Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2021-09-29 , DOI: 10.1007/s00216-021-03677-x Dawei Li 1 , Shen Ling 1 , Xinru Cheng 1 , Zhaoqi Yang 2 , Bei Lv 3
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A colorimetric biosensor assay has been developed for Cd2+ and Hg2+ detection based on Cd2+-dependent DNAzyme cleavage and Hg2+-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 Cd2+ detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd2+-specific DNAzyme (Cdzyme). In the presence of Cd2+, 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 H2O2, 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 Hg2+ accumulation probes during the course of Cd2+ detection. On the other hand, Hg-MBs can perform their second function in Hg2+ detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg2+, the G-quadruplex structure in Hg-MBs is disrupted upon Hg2+ binding. In the absence of Hg2+, 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 Cd2+ and Hg2+ 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.
Graphical abstract
中文翻译：
开发基于 DNAzyme 的比色生物传感器测定法，用于双重检测 Cd2+ 和 Hg2+
基于Cd 2+依赖性脱氧核糖核酸酶切割和Hg 2+结合诱导的G-四链体片段构象转换，开发了一种用于Cd 2+和Hg 2+检测的比色生物传感器测定法。通过亲和素-生物素化学将两个功能化的 DNA 序列固定在磁珠上，合成了两种类型的多功能磁珠（Cd-MBs 和 Hg-MBs）。对于 Cd 2+检测，Cd-MBs 用作识别探针，其用单个硫代磷酸核糖核碱基 (rA) 底物 (PS 底物) 和 Cd 2+特异性脱氧核糖核酸酶 (Cdzyme) 进行修饰。在 Cd 2+的存在下，PS底物被Cdzyme裂解，释放出单链DNA作为信号转导序列。与其他两个寡核苷酸分子组装后，产生双链 DNA，它可以被 FokI 核酸内切酶识别和切割。因此，由G-四链体片段组成的信号输出成分被释放出来，它通过添加血红素和H 2 O 2催化ABTS的氧化，从而诱导显着放大的比色信号。为了排除假阳性结果并减少干扰信号，在Cd 2+检测过程中，用poly-T片段修饰的Hg-MBs作为Hg 2+积累探针。另一方面，Hg-MBs 可以在 Hg 2+中发挥其第二个功能通过改变 G-四链体/血红素 DNAzyme 的催化活性进行检测。在 Hg 2+存在的情况下， Hg-MBs 中的 G-四链体结构在 Hg 2+结合时被破坏。在没有 Hg 2+的情况下，肉眼可以观察到颜色变化增强，以形成完整的 G-四链体/血红素 DNA 酶。生物传感器测定显示出优异的选择性和高灵敏度。Cd 2+和 Hg 2+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。本研究报告的设计策略可以广泛扩展基于金属离子特异性脱氧核糖核酸酶的生物传感器的应用。
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