Cytosine and protonated cytosine base pairs (C·CH⁺)-supported i-motif conformation has been widely employed in some interdisciplinary fields such as biology, medicine and chemistry. In this work, we report a new electrochemical biosensing method for the detection of glucose oxidase (GOx) and urease based on pH-induced DNA conformational-change. The constructed platform mainly includes TdT-mediated catalytic synthesis, GOx- or urease-catalyzed biological reaction and pH-induced DNA conformational-change. In the beginning, a kind of C-rich DNA is produced by TdT catalysis, and multiple C·CH⁺-supported i-motif structures appear under acidic condition. Then, the oxidation of glucose catalyzed by GOx or the hydrolyzation of urea aroused by urease can result in a generation of acidic or alkaline environment owing to the generated gluconic acid or ammonia. Herein, protonation and deprotonation interaction in TdT-yielded C-rich DNA can lead to different electrochemical impedance spectroscopy (EIS) toward Fe(CN)₆^3−/4-. Based on it, the EIS response changes proportionally toward GOx concentrations from 0.01 to 20 U/L or urease concentrations from 0.01 to 50 U/L, and the detection limit of GOx or urease is 0.0061 U/L or 0.0028 U/L (S/N = 3), respectively. Beyond this, we also construct a series of molecular logic gates (YES, AND, NOT, and NAND) with good performance by altering inputs under long C-rich DNA substrate. These excellent properties indicate that the unique sensing platform is potential to monitor GOx or urease in practical biosystems and clinical medical examinations.

胞嘧啶和质子化胞嘧啶碱基对(C·CH ⁺ )支持的i-motif构象已广泛应用于生物学、医学和化学等交叉学科领域。在这项工作中，我们报告了一种基于 pH 诱导的 DNA 构象变化检测葡萄糖氧化酶 (GOx) 和脲酶的新电化学生物传感方法。构建的平台主要包括TdT介导的催化合成、GOx或脲酶催化的生物反应和pH诱导的DNA构象变化。开始时，TdT催化产生一种富含C的DNA，多个C·CH ⁺-支持的 i-motif 结构出现在酸性条件下。然后，GOx催化的葡萄糖氧化或脲酶引起的尿素水解可由于产生葡萄糖酸或氨而导致酸性或碱性环境的产生。在此，TdT 产生的富 C DNA 中的质子化和去质子化相互作用可导致对 Fe(CN) ₆ ^3-/4-产生不同的电化学阻抗谱 (EIS) 。以此为基础，EIS响应向GOx浓度从0.01到20 U/L或脲酶浓度从0.01到50 U/L成正比变化，GOx或脲酶的检测限为0.0061 U/L或0.0028 U/L（S / N = 3），分别。除此之外，我们还通过在富含 C 的长 DNA 底物下改变输入来构建一系列性能良好的分子逻辑门（YES、AND、NOT 和 NAND）。这些优异的特性表明，独特的传感平台有可能在实际生物系统和临床医学检查中监测 GOx 或脲酶。