Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong, Daejeon 305-806, South Korea.
Small. 2010 Jan;6(1):126-31. doi: 10.1002/smll.200901635.
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.
一种新的策略是通过介导蛋白酶解形成寡核苷酸双链体，并利用双链体进行信号放大，来实现对高度敏感和快速蛋白酶分析。在基质金属蛋白酶-2（MMP-2）存在的情况下，通过在底物中的肽键水解切割，使完整的 DNA-肽在金纳米粒子（GNP）上发生碎裂，从而允许 DNA 从 GNP 扩散，形成 DNA/RNA 杂双链体，导致 RNA 被 RNase H 消化。由于 GNP 对 RNA 中的荧光团具有高猝灭效率和对 RNA 的多次消化，因此荧光信号恢复被放大。该方法允许在 4 小时内以低至 10 pM 的浓度评估 MMP-2 的活性。与使用相同肽序列的量子点、GNP 和磁性纳米粒子的报道的蛋白酶纳米传感器相比，该方法的检测时间快 6 倍，检测限的灵敏度高 100 倍。在 GNP 上用于信号放大的寡核苷酸双链体的酶切形成的制剂可能会导致更敏感和快速的蛋白酶分析技术的发展，从而扩展蛋白酶作为治疗靶点和疾病标志物的作用。
