Proteolytic Fluorescent Signal Amplification on Gold Nanoparticles for a Highly Sensitive and Rapid Protease Assay

A new strategy for highly sensitive and rapid protease assay is developed by mediating proteolytic formation of oligonucleotide duplexes and using the duplexes for signal amplification. In the presence of matrix metalloprotease‐2 (MMP‐2), fragmentation of the intact DNA–peptide on gold nanoparticles (GNP) by hydrolytic cleavage of a peptide bond within the substrate allows diffusion of DNA away from the GNP and the formation of a DNA/RNA heteroduplex, leading to digestion of RNA by RNase H. Because of the high quenching efficacy of GNP to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence signal recovery is amplified. This method permits the assessment of the activity of MMP‐2 at concentrations as low as 10 pM within 4 h. Compared with the reported protease nanosensors using quantum dots, GNP, and magnetic nanoparticles with the same peptide sequence, the assay time of this method is sixfold faster and the limit of detection is 100‐fold more sensitive. The formulations for proteolytic formations of oligonucleotides duplexes for signal amplification on GNP could lead to the development of more sensitive and rapid protease assay techniques, thus extending the role of proteases as therapeutic targets and disease indicators. Highly sensitive and rapid protease assay is demonstrated through proteolytic fluorescence amplification using gold nanoparticles and RNase H. Fragmented DNA of DNA‐peptide on gold nanoparticles by a protease allows a formation of DNA/RNA heteroduplexe and multiple digestion of fluorogenic RNA on gold nanoparticles by RNase H, leading to linear fluorescence recovery from a highly quenched state on a gold nanoparticle (see image).