Herein, an allosteric kissing complex-based electrochemical biosensor was ingeniously proposed for the simple, sensitive, regenerative and versatile detection of proteins. Two hairpins (Hp1 and Hp2) were designed and the Hp1 was immobilized on the electrode surface, which could form a kissing complex with Hp2 through the apical loop-loop or kissing interaction of the RNA-RNA base sequences. The Hp2 possesses the appended single-stranded tails on each end, which hybridize with the recognition element-conjugated DNA strands to construct a protein responsive switch of Hp2 scaffold. After kissing complex formation between the Hp2 scaffold and the immobilized Hp1, the streptavidin-labeled alkaline phosphatase (SA-ALP) can be introduced onto the electrode surface for the generation of electrochemical signal. In the presence of target protein, its binding to the recognition elements linked onto the Hp2 scaffold endows the steric strain to open the Hp2 stem, propagated by the disruption of the kissing complex structure, resulting into a decreased electrochemical signal related with the protein quantification. Also, the Hp1 immobilized electrode can be directly regenerated after protein-induced kissing complex dissociation. The current kissing complex-based electrochemical biosensing strategy can be easily extended for the detection toward different protein targets of interest by simply changing the recognition elements conjugated onto the Hp2 scaffold. The sensitive and selective detection toward proteins could be achieved with the detection limits toward Anti-Dig antibody and thrombin of about 1 ng/mL and 10 pM, respectively. The developed kissing complex-based protein biosensing strategy should be a beneficial supplement in current biosensor field, providing a promising means for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

在此，巧妙地提出了一种基于变构亲吻复合物的电化学生物传感器，用于简单，灵敏，再生和通用的蛋白质检测。设计了两个发夹（Hp1和Hp2），并将Hp1固定在电极表面上，这可以通过RNA的RNA碱基序列的顶端环环或接吻相互作用而与Hp2形成接吻复合体。Hp2在每个末端具有附加的单链尾巴，它们与结合识别元件的DNA链杂交以构建Hp2支架的蛋白质响应开关。在Hp2支架和固定的Hp1之间形成复合物后，可将链霉亲和素标记的碱性磷酸酶（SA-ALP）引入电极表面以产生电化学信号。在目标蛋白存在的情况下，其与连接到Hp2支架上的识别元件的结合赋予了空间应变以打开Hp2茎，该Hp2茎通​​过接吻复合物结构的破坏而传播，从而导致与蛋白质定量相关的电化学信号降低。同样，Hp1固定的电极可以在蛋白质诱导的接吻复合物解离后直接再生。通过简单地改变结合到Hp2支架上的识别元件，可以轻松地将当前基于接吻复合物的电化学生物传感策略扩展到不同的目标蛋白质检测目标。对蛋白质的敏感和选择性检测可以通过对Anti-Dig抗体和凝血酶的检测极限分别达到约1 ng / mL和10 pM来实现。