Metallothionein (MT) is a protein biomarker secreted by liver in response to the treatment for heavy metal toxicity and oncological diseases. On the basis of a new Ag⁺-stabilized DNA triplex probe (Ag⁺-SDTP), we establish a fluorescent biosensing system for high sensitivity detection of MT by combining catalytic hairpin assembly (CHA) and the CRISPR/Cas12a signal enhancements. The MT analyte complexes with Ag⁺ in Ag⁺-SDTP to disrupt the triplex structure and to release the ssDNA strands, which trigger subsequent CHA formation of many protospacer adjacent motif (PAM)-containing dsDNAs from two hairpins. Cas12a/crRNA further recognizes these PAM sequences to activate its trans-catalytic activity to cyclically cleave the fluorescently quenched ssDNA reporters to recovery drastically amplified fluorescence for detecting MT down to 0.34 nM within the dynamic range of 1∼800 nM. Moreover, the sensing method is able to selectively discriminate MT from other non-specific molecules and can realize low level detection of MT in diluted human serums, manifesting its potentiality for monitoring the disease-specific MT biomarker at trace levels.

金属硫蛋白（MT）是肝脏响应重金属毒性和肿瘤疾病治疗而分泌的蛋白质生物标志物。^{在新型Ag +}稳定的DNA三链体探针（Ag ⁺ -SDTP）的基础上，我们通过结合催化发夹组装（CHA）和CRISPR/Cas12a信号增强，建立了用于高灵敏度检测MT的荧光生物传感系统。MT 分析物与Ag ^{+ -SDTP 中的 Ag +}复合，破坏三链体结构并释放 ssDNA 链，从而触发随后从两个发夹形成许多包含原型间隔子相邻基序 (PAM) 的 dsDNA 的 CHA。Cas12a/crRNA进一步识别这些PAM序列，激活其反式催化活性，循环切割荧光猝灭的ssDNA报告基因，恢复大幅放大的荧光，从而在1∼800 nM的动态范围内检测低至0.34 nM的MT。此外，该传感方法能够选择性地区分MT与其他非特异性分子，并可实现稀释人血清中MT的低水平检测，显示出其在微量水平监测疾病特异性MT生物标志物的潜力。