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

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 fragment molecular interpretation, can be effectively solved. Meanwhile, the method also possesses high specificity and sensitivity, quick and simple operation and high-throughput for multiplex detection. Therefore, we plan to further optimize the experimental parameters in various steps, thoroughly check this method by applying it in the detection of necleic acid in breast cancer tissue and normal tissue samples, and finally provide a novel way for the quantitative detection of nucleic acid.

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。

迄今为止检测核苷酸的常规方法一直都是聚合酶链反应（Polymerase Chain Reaction，PCR）及其衍生方法，再与电泳等其他分析手段相结合达到分析检测核酸的目的。但该方法存在繁琐费时，特异性低，易发生交叉污染，“假阳性”率较高等。所以针对核酸检测，无论实验室基础研究还是实际临床应用，建立原理简单、操作快速、结果可靠、高通量及可视化的检测技术成为越来越需要迫切解决的问题。本课题组在本次基金的资助下，主要研究内容集中在以下4个方面：1. BioTag选择标准的建立；2. 优化捕捉寡核苷酸链和BioTag及stabler的浓度和加入量及比例；3. MS测定的优化及结果判读；4. 该方法的临床应用效果评价。三年时间里，本课题组初步完成了上述研究内容的各项任务，建立了BioTag小分子多肽库及其选择标准（包括①分子量在1000D左右的合成小分子多肽及小分子量40000D左右的商品化的单克隆抗体；②合成的小分子多肽不包含Met, Leu等性质不稳定的AA；③Tag分子末端的linker成分与stabler一致；④Tag内部尽量减少分子内作用力，例如形成茎环结构及二级结构，⑤尽量选择链状而非芳香类AA制备小分子多肽，以便减少个体内的空间位阻。），同时利用杂交原理及不同待测正常核苷酸与突变核苷酸所形成的三明治”夹心式复合结构的沉降系数不同，建立了较稳定的BioTag-MS核酸测定MS方法，可达到区分单碱基突变的目的，检测方法特异性高，灵敏度达到aM水平（10-21mol/L），并且利用15-nm功能化的胶体金颗粒固有的酒红色变化，增加了可视化（the naked eye readout）结果判读模式，且正在申请该部分内容的专利。在临床评价方面，成功检测了单基因病患者的耳聋基因1555A->G来评价该方法。现已发表1篇相关SCI文章（第一作者1篇）及2篇SCI文章（均第一作者）在投稿under review状态（其中包括1篇JACS）。BioTag-MS核酸检测方法其优点在于，通过Tag实现了信号放大功能，规避了直接检测靶核苷酸的离子化效率低的缺陷，甚至有文献报道其灵敏度可以达到500 zM（10-21）水平，所以带Tag的胶体金纳米探针（NP）与现代化色谱分析方法MS相结合，是一种优良的具有高特异性和高灵敏度的基因检测技术，具备了具有非常广泛应用前景的物质基础。