基于适配体的生物膜干涉检测重组人促红细胞生成素α新方法  

An Aptameric Biolayer Interferometric Assay for Detection of Recombinant Human Erythropoietin-α

作　　者：贺小琴 罗黎 郭磊 谢剑炜 HE Xiao-Qin;LUO Li;GUO Lei;XIE Jian-Wei((State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Toxicology and Pharmacology,Academy of Military Medical Sciences,Beijing 100850,China;Department of Spine Surgery and Institute for Orthopaedic Research,the 2 nd Clinical Medical College of Jinan University and the 1 st Affiliated Hospital of Southern University of Science and Technology(Shenzhen People’s Hospital),Shenzhen 518020,China;School of Public Health,Hebei Medical University,Shijiazhuang 050017,China)

机构地区：[1]军事科学院军事医学研究院毒物药物研究所,抗毒药物与毒理学国家重点实验室,北京100850 [2]暨南大学第二临床附属医院,南方科技大学第一附属医院(深圳市人民医院)脊柱外科,深圳市骨科研究所,深圳518020 [3]河北医科大学公共卫生学院,石家庄050017

出　　处：《分析化学》2020年第5期670-675,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21175152,21974152)资助。

摘　　要：采用生物膜干涉(BLI)表面敏感光谱技术,建立了一种基于链DNA适配体39 nt的传感分析方法,实现了重组人促红细胞生成素α(EPO-α)的灵敏检测。将生物素化的39 nt固定于链霉亲和素传感芯片上,经2200 r/min高速振荡实现微升体积分析物的快速扩散。对适配体39 nt的取向、在BLI传感芯片上的固定浓度和非特异性吸附等影响因素进行了考察,发现50 nmol/L 3’末端生物素化39 nt具有最佳响应信号;加入Tween 20和牛血清白蛋白(BSA)可有效克服非特异吸附。基于麦胚凝集素构建了夹心法信号放大体系,在Tris-TB生理缓冲溶液中,检测EPO-α的线性范围为10~200 nmol/L,检出限为5 nmol/L。应用于人血浆等复杂基质中的EPO-α检测,回收率为86.7%~104.2%,RSD<11%,结果令人满意。此BLI传感体系为免标记蛋白相互作用和复杂生物样品检测等实际应用提供了有益参考。The biolayer interferometry(BLI),one promising biosensing technique for real-time,high-sensitive detection of analytes,is a new surface-sensitive spectrometric technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections.In our previous research,we selected an ssDNA aptamer,39 nt,with definite interaction characteristics for recombinant human erythropoietin-α(EPO-α).In this work,a new aptameric BLI assay of EPO-αbased on this aptamer 39 nt was established.Firstly,the biotinylated aptamer 39 nt was immobilized onto the optical fiber streptavidin biosensor tip,and the analytes in microliter volume were quickly diffused via high-speed oscillation at 2200 r/min.Then,the key parameters such as the orientation of aptamer sequence,the concentration for immobilization,and the overcome of nonspecific adsorption were investigated and optimized to exhibit the affinity recognition of aptamers,and it was found that the 3’-end biotinylated aptamer 39 nt with 50 nmol/L provided the best sensitivity,and the addition of Tween 20 and bovine serum albumin(BSA)could efficiently avoid nonspecific adsorption effect.Besides,towards the original designed disposable biosensor tip,the affinity between EPO-αand aptamer 39 nt for ca.10 times was maintained by optimizing the regeneration condition.Furthermore,a signal amplification approach of a sandwiched format for detection of EPO-αvia wheat germ agglutinin(WGA)was established,with the linear range of 10-200 nmol/L,and a limit of detection of 5 nmol/L.This assay was further applied to the measurement of EPO-αin 50%human plasma with recoveries of 86.7%-104.2%and RSD less than 11%,providing a satisfied result.The BLI sensing system developed here provides a helpful way for practical applications for label-free protein interaction and detection.

关 键 词：适配体 重组人促红细胞生成素 生物膜干涉 麦胚凝集素 信号放大 

分 类 号：O652[理学—分析化学]