基于DNA介导的固定化纳米酶制备及其在蛋白质快速酶解分析中的应用  

Preparation of Immobilized Nano-Enzyme by DNA Direct Immobilization Strategy and Its Application in the Fast Enzymatic Hydrolysis for Protein Analysis

作　　者：杨星杰 肖鹏 杨丹妮 刘思瑶 YANG Xingjie;XIAO Peng;YANG Danni;LIU Siyao(School of Pharmacy,Jiangsu University,Zhenjiang 212013)

机构地区：[1]江苏大学药学院,江苏镇江212013

出　　处：《分析科学学报》2025年第1期7-13,共7页Journal of Analytical Science

基　　金：江苏省自然科学基金青年项目(BK20200908);江苏大学高级人才科研启动基金(20JDG074)。

摘　　要：以修饰了单链DNA的金纳米粒子(AuNPs)为载体,通过DNA介导的固定化策略(DDI)制备系列固定化纳米酶,用于蛋白质样品的快速酶解分析。所制备的固定化纳米酶具有纳米级尺寸,可以较好分散在溶液中进行酶解反应,减小固液界面的传质阻力,使酶与底物蛋白质较好地结合。因此该纳米酶具备分散性好,催化效率高的特点。系统研究了酶解反应温度与pH值对固定化纳米酶解活性的影响。性能考察结果表明,固定化纳米酶具有良好的储存稳定性与重复性。固定化纳米酶可有效水解牛血清蛋白(BSA),酶解时间仅需30 min即可获得91%的氨基酸序列覆盖率,酶解效率明显高于溶液酶。进一步将其应用于Hela细胞总蛋白质组的快速分析,得到了良好的酶解效果,展现了该固定化纳米酶在复杂蛋白质样品分析中的应用潜力。Immobilized trypsin on the single-stranded modified gold nanoparticles as carriers has been prepared with DNA direct immobilization(DDI)strategy.The prepared immobilized nano-enzyme has nanoscale dimensions,allowing them to disperse well in solution as the in-solution heterogeneous catalyst for the enzymatic digestion.Good dispersion reduces mass transfer resistance at the solid-liquid interface,facilitating a better interaction between the enzyme and substrate proteins.As a result,the immobilized trypsin exhibits good dispersibility and high catalytic efficiency.The influence of enzymatic reaction temperature and pH on the immobilized enzymatic activity has been systematically investigated.The results indicate that the immobilized nano-enzyme possesses good storage stability and reproducibility.It effectively hydrolyzes substrate BSA with a digestion time of only 30 minutes,achieving a 91% amino acid sequence coverage,significantly surpassing the digestion efficiency of free trypsin.Furthermore,it has been applied to the rapid analysis of the HeLa cell protein,and the enzymatic digestion results demonstrate excellent performance of the immobilized nano-enzyme,showing its potential in the analysis of complex protein samples.

关 键 词：金纳米粒子 固定化纳米酶 蛋白质 酶解 

分 类 号：Q503[生物学—生物化学]