微流芯片表面生物改性用于重组人骨形态发生蛋白-2的检测  

Bioactive modification of microfluidic chip surface for detecting recombinant human bone morphogenetic protein-2

作　　者：陈婧 何宏燕[1,2] 刘昌胜 CHEN Jing;HE Hongyan;LIU Changsheng(School of Material Science and Engineering,East China University of Science and Technology,Shanghai 200237,China;Medical Biomaterials Engineering Research Center of the Ministry of Education,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学材料科学与工程学院,上海200237 [2]华东理工大学教育部医用生物材料工程研究中心,上海200237

出　　处：《浙江大学学报（农业与生命科学版）》2020年第4期391-399,共9页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：上海市国际科技合作项目(18520710100);中国科学院-威高研究发展计划攻关项目(〔2017〕005);国家重点研发计划战略性国际科技创新合作重点专项(SQ2018YF020328);上海市浦江人才计划项目(18PJ1402400);国家自然科学基金创新群体项目(51621002);国家高等学校学科创新引智基地“111计划”项目(B14018)。

摘　　要：为了开发一种快速、特异、精准的基于大肠埃希菌表达系统制备的重组人骨形态发生蛋白-2(recombinant human bone morphogenetic protein-2, rhBMP-2)质量浓度检测方法,将酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)与微流控技术相结合,筛选合适的用于rhBMP-2的特异抗体,并基于调控抗体的特异性方向的策略,采用等离子体-蛋白A方法对高分子微流芯片的检测微流道孔进行修饰,以评价抗体蛋白在微流芯片表面的吸附效率以及最终的检测信号强度。结果表明:用等离子体处理微流道后,蛋白A和筛选的抗体成功包埋到微流芯片的检测孔表面。等离子体处理功率越高,抗体的吸附效果越好;当等离子体功率为100 W、处理时间为30 s时,第1抗体的吸附效果最好。改性后的微流孔可以有效地检测rhBMP-2质量浓度,在0~2 000 pg/mL范围内,rhBMP-2的质量浓度与荧光平均密度呈线性相关,而传统96孔ELISA法的线性区间范围是0~250 pg/mL。与传统ELISA法的试剂用量(600μL/样品)相比,微流芯片-ELISA方法试剂用量减少约97.3%。可见,本文开发的微流芯片表面改性技术具备应用于生物医药、食品安全及环境检测的潜力。In order to develop a rapid,specific,and accurate detection method for the concentration of recombinant human bone morphogenetic protein-2(rhBMP-2)through Escherichia coli-based expression systems,enzyme-linked immunosorbent assay(ELISA)was combined with microfluidic chip.The specific first antibody for rhBMP-2 was screened from several commercial products.Based on the regulation strategy of antibody orientation for enhancing detection signal,plasma-protein A method was then used to modify the detection microwells of the microfluidic chip.After tuning the conditions of plasma treatment on the detection microwells,adsorption efficiency of the first antibody and strength of the final detection signal were evaluated.It was found that the better capture efficiency of the first antibody could be obtained by using the higher power in the plasma treatment process.The best plasma condition was the power of 100 W and treatment time of 30 s.After the optimized modification conditions were applied for the microfluidic chip,the dilute concentrations of rhBMP-2 in a range of 0-2000 pg/mL were achieved.In comparison with the standard assay carried out in the96-well microtiter plate,the microwells of microfluidic chip exhibited a broader linear detection range(0-2000 pg/m L vs.0-250 pg/mL)and a much less reagent consumption(Each sample needed 600μL reagent consumption in the standard assay,while about 16μL in the microwell assay,which was 97.3%reduction in dosage).Clearly,this plasma-protein A immobilization strategy holds a great potential for polymeric microfluidic chip based assay in biomedical application,food safety,and environment monitoring.

关 键 词：酶联免疫吸附测定 重组人骨形态发生蛋白-2 微流控检测 表面改性 特异性抗体 

分 类 号：R318.08[医药卫生—生物医学工程] Q81[医药卫生—基础医学]