Sensor materials for the detection of proteases.
Sensor materials for the detection of proteases.
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DOI：
10.1016/j.bios.2008.11.002
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发表时间：
2009-03
影响因子：
12.6
通讯作者：
J. Stair;M. Watkinson;S. Krause
中科院分区：
文献类型：
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作者：
J. Stair;M. Watkinson;S. Krause
基于肽交联葡聚糖水凝胶的薄膜降解，探索了用于检测蛋白酶的通用和可调传感器材料的概念。水凝胶交联通过氧化葡聚糖中的醛基和对蛋白酶切割敏感的肽序列之间的简单亚胺键形成。在这项研究中，使用石英晶体微天平（QCM）的水凝胶膜的降解进行监测。使用人中性粒细胞弹性蛋白酶（HNE）和Ala-Ala-Pro-Val-Ala-Ala-Lys（AAPVAAK）的蛋白酶/肽对开发传感器材料。观察到水凝胶降解速率和蛋白酶活性之间的直接关系;使用25%交联膜检测到2.5至30 Uml − 1的HNE活性。薄膜降解迅速，HNE活性≥ 10 Uml −1时，薄膜在10分钟内完全降解。通过将交联密度从25%增加到75%，实现了降解速率增加3.5倍。QCM导纳数据与BVD等效电路拟合表明，在添加酶后，膜粘弹性增加。测试了组织蛋白酶G和Ala-Ala-Pro-Phe-Phe-Lys（AAPFFK）的第二蛋白酶/肽对，其中25%AAPFFK交联的水凝胶在100 mUml −1下表现出快速响应。将蛋白酶/肽对交换为HNE/AAPFFK和组织蛋白酶G/AAPVAAK显示出低水平的交叉敏感性，进一步证明了膜降解的特异性。
The concept of generic and tunable sensor materials for the detection of proteases based on the thin film degradation of peptide cross-linked dextran hydrogels was explored. Hydrogel cross-links were formed via simple imine linkages between aldehyde groups in oxidized dextran and a peptide sequence susceptible to protease cleavage. Degradation of the hydrogel films was monitored in this study using a quartz crystal microbalance (QCM). The sensor material was developed using the protease/peptide pair of human neutrophil elastase (HNE) and Ala-Ala-Pro-Val-Ala-Ala-Lys (AAPVAAK). A direct relationship between the hydrogel degradation rate and protease activity was observed; HNE activities from 2.5 to 30Uml−1were detected using 25% cross-linked films. Film degradation was rapid and was complete in less than 10min for HNE activities ≥10Uml−1. An increase in the rate of degradation by a factor of 3.5 was achieved by increasing the cross-linking density from 25% to 75%. QCM admittance data fitted with a BVD equivalent circuit showed increases in film viscoelasticity upon enzyme addition. A second protease/peptide pair of cathepsin G and Ala-Ala-Pro-Phe-Phe-Lys (AAPFFK) was tested where 25% AAPFFK cross-linked hydrogels demonstrated a rapid response at 100mUml−1. Swapping the protease/peptide pairs to HNE/AAPFFK and cathepsin G/AAPVAAK showed low levels of cross-sensitivity further demonstrating the specificity of film degradation.