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

doi:10.3785/j.issn.1008-9209.2019.08.071

浙江大学学报（农业与生命科学版）

2020, Vol. 46

Issue (4): 391-399 DOI: 10.3785/j.issn.1008-9209.2019.08.071

生物科学与技术

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

陈婧¹(

),何宏燕^1,²(

),刘昌胜^1,²

^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

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

Jing CHEN¹(

),Hongyan HE^1,²(

),Changsheng LIU^1,²

^1.School of Material Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
^2.Medical Biomaterials Engineering Research Center of the Ministry of Education, East China University of Science and Technology, Shanghai 200237, China

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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（600 μL/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。

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

Abstract:

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 0-2 000 pg/mL were achieved. In comparison with the standard assay carried out in the 96-well microtiter plate, the microwells of microfluidic chip exhibited a broader linear detection range (0-2 000 pg/mL vs. 0-250 pg/mL) and a much less reagent consumption (Each sample needed 600 μL reagent consumption in the standard assay, while about 16 μL 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.

Key words: enzyme-linked immunosorbent assay recombinant human bone morphogenetic protein-2 (rhBMP-2) detection on microfluidic chip surface modification specific antibody

收稿日期: 2019-08-07 出版日期: 2020-09-11

CLC:

R 318.08

基金资助: 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)

通讯作者: 何宏燕 E-mail: 971456416@qq.com;hyhe@ecust.edu.cn

作者简介: 陈婧（https://orcid.org/0000-0002-2974-7146），E-mail：971456416@qq.com

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引用本文:

陈婧,何宏燕,刘昌胜. 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[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(4): 391-399.

Jing CHEN,Hongyan HE,Changsheng LIU. Bioactive modification of microfluidic chip surface for detecting recombinant human bone morphogenetic protein-2. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(4): 391-399.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.08.071 或 http://www.zjujournals.com/agr/CN/Y2020/V46/I4/391

图1 高分子微流芯片表面的等离子体-蛋白A处理工艺示意图编号1～5分别表示装载样品、洗液、第2抗体、洗液和底物。

图2 瑞邦自制rhBMP-2的碱性磷酸酶（ALP）活性（A）和用不同厂家试剂盒检测的η值（B）

表1 不同抗体的二级结构元件含量比较 (%)

图3 不同等离子体处理功率下微流芯片表面的接触角对比CK：未处理的表面。

图4 干燥和湿润情况下不同微流芯片表面对第1抗体的吸附效果比较处理时间30 s。CK：未处理的表面。

图5 微流芯片表面在ELISA的不同反应阶段的表面形貌比较CK：对照（未处理的表面）；M1：25 W-等离子体处理；M2：25 W-第1抗吸附；M3：25 W-rhBMP-2吸附；N1：100 W-等离子体处理；N2：100 W-第1抗吸附；N3：100 W-rhBMP-2吸附。

图6 微流芯片表面在ELISA不同反应阶段的元素表征比较A. X射线光电子能谱图；B. N（1s）峰。CK：对照（未处理的表面）；N1：100 W-等离子体处理；N2：100 W-第1抗吸附；N3：100 W-rhBMP-2吸附。

图7 改性微流芯片对不同质量浓度rhBMP-2的ELISA荧光显色对比

图8 改性微流芯片（A）和欣博盛96孔板（B）的rhBMP-2 ELISA标准曲线对比

表2 微流芯片-ELISA与传统ELISA试剂用量对比 (µL)

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