近红外长余辉纳米探针在体外检测肿瘤细胞中的应用

发布时间：2018-02-11 05:49

本文关键词： 纳米探针长余辉近红外体外检测肿瘤细胞　出处：《华侨大学》2015年硕士论文　论文类型：学位论文

【摘要】：由于在体内和体外成像方面具有高灵敏度,光学成像系统日益受到科研工作者的重视。然而传统的荧光探针由于光漂白性、发光不稳定性、低信噪比等缺点限制了光学成像的发展。最近十年以来研究者们设计出了用长余辉纳米材料做为光学探针。长余辉纳米材料的优势在于它只需要体外激发,这在很大程度上可以去除生物自发荧光对成像的干扰,从而提高了信噪比和灵敏度。将长余辉纳米探针运用于活体成像的研究已比较多,而相比较它在体外生物检测的报道却比较少。在本论文中我们提出了采用可见光激发的近红外长余辉纳米材料与单克隆抗体经过生物复合过程、形成具有靶向性的生物光学探针来检测肿瘤细胞。在此基础上,为了进一步拓展长余辉材料在生物医学检测领域的应用,我们用长余辉纳米材料与抗体的结合物做为标记物,设计了一种可以大批量、高灵敏度地检测癌细胞的方法。我们先用溶胶-凝胶法合成一种粒径30-100 nm、能发射高亮度近红外光的长余辉纳米材料Zn_3Ga_2Ge_2O_(10):Cr~(3+)(ZGG);进一步制作的功能化ZGG-NH_2纳米颗粒与anti-EpCAM经过生物复合修饰得到的ZGG-EpCAM近红外长余辉纳米探针、进行体外靶向识别乳腺癌细胞MCF7研究。结果表明,用ZGG-EpCAM纳米探针标记的MCF7细胞的光信号要明显强于来自用没有复合上单克隆抗体的ZGG纳米颗粒孵育的细胞的光信号;从而说明了ZGG-EpCAM纳米荧光探针具有在体外特异性识别MCF7细胞的特性。另外,它可持续发光1 h以上,使长时间进行细胞体外检测变得可行。所以,ZGG-EpCAM纳米探针不仅可特异性地体外检测乳腺癌细胞,而且还有长时间监测肿瘤的能力。因而,本研究开发的近红外长余辉纳米探针对癌症的早期诊断和研究提供了一个新的视角。为了拓展近红外长余辉纳米材料在生物医学检测领域的应用,我们同样使用溶胶-凝胶制备出另外一种近红外长余辉纳米材料Zn_3Ga_2O_6:Cr~(3+)(ZGO);ZGO通过表面氨基化修饰后与anti-EGFR肺癌单克隆抗体的不同基团反应结合、获得能够靶向性识别表达EGFR的肺癌细胞(A549)的近红外长余辉纳米探针ZGO-EGFR。进一步利用ZGO-EGFR对固定于96孔板底部的A549细胞进行定量和高通量检测,我们发现最低可检测到35个细胞;从而说明,这种纳米探针可以克服传统检测方法无法检测较少细胞的困境。本研究研发的长余辉纳米荧光探针也为高通量和高灵敏度地检测其它生物医学样品提供了一种新的检测方法。
[Abstract]:Because of its high sensitivity in vivo and in vitro imaging, optical imaging system has been paid more and more attention by researchers. However, the traditional fluorescence probe is unstable due to photobleaching. Shortcomings such as low signal-to-noise ratio limit the development of optical imaging. In the last decade, researchers have designed long afterglow nanomaterials as optical probes. The advantage of long afterglow nanomaterials is that they only need to be excited in vitro. To a large extent, this can remove the interference of biological autofluorescence on imaging, thus improving the signal-to-noise ratio (SNR) and sensitivity. Many studies have been done on the application of long-afterglow nanoprobes to imaging in vivo. In this paper, we propose that the near infrared long afterglow nanomaterials excited by visible light undergo biological recombination with monoclonal antibodies. On this basis, in order to further expand the application of long afterglow materials in biomedical detection, we use long afterglow nanomaterials and antibodies as markers. And designed a way to do it in large quantities, The method of high sensitivity detection of cancer cells. We first synthesized a long afterglow nanomaterial, Zn_3Ga_2Ge_2O_(10):Cr~(3 / ZGG, which can emit high luminance near infrared light by sol-gel method. Further, the functionalized ZGG-NH_2 nanoparticles and anti-EpCAM were prepared. The near infrared long afterglow nanoprobes of ZGG-EpCAM were prepared by biological composite modification. The results showed that the optical signal of MCF7 cells labeled with ZGG-EpCAM nanoparticles was significantly stronger than that of cells incubated with ZGG nanoparticles without complex monoclonal antibodies. The results show that the ZGG-EpCAM nanofluorescent probe can specifically recognize MCF7 cells in vitro, in addition, it can continuously emit light for more than 1 hour. So the ZGG-EpCAM nanoprobe not only can detect breast cancer cells specifically in vitro, but also has the ability to monitor tumor for a long time. The NIR long afterglow nanoprobes developed in this paper provide a new perspective for the early diagnosis and research of cancer. In order to expand the application of NIR long afterglow nanomaterials in biomedical detection, We also used sol-gel to prepare another near infrared long-afterglow nano-material, Zn_3Ga_2O_6:Cr~(3 ZGOGo, which reacted with different groups of monoclonal antibodies against anti-EGFR lung cancer by surface modification. The near infrared long afterglow probe ZGO-EGFRwas obtained, which could target the lung cancer cells expressing EGFR. The quantitative and high-throughput detection of A549 cells fixed at the bottom of the 96-well plate by ZGO-EGFR showed that the lowest 35 cells could be detected. Thus, The long afterglow fluorescent probe can also provide a new method for the detection of other biomedical samples with high throughput and high sensitivity.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.4;O657.33

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