Protease expression is closely linked to various pathological phenomena, and their accurate quantification is essential to clinical diagnosis and cancer therapy. Herein, we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification (NESA) for single-molecule detection of multiple matrix metalloproteinases (MMPs). This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore (Cy3 or Cy5) and a quencher (BHQ2). In the presence of specific MMPs, MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates. After the magnetic separation, the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction, releasing large amounts of Cy3/Cy5 fluorescent molecules which can be simply quantified by using total internal reflection fluorescence-based single-molecule detection. Taking advantage of the high specificity of proteolytic cleavage, the high amplification efficiency of cyclic NESA, and the high sensitivity of single-molecule detection, this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7, superior to the target peptide-based methods. Moreover, this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors, holding great promise in clinic diagnosis and drug discovery.

蛋白酶表达与各种病理现象密切相关，它们的准确定量对于临床诊断和癌症治疗至关重要。在这里，我们首次展示了通过将蛋白酶敏感的裂解与切口酶辅助信号放大（NESA）结合起来用于多基质金属蛋白酶（MMP）的单分子检测，来构建敏感蛋白酶传感器的方法。该蛋白酶传感器涉及两个DNA-肽共轭物，它们既包含特定的蛋白酶切割位点，又包含触发DNA和两个报告DNA，这些DNA被荧光团（Cy3或Cy5）和淬灭剂（BHQ2）修饰。在存在特定的MMP的情况下，MMP介导的裂解反应会导致相应的DNA肽结合物释放特定的触发DNA。磁选后 最终的触发DNA可以与相应的报告DNA杂交，以启动环状NESA反应，释放出大量Cy3 / Cy5荧光分子，可以使用基于全内反射荧光的单分子检测方法对其进行简单地定量。利用蛋白酶切割的高特异性，环状NESA的高扩增效率和单分子检测的高灵敏度，此蛋白酶传感器可以同时检测多个MMP，MMP-2和MMP-2的检出限分别为3.33 pM和1.71 pM对于MMP-7，优于基于靶肽的方法。此外，该蛋白酶传感器可用于癌细胞中MMP-2和MMP-7的测定以及蛋白酶抑制剂的筛选，在临床诊断和药物开发中具有广阔的前景。