功能化核酸折纸用于生物活性分子检测的研究

发布时间：2018-05-28 19:18

本文选题：RNA纳米花 + 癌细胞　；参考：《曲阜师范大学》2017年硕士论文

【摘要】：本论文在利用荧光、紫外吸收和激光扫描共聚焦显微镜等技术下,利用功能化核酸折纸方法和基于杂交链式反应的比色放大法分别对细胞进行联合给药、靶向治疗及miRNA、ATP的检测。本文主要内容如下:1.基于磁性RNA纳米花用于复合给药和靶向治疗的诊断-治疗一体化系。本部分开发了一种基于磁性RNA纳米花用于复合给药和靶向治疗的诊断-治疗一体化系统。与传统的核酸结构相比之下,通过将磁性纳米粒子(MNP)引入到中可使得RNA NF分离方便。叶酸(FA)修饰的MNP/RNA NF(FA/MNP/RNA NF)作为一种具有优良的生物相容性纳米靶向载体克服了MNP/RNA NF的非选择性。然后,抗癌药物阿霉素(DOX)和光敏剂四甲基吡啶卟啉(TMPyP4)结合RNA NF被用作共同药物运输模型,RNA NF首次被用于联合给药。因此,成像荧光标记,目标识别元素和药物分子都聚集在MNP/RNA NF的表面。实验结果表明,联合给药平台(FA/MNP/RNA NF/D/T)的疗效优于单一给药平台(FA/MNP/RNA NF/D)。此外,FA/MNP/RNA NF被作为探针用于癌细胞的检测,检出限为500 cell/m L。综上所述,对于其他生物分子的细胞内定量,基于FA/MNP/RNA NF的共给药平台以及诊断-治疗一体化系统是一种很有前途的方法。2.一种通用的基于杂交链式反应的比色放大法对核酸和适体特异性配体的检测。我们提出了一种基于金纳米粒子-DNA(GNP-DNA)杂交链式反应(HCR)并用于检测核酸和适体特异性配体的通用放大比色法。这种通用的阵列由捕获探针和发夹DNA-GNP组成。首先,捕获探针特异性地识别目标分子,并导致引发剂序列的释放。然后,发夹DNA修饰的金纳米粒子通过引发剂引发的HCR进而由分散态聚集成相互交联的聚集体。我们采用miRNA的靶序列(miRNA-203)和核酸适体的特异性配体(ATP)作为靶分子来验证这种方法。在miRNA-203的强竞争力下,引发序列(DNA 2)从捕获探针(MNP/DNA1/2共轭物)释放,发夹DNA(H 1和H 2)可以与DNA 2互补从而形成GNP-H1/GNP-H2聚集体,所用miRNA-203浓度在1.0×10-11-3.0×10-10M范围内时,记录GNP-H1/GNP-H2聚集体溶液在620 nm和520 nm处的吸光度比值(A620/A520),所得检出限为1.0×10-11M。与此同时,溶液中的颜色由淡红色变为紫色,然后再变为淡蓝色。对于ATP,在适配体与ATP的强的相互作用下,引发序列(DNA 3的5’端)从捕获探针(DNA3)释放,我们所设计的比色法对ATP检测具有良好的灵敏性,检测限为1.0×10-8M ATP。该策略对于细胞内核酸和核酸适配体的特异性配体的定性分析与定量分析也显示出良好的性能。
[Abstract]:In this paper, using fluorescence, ultraviolet absorption and laser scanning confocal microscope, we used functional nucleic acid origami method and colorimetric amplification method based on hybrid chain reaction to carry out combined administration, targeted therapy and the detection of miRNA-ATP. The main contents of this paper are as follows: 1. A Diagnostic and Therapeutic Integration system based on Magnetic RNA Nanoflowers for combined Administration and targeted Therapy. In this part, an integrated diagnostic and therapeutic system based on magnetic RNA nanosplast for combined drug delivery and targeted therapy is developed. Compared with the traditional nucleic acid structure, RNA NF can be separated easily by introducing magnetic nanoparticles into the nucleic acid structure. Folate FA-modified MNP/RNA NF(FA/MNP/RNA NFS as a good biocompatible nano-target carrier overcomes the nonselectivity of MNP/RNA NF. Then, the anticancer drug dox) and the Guang Min agent TMPyP4) combined with RNA NF were used as a common drug transport model for the first time. Therefore, imaging fluorescent labeling, target recognition elements and drug molecules are clustered on the surface of MNP/RNA NF. The results showed that the combined drug delivery platform, FAR / MNPR / RNA NFR / D / T, was more effective than the single drug delivery platform, namely, FAR / MNPR / RNA NFR / DX. In addition, FAP / MNPrRNA NF was used as a probe to detect cancer cells with a detection limit of 500 cell/m / L. In conclusion, for the intracellular quantification of other biomolecules, the co-delivery platform based on FA/MNP/RNA NF and the integrative system of diagnostics and therapy are promising methods. A universal colorimetric amplification method based on hybrid chain reaction for detection of nucleic acid and aptamer specific ligands. We propose a universal amplification colorimetric method based on gold nanoparticles (DNA-GNP-DNA) hybridization chain reaction (HCR) to detect nucleic acid and aptamer specific ligands. The universal array consists of a capture probe and a hairpin DNA-GNP. First, the target molecule is specifically identified by the capture probe, which leads to the release of the initiator sequence. Then, the gold nanoparticles modified by hairpin DNA were synthesized into cross-linked aggregates by HCR initiated by the initiator. The target sequence of miRNA and the aptamer specific ligand ATP) were used as target molecules to verify this method. Under the strong competitiveness of miRNA-203, the initiation sequence DNA2) was released from the capture probe MNP / DNA 1 / 2 conjugate. Hairpin DNA(H _ 1 and H _ 2) could complement DNA _ 2 to form GNP-H1/GNP-H2 aggregates. When the miRNA-203 concentration was 1.0 脳 10 ~ (-11) to 3.0 脳 10 ~ (-10) M, The absorbance ratio of GNP-H1/GNP-H2 aggregates at 620nm and 520nm was recorded. The detection limit was 1.0 脳 10-11M. At the same time, the color in the solution changed from light red to purple and then to pale blue. For ATP, the 5 'end of the initiation sequence of ATP is released from the capture probe DNA3 under the strong interaction between aptamer and ATP. Our colorimetric method is sensitive to ATP detection with a detection limit of 1.0 脳 10 ~ (-8) M ATP. This strategy also shows good performance for qualitative analysis and quantitative analysis of specific ligands of nucleic acid and aptamer.
【学位授予单位】：曲阜师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318;O657.3

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