Lu Ling-Fei, Li Yan-Yun, Zhang Min, Shi Guoyue
Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
Analyst. 2015 May 21;140(10):3642-7. doi: 10.1039/c5an00499c. Epub 2015 Apr 14.
In this work, a label-free molecular beacon (MB)-like biosensor is designed for the determination of H2O2 and glucose based on the fluorescence regulation of Hoechst dyes hosted by the designed AT-rich single-stranded DNA (ssDNA), in which Hg(2+) and cysteine (Cys) act as activators. The designed AT-rich ssDNA (ATprobe) can be directed to form a hairpin with an Hg(2+)-induced T-Hg(2+)-T complex, which provides a medium for enhancing the fluorescence of Hoechst dyes significantly. On the other hand, Cys can effectively grab Hg(2+) from the T-Hg(2+)-T complex by thiol-Hg(2+) interactions, destructing the hairpin and then switching the Hoechst dyes to the fluorescence "off" state. Combined with these properties, we have demonstrated its application for label-free fluorescence "turn on" detection of H2O2. The sensing mechanism is based on the specific reaction between H2O2 and Cys catalyzed by I(-), the resulting disulfide reverses the Cys-mediated fluorescence decrease of the MB-hosted Hoechst dyes. The approach achieves a low detection limit of 0.1 μM for H2O2. Moreover, this method is further applied to the noninvasive detection of glucose in artificial saliva and urine samples, combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. Compared to traditional methods, the proposed design is cost-effective, simple to prepare and manipulate without fluorescence labeling or chemical modification.
在这项工作中，设计了一种无标记的类分子信标（MB）生物传感器，用于基于由设计的富含AT的单链DNA（ssDNA）所承载的Hoechst染料的荧光调节来测定过氧化氢（H₂O₂）和葡萄糖，其中汞离子（Hg²⁺）和半胱氨酸（Cys）作为激活剂。所设计的富含AT的ssDNA（AT探针）可在Hg²⁺诱导下形成含T-Hg²⁺-T复合物的发夹结构，这为显著增强Hoechst染料的荧光提供了介质。另一方面，Cys可通过硫醇-Hg²⁺相互作用从T-Hg²⁺-T复合物中有效夺取Hg²⁺，破坏发夹结构，进而将Hoechst染料切换至荧光“关闭”状态。结合这些特性，我们展示了其用于H₂O₂的无标记荧光“开启”检测的应用。传感机制基于I⁻催化的H₂O₂与Cys之间的特异性反应，生成的二硫键逆转了Cys介导的MB所承载的Hoechst染料的荧光降低。该方法实现了对H₂O₂的低检测限为0.1 μM。此外，该方法进一步应用于人工唾液和尿液样本中葡萄糖的无创检测，与葡萄糖氧化酶（GOx）结合用于葡萄糖氧化并生成H₂O₂。与传统方法相比，所提出的设计具有成本效益，制备和操作简单，无需荧光标记或化学修饰。
