Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinases.

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.