基于双光子激发荧光和生物素-亲合素放大体系的多种肿瘤标志物的可视化实时动态三维活检

Tumor markers are a class of biochemical substances which can reflect the existence and growth of the tumor, and in cancer early screening, diagnosis, prognosis, auxiliary curative effect evaluation, monitoring recurrence and metastasis have important significance. Therefore, the accurate detection and analysis of tumor markers is always the researching focus; and with the help of two-photon fluorescence immuno probe which can achieve the three dimensional (3D) dynamic holographic depth imaging of tumor marker, in the tumor occurrence and development process, not only some key single molecular events and the cancer cell metastasis process will be dynamically monitored, but also the sharp tools will be provided to reveal the cancer occurrence, development and metastasis mechanisms. Under the basis of the early development of the immune two-photon fluorescence probes with specific recognition carcinoembryonic antigen and carbohydrate antigen, the two-photon fluorescence generation mechanism, the biotin-avidin multistage amplification effect (the exponential improvement of the sensitivity) and the antigen-antibody specificity affinity principle will be first combined into the fluorescence immunohistochemistry system, and then two-photon fluorescence immuno probes will be constructed. Therefore, the visualized real-time dynamic 3D biopsy of the multiple tumor markers will have been implemented, and this can completely achieve the accurate diagnosis of tumors and cancer. Two-photon fluorescence imaging can greatly improve the definition and resolution of imaging sensitivity, and can avoid the light induced toxicity, photobleaching and tissue autofluorescence interference.

肿瘤标志物是一类能反映肿瘤存在与生长状态的生化物质，它们在肿瘤的早期筛查、辅助诊断、预后判断、疗效评价、复发和转移监测中具有重要意义。因此对肿瘤标志物的准确检测与分析始终是人们努力的焦点；而借助双光子荧光免疫探针可以对肿瘤标志物进行动态全息三维深度成像，不但能够在肿瘤发生和发展过程中对某些关键单分子事件和癌细胞转移过程实施动态监测，而且能为揭示癌症发生、发展及转移机制提供锐利工具。在前期开发癌胚抗原与糖链抗原的免疫双光子荧光探针的基础上，本申请拟首次通过将双光子荧光发生机制、生物素-亲和素多级放大效应（呈几何级数提高灵敏度）与抗原-抗体特异性亲和原理三者统一到荧光免疫组化体系中，构建双光子荧光免疫探针，实现对多种肿瘤标志物的可视化实时动态三维活检，进而实现对肿瘤及癌症的准确诊断。双光子成像可以大大提高成像灵敏度、清晰度与分辨率，且可避免光致毒、光漂白与组织自发荧光干扰。

肿瘤标志物（糖链抗原：CA19-9和CA125）是一类能反映肿瘤存在与生长状态的生化物质，CA19-9的测定广泛用于胰腺癌、胆囊癌、结肠癌和胃癌的临床监测与诊断，而CA125则用于卵巢癌、输卵管腺癌、子宫内膜癌和宫颈癌的诊断。CA19-9和CA125在肿瘤的早期筛查、辅助诊断、预后判断、疗效评价、复发和转移监测中具有重要意义。. 本研究利用合成的双光子荧光标记探针LP标记糖链抗原的抗体（CA19-9和CA125的抗体分别：Ab199和Ab125）, 并成功地用于糖链抗原的实时动态显微与检测。本研究的创新点有三：首次用双光子荧光标记探针LP标记抗糖链抗原的抗体Ab199和Ab125, 制备了免疫双光子荧光抗体LP-Ab199和LP-Ab125；首次实现对肿瘤标志物CA19-9和CA125的高清晰度、高灵敏度与高分辨率的实时动态活检；首次制成简单便捷的用于CA19-9和CA125检测的双光子荧光标记试剂盒, 方便快速地进行门诊化验与筛选。. 对肿瘤标志物的准确检测与分析始终是人们努力的焦点；而借助双光子荧光免疫探针可以对肿瘤标志物进行动态全息三维深度成像, 不但能够在肿瘤发生和发展过程中对某些关键单分子事件和癌细胞转移过程实施动态监测, 而且能为揭示癌症发生、发展及转移机制提供锐利工具。本研究解决了肿瘤标识物（CA19-9和CA125）活检的关键技术难题，也破解了肿瘤或癌症阴阳性化验确定这一共性技术。双光子荧光标记试剂盒能准确诊断肿瘤/癌症发生与否，解决了过去肿瘤/癌症早期诊断难的严重问题，为肿瘤/癌症的早发现早治疗提供了锐利工具，从而大大减少广大群众罹患肿瘤/癌症的机率。

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