BioTag-MS法── 一种新的核酸定量分析方法的建立及临床应用效果评价

项目名称： BioTag-MS法── 一种新的核酸定量分析方法的建立及临床应用效果评价

项目编号： No.21205063

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 分析化学

项目作者： 杨冰

作者单位： 南京医科大学

项目金额： 25万元

中文摘要： 质谱的问世及软电离离子化技术的发展，对现代生物分析化学的进步起着巨大作用，但在核酸检测还存在灵敏度低、特异性差等难题。前期工作中，我们选择检测信号高、分子结构简单和易于组装的多肽片段为生物标签，制备了双功能化金纳米探针- - 表面同时组装寡核苷酸和多肽，首次建立了以生物磁性纳米探针与质谱技术相结合定量测定核酸的方法（BioTag-MS法）。预实验结果提示该方法将质谱、纳米标记和生物信息学相结合的研究思路具有可行性。该方法以生物标签作为测定对象，不但放大靶目标信号，无需PCR即可测定核酸，而且按照其与靶目标的唯一对应关系，间接测定核酸，有效避免了核酸分子因离子化效率低和缺乏解读其碎片分子的数据库等问题。同时该方法还兼具高特异性与灵敏度、操作简单快速和可实现高通量多重检测等优点。本研究拟进一步优化各步骤实验参数，并在一定数量乳腺癌癌灶组织和正常组织样本中进行论证，将为核酸定量检测提供新方法。

中文关键词： 胶体金；探针；质谱；核苷酸；

英文摘要： With the advent of mass spectrometry (MS) and the progress in soft ionization technology, tremendous changes have taken place in modern bio-analytical chemistry. However, problems, such as low sensitivity and unsatified specificity, still remain in nucleic acid detection. In order to solve these problems, in the present study we established a BioTag-MS method for the quantitative determination of nucleic acid by combining bio-magnetic nanoprobes and MS technique. In this method, peptide fragments with high detection signal, simple molecular structure and excellent characteristics for assembling were chosen as biological lables to prepare a dual-functionalized gold nanoparticle probe, the surface of which was loaded with oligonucleotides and peptides. Preliminary experimental results obtained by applying this method indicated the feasibilty of combing MS, nanoparticle labeling and bioinformatics as a novel way in nucleic acid determination. Since bio-tag is used as a detection object in this method, detection signal is amplified and the determination of necleic acid can be carried out without preforming PCR. Also, because determination of nucleic acid were performed in accordance with its corresponding relationship to the target, problems, such as difficulty in nucleic acid ionization and lack of database in its

英文关键词： Colloid gold；Probe；MS；Nucleotide；