本文主要研究内容
作者粟莎莎（2019）在《基于MeCP2蛋白和双酶信号放大策略的电化学生物传感器检测DNA甲基化水平的研究》一文中研究指出：目的:本研究以甲基化CpG结合蛋白2(Methyl-CpG-Binding protein2,MeCP2)作为DNA甲基化位点特异性识别单元,以纳米金(Gold nanoparticles,AuNPs)修饰电极和双酶标记抗体作为信号放大体系,构建一种简单灵敏的电化学生物感器用于DNA甲基化的定量检测。方法:1.双酶标记抗体的制备。采用戊二醛交联法和过碘酸钠法将葡萄糖氧化酶(Glucose oxidase,GOD)和辣根过氧化物酶(Horseradish peroxidase,HRP)共同修饰到抗6×His鼠单克隆抗体上,并用紫外-可见分光光谱法对所制备的双酶标记抗体GOD-HRP/IgG进行表征。2.DNA电化学生物传感器的构建。采用电沉积法在电极表面修饰上AuNPs,以自组装的方式依次将DNA探针、巯基己醇和靶DNA连接到电极上,构建DNA电化学生物传感器。通过扫描电子显微镜和原子力显微镜对电沉积纳米金和探针固定的形貌进行表征鉴定,并用循环伏安法和电化学阻抗法对每一步组装效果进行电化学表征。3.可行性分析。设计相同碱基序列的非甲基化和含1个甲基化位点的靶DNA,制备DNA电化学生物传感器,与MeCP2作用后,再和GOD-HRP/IgG反应,在含葡萄糖和对苯二酚的检测液中进行差分脉冲伏安扫描,对比甲基化与非甲基化靶DNA检测结果;同时考察实验所设计的双酶信号放大策略的放大效果,进行方法可行性的分析。4.优化重要实验参数。对杂交时间、MeCP2浓度、最适抗体量以及底物葡萄糖浓度等实验条件进行优化。5.DNA甲基化位点定量分析。设计相同碱基序列不同数量甲基化位点的靶DNA,采用所制备的电化学生物传感器分析对应的电流信号,建立不同数量甲基化位点与电信号之间的相关关系。6.考察电化学生物传感器的分析性能,灵敏度、重复性和稳定性。结果:1.紫外可见分光光谱扫描曲线中可见GOD-HRP/IgG的吸收峰分别向GOD和HRP最大吸收峰处移动,表明双酶修饰抗体成功。2.扫描电子显微镜显示AuNPs呈球状、分布均匀;原子力显微镜鉴定结果表明,AuNPs大小与扫描电子显微镜显示结果一致,DNA分布有序;循环伏安法和电化学阻抗法共同表明各成分均成功组装在电极表面。3.可行性验证结果表明,该方法可有效区分甲基化与非甲基化序列,同时实验设计的双酶标记体系产生的电化学响应信号明显强于单酶标记体系,具有较好的信号增强效果。4.基于实验结果,综合考虑采用的最佳杂交时间为90 min、MeCP2浓度为200 mg/L、抗体浓度为10mg/L、葡萄糖浓度为0.5 mmol/L。5.采用所制备的电化学生物传感器分析不同甲基化数量靶序列DNA的电流信号,结果显示,在1~5个甲基化位点范围内,峰电流的强度与靶DNA甲基化位点的数量呈线性关系,线性方程为I(μA)=7.550 N+1.328,相关系数r为0.984。6.该方法可检到低至0.1 fmol/L的甲基化靶序列DNA,灵敏度高,线性范围宽,重复性、稳定性均良好。结论:成功构建用于DNA甲基化检测的电化学生物传感,能有效区分多个甲基化位点的靶序列DNA,有望成为临床DNA甲基化定量分析的有效手段。
Abstract
mu de :ben yan jiu yi jia ji hua CpGjie ge dan bai 2(Methyl-CpG-Binding protein2,MeCP2)zuo wei DNAjia ji hua wei dian te yi xing shi bie chan yuan ,yi na mi jin (Gold nanoparticles,AuNPs)xiu shi dian ji he shuang mei biao ji kang ti zuo wei xin hao fang da ti ji ,gou jian yi chong jian chan ling min de dian hua xue sheng wu gan qi yong yu DNAjia ji hua de ding liang jian ce 。fang fa :1.shuang mei biao ji kang ti de zhi bei 。cai yong wu er quan jiao lian fa he guo dian suan na fa jiang pu tao tang yang hua mei (Glucose oxidase,GOD)he la gen guo yang hua wu mei (Horseradish peroxidase,HRP)gong tong xiu shi dao kang 6×Hisshu chan ke long kang ti shang ,bing yong zi wai -ke jian fen guang guang pu fa dui suo zhi bei de shuang mei biao ji kang ti GOD-HRP/IgGjin hang biao zheng 。2.DNAdian hua xue sheng wu chuan gan qi de gou jian 。cai yong dian chen ji fa zai dian ji biao mian xiu shi shang AuNPs,yi zi zu zhuang de fang shi yi ci jiang DNAtan zhen 、qiu ji ji chun he ba DNAlian jie dao dian ji shang ,gou jian DNAdian hua xue sheng wu chuan gan qi 。tong guo sao miao dian zi xian wei jing he yuan zi li xian wei jing dui dian chen ji na mi jin he tan zhen gu ding de xing mao jin hang biao zheng jian ding ,bing yong xun huan fu an fa he dian hua xue zu kang fa dui mei yi bu zu zhuang xiao guo jin hang dian hua xue biao zheng 。3.ke hang xing fen xi 。she ji xiang tong jian ji xu lie de fei jia ji hua he han 1ge jia ji hua wei dian de ba DNA,zhi bei DNAdian hua xue sheng wu chuan gan qi ,yu MeCP2zuo yong hou ,zai he GOD-HRP/IgGfan ying ,zai han pu tao tang he dui ben er fen de jian ce ye zhong jin hang cha fen mai chong fu an sao miao ,dui bi jia ji hua yu fei jia ji hua ba DNAjian ce jie guo ;tong shi kao cha shi yan suo she ji de shuang mei xin hao fang da ce lve de fang da xiao guo ,jin hang fang fa ke hang xing de fen xi 。4.you hua chong yao shi yan can shu 。dui za jiao shi jian 、MeCP2nong du 、zui kuo kang ti liang yi ji de wu pu tao tang nong du deng shi yan tiao jian jin hang you hua 。5.DNAjia ji hua wei dian ding liang fen xi 。she ji xiang tong jian ji xu lie bu tong shu liang jia ji hua wei dian de ba DNA,cai yong suo zhi bei de dian hua xue sheng wu chuan gan qi fen xi dui ying de dian liu xin hao ,jian li bu tong shu liang jia ji hua wei dian yu dian xin hao zhi jian de xiang guan guan ji 。6.kao cha dian hua xue sheng wu chuan gan qi de fen xi xing neng ,ling min du 、chong fu xing he wen ding xing 。jie guo :1.zi wai ke jian fen guang guang pu sao miao qu xian zhong ke jian GOD-HRP/IgGde xi shou feng fen bie xiang GODhe HRPzui da xi shou feng chu yi dong ,biao ming shuang mei xiu shi kang ti cheng gong 。2.sao miao dian zi xian wei jing xian shi AuNPscheng qiu zhuang 、fen bu jun yun ;yuan zi li xian wei jing jian ding jie guo biao ming ,AuNPsda xiao yu sao miao dian zi xian wei jing xian shi jie guo yi zhi ,DNAfen bu you xu ;xun huan fu an fa he dian hua xue zu kang fa gong tong biao ming ge cheng fen jun cheng gong zu zhuang zai dian ji biao mian 。3.ke hang xing yan zheng jie guo biao ming ,gai fang fa ke you xiao ou fen jia ji hua yu fei jia ji hua xu lie ,tong shi shi yan she ji de shuang mei biao ji ti ji chan sheng de dian hua xue xiang ying xin hao ming xian jiang yu chan mei biao ji ti ji ,ju you jiao hao de xin hao zeng jiang xiao guo 。4.ji yu shi yan jie guo ,zeng ge kao lv cai yong de zui jia za jiao shi jian wei 90 min、MeCP2nong du wei 200 mg/L、kang ti nong du wei 10mg/L、pu tao tang nong du wei 0.5 mmol/L。5.cai yong suo zhi bei de dian hua xue sheng wu chuan gan qi fen xi bu tong jia ji hua shu liang ba xu lie DNAde dian liu xin hao ,jie guo xian shi ,zai 1~5ge jia ji hua wei dian fan wei nei ,feng dian liu de jiang du yu ba DNAjia ji hua wei dian de shu liang cheng xian xing guan ji ,xian xing fang cheng wei I(μA)=7.550 N+1.328,xiang guan ji shu rwei 0.984。6.gai fang fa ke jian dao di zhi 0.1 fmol/Lde jia ji hua ba xu lie DNA,ling min du gao ,xian xing fan wei kuan ,chong fu xing 、wen ding xing jun liang hao 。jie lun :cheng gong gou jian yong yu DNAjia ji hua jian ce de dian hua xue sheng wu chuan gan ,neng you xiao ou fen duo ge jia ji hua wei dian de ba xu lie DNA,you wang cheng wei lin chuang DNAjia ji hua ding liang fen xi de you xiao shou duan 。
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论文作者分别是来自贵州医科大学的粟莎莎，发表于刊物贵州医科大学2019-07-01论文，是一篇关于甲基化论文,电化学论文,传感器论文,双酶催化论文,贵州医科大学2019-07-01论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自贵州医科大学2019-07-01论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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