无标记的化学发光法检测蛋白酶活性

发布时间：2018-06-08 21:46

本文选题：化学发光 + 胰蛋白酶　；参考：《天津大学》2015年硕士论文

【摘要】：蛋白酶,是水解蛋白质或多肽的一类酶,在许多人体生理过程中具有非常重要的作用。研究发现,在许多如炎症、心血管疾病、癌症等的病理条件下,蛋白酶活性也会发生改变。因此简便、快速、高灵敏度的蛋白酶检测方法在医学诊断领域具有重要意义。本文发展了无标记的化学发光蛋白酶活性检测新方法。本文由以下两部分组成:第一部分建立了基于链霉亲和素修饰磁珠(MB-SAs)的无标记化学发光(CL)法检测蛋白酶活性。设计了一条生物素化的多肽(bio-P1),其包含精氨酸(Arg)和一个末端半光氨酸(Cys),作为胰蛋白酶的水解底物。将bio-P1加入到碱性鲁米诺(luminol)-NaIO4溶液,bio-P1末端的Cys能够催化溶液反应产生很强的CL信号。当通过链霉亲和素-生物素反应将bio-P1固定在MB-SAs表面后,再加入到碱性luminol-NaIO4溶液中,溶液仅发出微弱的CL信号。基于此,我们建立了基于CL信号恢复的CL法检测蛋白酶活性及其抑制剂。该方法不需要洗涤或分离过程,线性范围为0.01 nM-2 nM,检测限为10 pM。通过检测胰蛋白酶抑制剂,我们发现胰蛋白酶抑制剂SBTI、PMSF和EDTA对胰蛋白酶抑制的IC50(抑制效率为50%时所对应的的抑制剂浓度)分别为1 nM、0.01 mM和3.28 mM。另外,通过简单的改变多肽底物,此方法能够拓宽至其他蛋白酶的检测。第二部分,我们设计了含有Arg和末端Cys的多肽(P),利用氧化石墨烯(GO)和luminol-NaIO4-P CL系统,发展了无标记的CL传感器检测胰蛋白酶活性及其抑制剂。该方法依据P和P-GO复合物对luminol-NaIO4 CL反应的催化作用不同而建立。将P和luminol-NaIO4碱性溶液混合,检测到很强的CL信号。然而,将P-GO复合物和luminol-Na IO4溶液混合,只能检测到微弱的CL信号。基于上述发现,我们建立了无标记的CL法检测胰蛋白酶活性。检测线性范围为0.02-10 nM,检测限可达7.3 pM,并且能够用于胰蛋白酶抑制剂的筛选。胰蛋白酶抑制剂SBTI和EDTA对胰蛋白酶抑制的IC50分别为0.7 nM和12 mM。通过改变多肽底物,此方法可以拓宽至其他蛋白酶的检测。
[Abstract]:Protease is a kind of enzyme that hydrolyzes protein or polypeptide. It plays a very important role in many physiological processes of human body. Studies have found that protease activity also changes in many pathological conditions, such as inflammation, cardiovascular disease, and cancer. Therefore, simple, rapid and sensitive detection of protease is of great significance in the field of medical diagnosis. A new method for the detection of chemiluminescence protease activity without labeling was developed. This paper is composed of the following two parts: in the first part, an unlabeled chemiluminescence (CLL) method based on streptavidin modified MB-SAs) was developed for the detection of protease activity. A biotinylated polypeptide named Bio-P _ (1) containing arginine (Arg) and a terminal hemiphosminated cystatin (Cysn) was designed as a substrate for hydrolysis of trypsin. The Cys at the end of bio-P1 can catalyze the solution reaction to produce a strong CL signal. Bio-P1 was immobilized on the surface of MB-SAs by streptavidin-biotin reaction, and then added to alkaline luminol-NaIO4 solution, which only gave off a weak CL signal. Based on this, we established CL method based on CL signal recovery to detect protease activity and its inhibitors. The linear range of the method is 0.01 nm -2 nm and the detection limit is 10 pm. Through the detection of trypsin inhibitors, we found that the inhibition of trypsin inhibitor SBTIPMSF and EDTA on the inhibition of trypsin IC50 (inhibition efficiency is 50 corresponding to the inhibitor concentration) is 1 nM0. 01 mm and 3. 28 mm respectively. In addition, by simply changing the peptide substrate, the method can be extended to other protease detection. In the second part, we developed an unlabeled CL sensor for the detection of trypsin activity and its inhibitors using graphene oxide (GOO) and luminol-NaIO4-PCL systems. The method is based on the different catalytic effects of P and P-GO complexes on luminol-NaIO4CL reaction. A strong CL signal was detected by mixing P with luminol-NaIO4 alkaline solution. However, only weak CL signals could be detected by mixing P-GO complex with luminol-NaIO4 solution. Based on the above findings, we developed a non-labeled CL assay for the detection of trypsin activity. The linear range of detection was 0.02-10 nm, the detection limit was 7.3 pm, and could be used for screening trypsin inhibitors. The IC50 of trypsin inhibitor SBTI and EDTA were 0.7 nm and 12 mm respectively. By changing the peptide substrate, this method can be extended to other protease detection.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O657.3;R446.1

【相似文献】