基于DNA银纳米簇荧光探针的核酸检测研究

发布时间：2018-07-24 12:08

【摘要】：DNA检测涉及到疾病诊断、基因识别、环境分析、指纹鉴定等很多方面,发展灵敏的、特异性的DNA传感器具有非常重要的意义。现有的检测DNA的方法包括了化学发光法、电化学方法、比色法、荧光法等等。荧光探针具有操作方便、快速检测、容易自动化等优势,在分析检测方面得到广泛的应用。对于检测DNA的荧光探针,如何发展出具备合适尺寸、低毒性、高亮度、稳定性强、灵敏度高等要求的荧光探针仍然是这一领域发展的关键问题之一。已经研究报道的荧光探针诸如有机荧光染料和半导体量子点已经在生物化学领域得到了广泛应用,但是有机荧光染料缺乏稳定性,而半导体量子点的体积大、毒性大缺陷都限制了其应用及发展。纳米材料的研究发展则极大推进了分析化学领域的应用,以DNA为模板的DNA银纳米簇具有良好的性质。DNA银纳米簇具有强的荧光发射强度、高稳定性、较小的尺寸。除此之外,可以通过调整模板DNA的序列或长度来调整DNA银纳米簇的荧光发射波长范围。DNA银纳米簇为荧光探针领域的发展带来了新的方向,成为分析化学领域一个有力的研究工具。在过去的十年中,DNA银纳米簇作为荧光探针已经在核酸、蛋白、多肽、金属离子等物质检测,以及细胞成像等领域得到推广应用。研究表明,以部分互补的两条DNA链为模板合成的银纳米簇其本身具有微弱的荧光,互补杂交之后形成DNA银纳米簇对荧光得到显著增强。本工作主要基于DNA银纳米簇的特殊荧光性质建立了一种快速、简单、高选择性检测DNA的方法,主要开展了以下研究工作:通过设计挑选特定碱基序列和长度的DNA,得到了可互补的DNA银纳米簇对荧光探针。系统地研究了该DNA银纳米簇对荧光探针对于H1N1待测DNA的响应特性。研究发现,当H1N1待测DNA存在时,由于互补杂交竞争反应的存在,两种DNA银纳米簇杂交受阻,单独存在的DNA银纳米簇本身呈现微弱的荧光。随着H1N1待测DNA浓度的增加,DNA银纳米簇对与待测DNA共存溶液的荧光强度降低,待测DNA浓度与荧光强度在一定范围内存在良好的线性。而据此设计了检测H1N1待测DNA的方法,推广了DNA银纳米簇在DNA检测方面的应用。
[Abstract]:DNA detection involves many aspects, such as disease diagnosis, gene identification, environmental analysis, fingerprint identification and so on. It is of great significance to develop sensitive and specific DNA sensors. The existing methods for detecting DNA include chemiluminescence, electrochemistry, colorimetry, fluorescence and so on. Fluorescent probe has been widely used in analysis and detection because of its advantages of easy operation, rapid detection and easy automation. How to develop fluorescent probes with suitable size, low toxicity, high brightness, high stability and high sensitivity is still one of the key problems in the development of DNA. Fluorescent probes, such as organic fluorescent dyes and semiconductor quantum dots, have been widely used in biochemistry, but organic fluorescent dyes lack stability and semiconductor quantum dots have large volume. Its application and development are limited by its toxic defects. The research and development of nanomaterials have greatly promoted the application of analytical chemistry. DNA silver nanoclusters with DNA as template have good properties. DNA silver nanoclusters have strong fluorescence emission intensity, high stability and small size. In addition, the fluorescence emission wavelength range of DNA silver nanoclusters can be adjusted by adjusting the sequence or length of template DNA, which brings a new direction to the development of fluorescent probes. To become a powerful research tool in the field of analytical chemistry. In the past decade, silver nanoclusters have been widely used as fluorescent probes in the fields of nucleic acid, protein, polypeptide, metal ions and cell imaging. The results show that the silver nanoclusters synthesized by using two partially complementary DNA chains as templates have weak fluorescence, and the fluorescence of silver nanoclusters formed by complementary hybridization is enhanced significantly. Based on the special fluorescence properties of DNA silver nanoclusters, a rapid, simple and highly selective method for the detection of DNA was developed. The main work of this paper is as follows: a complementary DNA silver nanocluster pair fluorescence probe was obtained by selecting the specific base sequence and length of DNA. The response of the DNA silver nanoclusters to the fluorescence probe to the DNA to be tested by H1N1 was systematically studied. It is found that when H1N1 is in the presence of DNA, the hybridization of two DNA silver nanoclusters is blocked due to the existence of complementary hybridization competition reaction, and the single DNA silver nanoclusters exhibit weak fluorescence. With the increase of the concentration of DNA, the fluorescence intensity of the solution coexisting with the DNA is decreased, and there is a good linearity between the concentration of DNA and the intensity of fluorescence in a certain range. Based on this, the method of detecting H1N1 DNA was designed, and the application of DNA silver nanoclusters in DNA detection was extended.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O657.3;TB383.1

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