基于二维纳米探针检测血清exosomes内miRNA标志物进行结直肠癌筛查与诊断的新技术研究

Colorectal cancer is a malignant tumor with high morbidity and mortality in China. The screening and diagnosis of colorectal cancer is key for its prevention and treatment. However, current clinical diagnostic technologies of colorectal cancer have drawbacks including low sensitivity and poor specificity. Therefore, it is essential to develop novel, accurate, and sensitive technologies for the screening and diagnosis of colorectal cancer. Previous studies demonstrated the unique expression of miRNA biomarkers in serum exosomes of colorectal cancer patients. The biomarkers can be used for screening and diagnosing colorectal cancer. Based on our previous experiences of applying graphene oxide material and developing a serum circulating miRNA detection technology, we will develop a novel screening and diagnostic technology of colorectal cancer using a two-dimensional nanosized graphene oxide (NGO) nanoprobe in this study. This technology involved using the NGO material and fluorescence-labeled oligonucleotide probes to produce the NGO nanoprobe for the miRNA biomarkers detection in serum exosomes. The NGO nanoprobe can penetrate the lipid bilayer membranes of exosomes. Then, the oligonucleotide probes adsorbed on the nanoprobe hybridize with the target miRNAs and subsequently dissociate from the nanoprobe surface to generate a fluorescent signal. According to the signal intensity, the expression of miRNA biomarkers in serum exosomes can be determined accurately. Hence, the screening and diagnosis of colorectal cancer can be made by the results. The results of this study provide new ideas and directions for the development of colorectal cancer diagnostic technology.

结直肠癌已成为我国高发病率和高死亡率的恶性肿瘤。结直肠癌的筛查与诊断是其防治的关键。目前结直肠癌的临床诊断技术存在灵敏度低与特异性差等缺陷，因此需要研发准确且灵敏的结直肠癌筛查和诊断新技术。研究发现，结直肠癌患者血清外泌体(exosomes)中存在特定表达的miRNA标志物，可用于结直肠癌的筛查与诊断。本项目在前期应用氧化石墨烯和开发血清循环miRNA检测技术的基础上，拟研发一项利用二维纳米氧化石墨烯(NGO)材料与荧光标记核酸探针所构建的NGO纳米探针检测血清exosomes内miRNA标志物进行结直肠癌筛查和诊断的新技术。新技术中的NGO纳米探针在穿透exosomes双层膜后，其吸附的核酸探针与目标miRNA杂交互补，脱离其表面后产生荧光信号；根据信号强度准确测定血清exosomes内miRNA标志物，由此进行结直肠癌的筛查和诊断。本研究将为结直肠癌诊断技术的发展提供新思路与新方向。

结直肠癌已成为我国高发病率和高死亡率的恶性肿瘤。结直肠癌的筛查与诊断是其防治的关键。目前结直肠癌的临床诊断技术存在灵敏度低与特异性差等缺陷，因此需要研发准确且灵敏的结直肠癌筛查和诊断新技术。研究发现，结直肠癌患者血浆外泌体的含量以及其内特定表达的miRNA标志物，可用于结直肠癌的筛查与诊断。本项目在前期应用氧化石墨烯和开发血清循环miRNA检测技术的基础上，研发利用二维纳米材料黑磷(BP)与外泌体特定蛋白CD63、EpCAM的适配体(aptamer)构建BP纳米探针用于外泌体水平的检测，并利用结肠癌患者血浆验证该探针的准确性，(线性范围：5×102-2×104 particles/μL，检测限分别为3.38×102 particles/μL，1.86×102 particles/μL)。同时我们还利用BP与荧光标记核酸探针构建另外一种BP纳米探针用于检测临床血浆exosomes内miRNA标志物，(线性范围： 1×105-1×106 particles/μL，检测限为2.5×104 particles/μL)，通过激光共聚焦验证了纳米探针检测的可靠性。本项目通过检测外泌体以及其内特定表达的miRNA可以进行结直肠癌筛查和诊断。因此，本研究将为结直肠癌诊断技术的发展提供新思路与新方向。.另外，本项目还使用了掺杂钙离子的聚多巴胺(PDA)修饰二维黑磷(BP)纳米片(BP@PDA)作为检测复杂生物样品中核酸和蛋白质的纳米探针平台。荧光标记的单链DNA适配体或探针通过钙离子的配位作用被稳定吸附在含有钙离子的BP@PDA表面。BP@PDA的PDA外壳包裹增强了内部BP的稳定性，同时也提供了与DNA碱基结合的位点，并能够淬灭DNA链上标记的荧光。无需任何化学偶联，该纳米探针就可高特异性与选择性地检测1%稀释血清样品中的靶标蛋白(人凝血酶，线性范围:10至25 nM，检测限:0.02 nM)、靶标单链DNA(线性范围:1至10 nM，检测限:0.52 nM)，并可分析检测活细胞中的胞内信使RNA。这种纳米探针平台的设计与开发将有助于未来基于聚合物修饰或改性二维纳米材料的生物分子分析技术的发展。