基于纳米生物条形码和杂交链式反应构建电化学生物传感器用于蛋白激酶活性分析  

An Electrochemical Biosensor for Detection of Protein Kinase Activity Based on Nanoparticle-based Bio-barcode and Hybridication Chain Reaction Amplification Strategy

作　　者：张慧莲 苏爱雯 吴永菊 张艳丽 王红斌 杨文荣 庞鹏飞[1] ZHANG Hui-lian;SU Ai-wen;WU Yong-ju;ZHANG Yan-li;WANG Hong-bin;YANG Wen-rong;PANG Peng-fei(Key Laboratory of Environmental Functional Materials of Yunnan Province Education Department,School of Chemistry and Environment,Yunnan Minzu University,Kunming 650504,China;School of Life and Environmental Sciences,Deakin University,Geelong VIC 3217,Australia)

机构地区：[1]云南民族大学化学与环境学院,云南省教育厅环境功能材料重点实验室,云南昆明650504 [2]迪肯大学生命与环境科学学院,澳大利亚维多利亚州吉朗3217

出　　处：《分析测试学报》2023年第11期1452-1460,共9页Journal of Instrumental Analysis

基　　金：国家自然科学基金资助项目(21665027,21565031);云南省科技厅应用基础研究项目(202001AT070012);云南民族大学研究生科研创新基金项目(2023SKY028,2023SKY027)。

摘　　要：该文结合纳米生物条形码和杂交链式反应(HCR)双重信号放大策略构建了一种电化学生物传感器用于蛋白激酶A(PKA)的活性分析。以MoS2/AuNPs纳米复合材料作为玻碳电极修饰材料,半胱氨酸修饰的底物肽链通过Au-S键自组装到修饰电极表面。当存在目标物PKA时,底物肽链被磷酸化,可与纳米生物条形码(S1-AuNPs-Ab)特异性结合,以S1-AuNPs-Ab探针中S1链作为引发链,可诱导发夹DNA(H1和H2)发生杂交链式反应。HCR产物吸附亚甲基蓝(MB)电活性分子,产生放大的电化学响应信号,实现对PKA活性的定量分析。该电化学传感器检测PKA的线性范围为10^(-3)~20 U/mL,检出限(S/N=3)为3×10^(-4) U/mL。构建的电化学传感器具有良好的选择性、重现性和稳定性,可用于实际样品细胞裂解液中PKA的活性测定和蛋白激酶抑制剂的筛选及激酶相关药物的发现。An electrochemical biosensor was developed for the detection of protein kinase A(PKA)activity based on dual signal amplification strategy of nanoparticle-based bio-barcode and hybridization chain reaction(HCR).MoS2/AuNPs nanocomposite was used as modified nanomaterial for glassy carbon electrode(GCE).Cysteine-labeled substrate peptide was self-assembled on modified GCE surface by Au-S bonds,which was phosphorylated in the presence of PKA and ATP.Nanoparticlebased bio-barcode of S1-AuNPs-Ab was linked to phosphorylated substrate peptide through specific affinity.The initiator strand S1 of S1-AuNPs-Ab probe opened the hairpin DNA(H1 and H2)structures and triggered a cascade of hybridization chain reaction.Methylene blue(MB)molecules were captured on HCR product,resulting in an enhanced electrochemical response signal and achieving quantitative analysis of PKA activity.The response current of electrochemical biosensor is proportional to PKA concentrations in the range of 10^(-3)-20 U/mL,with a detection limit(S/N=3)of 3×10^(-4) U/mL.The proposed electrochemical biosensor exhibits good selectivity,reproducibility and stability,and could be applied for PKA activity assay in cell lysates samples,inhibitor screening and discovery of protein kinase related drugs.

关 键 词：蛋白激酶A 纳米生物条形码 杂交链式反应 电化学生物传感器 

分 类 号：O657.1[理学—分析化学] O629.7[理学—化学]