Weng Benrui, Wang Yifei, Zhang Qingqing, Jiang Yuqian, Shang Jinhua, Liu Xiaoqing, Wang Fuan
College of Chemistry and Molecular Sciences, Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430072, P.R. China.
Key Laboratory of Biological Effect, Beijing Life Science Academy, Beijing, 02209, P.R. China.
Angew Chem Int Ed Engl. 2025 Sep 26;64(40):e202507241. doi: 10.1002/anie.202507241. Epub 2025 Sep 6.
Peptide-based biosensors are widely used for in vitro detection of protease activity but often suffer from the limited sensitivity, poor accuracy, and incompatibility with point-of-care testing (POCT) devices. Herein, we developed a versatile deoxyribozyme (DNAzyme)-amplified protease-sensing (DP) platform that integrates the positively charged oligopeptides with a negatively charged DNAzyme biocatalyst for highly-sensitive protease detection. The system leverages the electrostatic peptide-DNAzyme interactions to inhibit DNAzyme catalytic activity, which is reactivated upon the protease-triggered peptide hydrolysis, thus enabling an efficient signal amplification via the successive cleavage of DNAzyme substrate. Compared to conventional peptide-based sensing platform, our DP system offers an enhanced sensitivity and signal-to-noise ratio and is highly modular for detecting various clinically relevant proteases through a simple replacement of the peptide blocker. By introducing a dual-enzyme recognition mechanism, we developed a dual-protease-triggered DP platform for enabling the accurate detection of SARS-CoV-2 proteases in saliva. We also applied the DP platform to differentiate between normal and cancerous colon cells and tissues by detecting colorectal cancer (CRC)-associated proteases. Overall, this work introduces a universal and scalable biosensing strategy for activity-based protease detection with potential applications in both infectious disease diagnostics and cancer classification, advancing the field of DNAzyme-based POCT technologies.
基于肽的生物传感器被广泛用于体外检测蛋白酶活性，但常常存在灵敏度有限、准确性差以及与即时检测（POCT）设备不兼容等问题。在此，我们开发了一种通用的脱氧核酶（DNAzyme）放大蛋白酶传感（DP）平台，该平台将带正电荷的寡肽与带负电荷的DNAzyme生物催化剂整合，用于高灵敏度的蛋白酶检测。该系统利用肽与DNAzyme之间的静电相互作用来抑制DNAzyme的催化活性，而这种活性在蛋白酶触发的肽水解后会重新激活，从而通过连续切割DNAzyme底物实现有效的信号放大。与传统的基于肽的传感平台相比，我们的DP系统具有更高的灵敏度和信噪比，并且高度模块化，通过简单更换肽阻断剂即可检测各种临床相关蛋白酶。通过引入双酶识别机制，我们开发了一种双蛋白酶触发的DP平台，用于准确检测唾液中的SARS-CoV-2蛋白酶。我们还将DP平台应用于通过检测结直肠癌（CRC）相关蛋白酶来区分正常和癌性结肠细胞及组织。总体而言，这项工作引入了一种通用且可扩展的生物传感策略，用于基于活性的蛋白酶检测，在传染病诊断和癌症分类中均有潜在应用，推动了基于DNAzyme的POCT技术领域的发展。
