纳米结构辅助的恒温扩增反应的设计及其在生物传感器中的应用

发布时间：2018-03-11 04:33

本文选题：恒温扩增反应　切入点：生物传感　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：实现高灵敏,高特异,快速,准确以及高通量的生物标志物检测,对癌症的诊断,治疗,预防,以及分子生物学的研究有着极其重要的科学意义。由于恒温扩增反应具有灵敏度高,操作简便,应用范围广等优势,近年来被科学家们广泛应用于生物传感器的制备和应用中。在这一研究背景下,本课题利用交叉学科的技术手段,致力于设计和开发高效能的生物传感器,并将其应用于临床检验中。通过利用一些纳米结构,比如分子信标探针,纳米金,和石墨烯等,可以极大的改善恒温扩增反应的性能,提高其应用能力。本论文的主要研究内容包括以下四个方面：1.设计了一种双标记的纳米金探针,首次提出了一个双向四态的分析理论用于检测尿液样本中的端粒酶,并进一步用于膀胱癌的诊断。本方法的多态性表现在,对于不同活性和浓度的端粒酶,纳米金会表现出四种状态：沉淀,红色透明,紫色,以及蓝色。双向性表现在,纳米金的四态和其对应的A650/A520的比值是双向的,类似于坐标轴。A650/A520比值最小的是红色的纳米金,代表原点。随着沉淀越来越明显,4650/A520比值增大,这个代表正方向。随着纳米金颜色由紫色变成蓝色,A650/A520比值也在增大,这个代表负方向。基于这一策略,我们检测了18例临床尿液样本(包括正常人,炎症患者,以及膀胱癌病人),证明了本方法具有很好的临床诊断能力。2.首次完成了利用纳米金监测复杂环境中的链置换扩增反应。随着链置换扩增反应的充分进行,肉眼可以观测到纳米金由蓝色到红色的变化。另外对靶分子参与反应的14个位点的碱基进行点改变,发现这14条具有点突变的序列并不能使得纳米金的颜色发生变化,说明本方法具有很好的检测点突变的能力。另外本方法在复杂环境中也可以顺利进行,具有较好的实用性。本研究极大地拓宽了链置换扩增反应的应用范围。3.为了能够提高癌症早期诊断的准确性,我们提出了单体系双标志物检测的方法。本方法可以同时实现对miRNA和端粒酶的检测。通过利用切刻酶引发的扩增反应,以及对DNA序列的巧妙设计,我们成功的将端粒酶的延伸反应以及miRNA诱发的滚环扩增反应合二为一,并且将检测信号进行了放大。另外本方法还用到了石墨烯来降低背景信号,以达到更好的检出效果。通过对近300例病人尿液样本的分析,结果证明,和miRNA检测或端粒酶的检测方法相比,miRNA和端粒酶双检的方法不仅具有较高的灵敏度,而且特异度也大大的提高了。尤其是本方法可以对浸润性和非浸润性的膀胱癌病人进行区分,从而为癌症的早期诊断提供了新思路。4.为了实现快速高灵敏度的检测miRNA,我们提出了一种基于恒温双循环扩增反应的一步分析法。我们设计了一条多功能的分子信标探针,并在切刻酶,消化酶,聚合酶的参与下完成一个双循环反应,并达到了指数扩增的能力。本方法在37℃的情况下可以检测到10 fM的miRNA。而在4℃下可以检测到1 aM的miRNA,相当于在15μL的样本中可以检测到9条DNA链。最后将本方法应用于癌细胞系甚至于癌组织中miRNA的检测时,也表现出了很好的特异性和灵敏度,为癌症的早期诊断提供了新方法。
[Abstract]:To achieve high sensitivity, high specificity, rapid, accurate and high-throughput biomarkers for cancer detection, diagnosis, treatment, prevention, has extremely important scientific significance and research of molecular biology. The isothermal amplification reaction has high sensitivity, simple operation, wide application range and other advantages, in recent years by scientists. Widely used in the fabrication of biosensors and application. In the context of this study, this paper uses technology of cross discipline, dedicated to the design and development of biosensors with high efficiency, and its application in clinical examination. Through the use of some nano structure, such as molecular beacon probe and gold nanoparticles. Graphene can greatly improve the performance, the constant temperature amplification reaction, improve the ability of application. The main research contents of this thesis include the following four aspects: 1. design of a double labeled gold nanoparticle probes for the first time. Theoretical analysis of a two-way four state for the detection of telomerase activity in urine samples is proposed, and further for the diagnosis of bladder cancer. The polymorphism of this method in the performance of, for different activity and concentration of telomerase, gold nanoparticles exhibit four states: precipitation, transparent red, purple, and blue. Bidirectional performance in the four state, the ratio of gold nanoparticles and their corresponding A650/A520 is two-way, similar to the.A650/A520 axis ratio is the smallest nano gold, red represents the origin. With the precipitation is more and more obvious, the ratio of 4650/A520 was increased, the generation of surface orientation. With nano gold color from purple to blue, the ratio of A650/A520 in this increase represents the negative direction. Based on this strategy, we detected 18 cases of clinical urine samples (including normal, inflammation, and bladder cancer patients), proved that this method has good clinical diagnosis .2. completed the first chain replacement by using gold nanoparticle monitoring in complex environment with sufficient amplification. Strand displacement amplification reaction, the naked eye can be observed from blue to red gold nanoparticles changes. In addition to the 14 sites of target molecules involved in the reaction of the base point of change, has found that the 14 point mutation the sequence that does not change the color of gold nanoparticles, this method has good ability to detect point mutations. Also this method can be carried out smoothly in the complex environment, has better practicability. This study greatly expands the application range of.3. strand displacement amplification reactions in order to improve the accuracy of early diagnosis of cancer, we put forward a new method of single system of double marker detection. This method can realize the detection of miRNA and telomerase. By using enzyme cutting by amplification reaction, and The ingenious design of DNA sequences, we successfully extend reaction telomerase, rolling circle amplification reaction induced by miRNA and will be made one, detection signal is amplified. This method is also used to reduce the background signal of graphene, in order to achieve better detection result. Through the analysis, nearly 300 cases of patient urine samples the results prove that, compared with the detection method and miRNA detection method of miRNA or telomerase, telomerase and double check not only has high sensitivity and specificity are greatly improved. Especially the method can distinguish between invasive and non invasive bladder cancer patients, so as to early diagnosis of cancer provides a new idea in order to realize the rapid detection of miRNA.4. with high sensitivity, we propose a step analysis method based on double circulation constant temperature amplification reaction. We design a multifunctional molecular letter Probe, and engraved in enzyme digestion enzymes, to complete a double cycle reaction polymerase participation, and ability to achieve exponential amplification. This method can detect 10 fM miRNA. at 4 DEG C can be detected by 1 aM miRNA at 37 DEG C under the condition of the equivalent of 15. The L sample was detected in 9 DNA strands. Finally this method is applied to cancer cells even in the detection of miRNA in cancer tissue, also showed good specificity and sensitivity, it provides a new method for early diagnosis of cancer.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TB383.1;TP212.3

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