传感器测定法表现出优异的选择性和高灵敏度。Cd 和 Hg 的检测限分别为 1.9 nM 和 19.5 nM。此外，构建的传感器用于检测环境水样，结果表明该检测系统可靠，可进一步用于环境监测。本研究中报道的设计策略可以广泛扩展基于金属离子特异性 DNA 酶的生物传感器的应用。
