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 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 hours. 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 6-fold faster and the limit of detection is 100-fold more sensitive. We believe that the formulations for proteolytic formations of oligonucleotides duplexes for signal amplification on GNP will lead to the development of more sensitive and rapid protease assay techniques, thus extend the role of proteases as therapeutic targets and disease indicators
한국생물공학회 [The Korean Society for Biotechnology and Bioengineering]
설립연도
1984
분야
공학>생물공학
소개
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