基于生物条形码的粘蛋白和特定细胞电化学发光适配体生物传感方法研究  

Electrogenerated chemiluminescence aptamer-based biosensing for the determination of mucin and specific cells based on bio-bar-code amplification

作　　者：王晓飞 漆红兰[1] 张成孝[1] Xiaofei Wang;Honglan Qi;Chengxiao Zhang(Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education,Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province,School of Chemistry and Chemical Engineering,Shaanxi Normal University,Xi’an 710062,China)

机构地区：[1]陕西师范大学化学化工学院,应用表面与胶体化学教育部重点实验室,陕西省生命分析化学重点实验室,西安710062

出　　处：《中国科学：化学》2021年第3期388-394,共7页SCIENTIA SINICA Chimica

基　　金：国家自然科学基金(编号:21775097,21775098);中央高校基础研究经费(编号:GK201801006)资助项目。

摘　　要：本文基于适配体识别和生物条形码放大策略,以MCF-7细胞和粘蛋白(MUC1)为目标物,MUC1的特异性适配体(rcDNA)为分子识别物质,Ru(phen)_(3)^(2+)为信号物质,rcDNA通过巯基自组装于金电极表面作为传感界面,发卡DNA(hpDNA)和rcDNA通过巯基自组装在金纳米粒子(AuNP)表面合成的hpDNA/AuNP/rcDNA为生物条形码探针,建立了测定MUC1和特定细胞的电化学发光适配体生物传感新方法.当目标物被传感界面上的rcDNA俘获后,进而与生物条形码探针形成夹心复合物,Ru(phen)32+嵌入hpDNA中.在共反应剂的存在和+0.95 V恒电位下测量电化学发光强度.电化学发光强度的变化值与MUC1和MCF-7细胞浓度的对数在4~800 pg/mL和30~5.0×10^(4 )cells/mL范围内呈良好的线性关系,检出限分别为0.5 pg/mL和9 cells/mL.将该法应用于监测两种物质刺激下MCF-7细胞表面MUC1含量的变化,发现芹黄素刺激下细胞表面MUC1含量降低,而过氧化氢刺激下细胞表面MUC1含量不变.A highly sensitive electrogenerated chemiluminescence(ECL)aptamer-based biosensing based on bio-barcode amplification was developed for the determination of cell surface proteins and specific cells.MCF-7 cells were used as target cells,mucin(MUC1)was used as target proteins,anti-MUC1 specific aptamers(rcDNA)were used as molecular recognition elements and Ru(phen)_(3)^(2+)was used as ECL signal molecules.A hairpin DNA(hpDNA)and a rcDNA were self-assembled on the surface of gold nanoparticles(AuNP)by self-assembly technique to obtain the biobar-code probe hpDNA/AuNP/rcDNA.rcDNA was modified onto the surface of gold electrodes to form a biosensing platform.A sandwich complex among rcDNA,target MUC1(or MCF-7 cells)and hpDNA/AuNP/rcDNA was formed on the surface of electrodes.After Ru(phen)32+was intercalated into hpDNA,strong ECL signals were generated in the presence of co-reactants at the constant potential of 0.95 V.The increased ECL intensity was proportional to the logarithm of MUC1 concentration in the range of 4~800 pg/mL with a detection limit of 0.5 pg/mL.The increased ECL intensity was proportional to the logarithm concentration of MCF-7 cells in the range of 30~5×10^(4) cells/mL with a detection limit of 9 cells/mL.In addition,the developed ECL method was used to discriminate the MUC1 expression on different types of the cells and to monitor the dynamics of MUC1 on the surface of MCF-7 cells under the external drug stimulation,including apigenin and H_(2)O_(2).The decline of MUC1 expression on the surface of MCF-7 cells was observed under apigenin treatment while inconspicuous MUC1 expression was observed under H_(2)O_(2) treatment.

关 键 词：电化学发光 生物传感 细胞分析 粘蛋白 药物刺激 

分 类 号：Q503[生物学—生物化学]