Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore.
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore.
Biosens Bioelectron. 2016 Mar 15;77:1126-33. doi: 10.1016/j.bios.2015.11.003. Epub 2015 Nov 3.
Polyethylene glycol (PEG) hydrogel is permeable to biomolecules, but its permeability depends on the molecular weight of monomers and the concentration of monomer solutions. In this study, we show that PEG hydrogel made from 20% to 30% of PEG700 monomer is permeable to amino acids yet impermeable to oligopeptides. Because of its unique permeability, the gel can be used to detect protease by separating amino acids from oligopeptides when proteases cleave the oligopeptides and release amino acids. Based on this principle, an UV crosslinked gel array is fabricated on a chip for simultaneous detection of protease in up to 40 samples with only 1 µl of volume required for each sample. As a proof of concept, the on-chip protease assays are used to detect trypsin in buffer and serum. The detection limits are 1.2 nM in buffer and 17.7 nM in serum, which are comparable to conventional protease assays. Moreover, because only 1 µl of liquid is required, as little as 1.2 fmol of trypsin can be detected by using the on-chip assay. The protease assay also shows good specificity for trypsin and chymotrypsin. The gel array chip could be a useful miniaturized platform for high-throughput detection of different proteases and screening of their inhibitors.
聚乙二醇（PEG）水凝胶可渗透生物分子，但它的渗透性取决于单体的分子量和单体溶液的浓度。在这项研究中，我们表明，由 20%至 30%PEG700 单体制成的 PEG 水凝胶可渗透氨基酸，但不可渗透寡肽。由于其独特的渗透性，当蛋白酶切割寡肽并释放氨基酸时，凝胶可用于通过将氨基酸与寡肽分离来检测蛋白酶。基于这一原理，在芯片上制作了一个 UV 交联的凝胶阵列，可同时检测多达 40 个样本中的蛋白酶，每个样本只需 1 µl 体积。作为概念验证，在芯片上进行了蛋白酶检测以检测缓冲液和血清中的胰蛋白酶。在缓冲液中的检测限为 1.2 nM，在血清中的检测限为 17.7 nM，与传统的蛋白酶检测相当。此外，由于仅需要 1 µl 的液体，使用芯片上的检测方法可以检测到低至 1.2 fmol 的胰蛋白酶。蛋白酶检测还显示出对胰蛋白酶和糜蛋白酶的良好特异性。凝胶阵列芯片可以成为用于高通量检测不同蛋白酶和筛选其抑制剂的有用的小型化平台。
