基于光学纳米探针的三磷酸腺苷、甲胎蛋白、microRNA和端粒酶检测

发布时间：2018-08-16 17:31

【摘要】：本论文利用功能性核酸分子将贵金属和上转换纳米材料构建成三种类型的纳米自组装生物传感器,此类传感器具有独特的圆二色性(CD)、表面增强拉曼散射(SERS)或荧光特性。基于良好的生物相容性以及适配体与目标物的特异性识别性能,将所构建的纳米生物传感器作为光学活性探针,成功建立了系列高灵敏度、高特异性的细胞内和血清中生物标志物检测的新原理和新方法。具体内容如下:1.以含有核酸适配体的DNA分子为骨架,将不同粒径的金纳米粒子构建成一种异质二聚体等离子手性传感器,实现肿瘤细胞内三磷酸腺苷(ATP)的高灵敏检测。在溶液中,异质二聚体对ATP有特异性识别,使二聚体结构发生解离,CD信号对ATP浓度的变化具有明显响应;进入细胞后,该传感器具有较高的生物相容性和稳定性,并能与细胞中ATP特异性结合进而产生CD信号的变化。对细胞内ATP的检测灵敏度达到0.2mmol/L,线性范围为1.5-4.2 mmol/L。建立了细胞内小分子生物标志物的手性传感检测方法。2.基于适配体碱基互补配对原则构建了银纳米粒子三聚体表面增强拉曼传感器,实现对血清中甲胎蛋白(AFP)的超灵敏检测。以修饰拉曼信标的银纳米粒子作为基底,可显著提高传感器的电磁场强度。实验结果证明银纳米粒子三聚体具有强烈的拉曼增强效应。成功构建了SERS传感检测AFP的体系,对AFP的最低检测限(LOD)达到0.097amol/L,线性范围为0.2-20 amol/L。该传感器有望作为一种通用的检测平台应用于其它生物标志物的定量检测。3.采用DNA驱动的金纳米棒二聚体和上转换纳米颗粒多级次卫星状纳米结构,实现了肿瘤细胞内microRNA含量和端粒酶活性的同时检测。该传感器进入细胞后,基于碱基互补配对和引物延伸原则,利用SERS和荧光信号分别实现细胞中miR-21和端粒酶同时定量分析。其中,拉曼强度与miR-21浓度之间具有良好的线性关系,线性范围是0.021-22.36 amol/ngRNA,LOD达到0.011 amol/ngRNA;荧光强度与端粒酶活性之间线性范围是0.6×10-12到31×10-12 IU,LOD达到3.2×10-13 IU。这方法实现了活细胞内双重信号介导的两种癌症标志物的同时检测。
[Abstract]:In this paper, three types of nano-self-assembled biosensors were constructed by using functional nucleic acid molecules to synthesize noble metals and up-converted nanomaterials. These sensors have unique circular dichroism (CD), surface-enhanced Raman scattering (SERS) or fluorescence characteristics. Based on the good biocompatibility and the specificity of the aptamer and the target, a series of high sensitivity nanosensors were successfully established by using the constructed nano-biosensors as optical active probes. New principles and methods for the detection of high specific biomarkers in cells and in serum. The details are as follows: 1. Using DNA molecule containing aptamer of nucleic acid as skeleton, a heterogeneous dimer plasma chiral sensor was constructed by using gold nanoparticles with different particle sizes to detect adenosine triphosphate (ATP) in tumor cells with high sensitivity. In the solution, the heterogeneous dimer has specific recognition to ATP, which makes the dissociation CD signal of the dimer structure have obvious response to the change of ATP concentration, and the biosensor has high biocompatibility and stability after entering the cell. And can specifically bind to ATP in cells to produce changes in CD signals. The sensitivity of intracellular ATP was 0.2 mmol / L and the linear range was 1.5-4.2 mmol / L. A chiral sensing method for detection of small molecular biomarkers in cells was established. Based on the principle of aptamer base complementary pairing, a surface-enhanced Raman sensor of silver nanoparticles trimer was constructed to detect alpha-fetoprotein (AFP) in serum. Using silver nanoparticles modified Raman beacons as the substrate, the electric magnetic field intensity of the sensor can be significantly increased. The experimental results show that the silver nanoparticles trimer has a strong Raman enhancement effect. The SERS sensing system for AFP detection is successfully constructed. The minimum detection limit for AFP is 0.097 amol / L, and the linear range is 0.2-20 amol / L. The sensor is expected to be used as a general detection platform for quantitative detection of other biomarkers. DNA driven gold nanorods dimer and up-converted nanocrystalline multistage subsatellite nanostructures were used to detect microRNA content and telomerase activity in tumor cells at the same time. Based on the principle of base complementary pairing and primer extension, the SERS and fluorescence signals were used to realize the simultaneous quantitative analysis of miR-21 and telomerase in the cells. The linear range between Raman intensity and miR-21 concentration was 0.021-22.36 amol / ngRNA-LOD was 0.011 amol / ngRNAs, and the linear range between fluorescence intensity and telomerase activity was 0. 6 脳 10-12 to 31 脳 10-12 IULD was 3. 2 脳 10-13 IU. This method realizes the simultaneous detection of two cancer markers mediated by double signals in living cells.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3;R446.1

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