【摘要】：DNA檢測涉及到疾病診斷、基因識(shí)別、環(huán)境分析、指紋鑒定等很多方面,發(fā)展靈敏的、特異性的DNA傳感器具有非常重要的意義�，F(xiàn)有的檢測DNA的方法包括了化學(xué)發(fā)光法、電化學(xué)方法、比色法、熒光法等等。熒光探針具有操作方便、快速檢測、容易自動(dòng)化等優(yōu)勢,在分析檢測方面得到廣泛的應(yīng)用。對(duì)于檢測DNA的熒光探針,如何發(fā)展出具備合適尺寸、低毒性、高亮度、穩(wěn)定性強(qiáng)、靈敏度高等要求的熒光探針仍然是這一領(lǐng)域發(fā)展的關(guān)鍵問題之一。已經(jīng)研究報(bào)道的熒光探針諸如有機(jī)熒光染料和半導(dǎo)體量子點(diǎn)已經(jīng)在生物化學(xué)領(lǐng)域得到了廣泛應(yīng)用,但是有機(jī)熒光染料缺乏穩(wěn)定性,而半導(dǎo)體量子點(diǎn)的體積大、毒性大缺陷都限制了其應(yīng)用及發(fā)展。納米材料的研究發(fā)展則極大推進(jìn)了分析化學(xué)領(lǐng)域的應(yīng)用,以DNA為模板的DNA銀納米簇具有良好的性質(zhì)。DNA銀納米簇具有強(qiáng)的熒光發(fā)射強(qiáng)度、高穩(wěn)定性、較小的尺寸。除此之外,可以通過調(diào)整模板DNA的序列或長度來調(diào)整DNA銀納米簇的熒光發(fā)射波長范圍。DNA銀納米簇為熒光探針領(lǐng)域的發(fā)展帶來了新的方向,成為分析化學(xué)領(lǐng)域一個(gè)有力的研究工具。在過去的十年中,DNA銀納米簇作為熒光探針已經(jīng)在核酸、蛋白、多肽、金屬離子等物質(zhì)檢測,以及細(xì)胞成像等領(lǐng)域得到推廣應(yīng)用。研究表明,以部分互補(bǔ)的兩條DNA鏈為模板合成的銀納米簇其本身具有微弱的熒光,互補(bǔ)雜交之后形成DNA銀納米簇對(duì)熒光得到顯著增強(qiáng)。本工作主要基于DNA銀納米簇的特殊熒光性質(zhì)建立了一種快速、簡單、高選擇性檢測DNA的方法,主要開展了以下研究工作:通過設(shè)計(jì)挑選特定堿基序列和長度的DNA,得到了可互補(bǔ)的DNA銀納米簇對(duì)熒光探針。系統(tǒng)地研究了該DNA銀納米簇對(duì)熒光探針對(duì)于H1N1待測DNA的響應(yīng)特性。研究發(fā)現(xiàn),當(dāng)H1N1待測DNA存在時(shí),由于互補(bǔ)雜交競爭反應(yīng)的存在,兩種DNA銀納米簇雜交受阻,單獨(dú)存在的DNA銀納米簇本身呈現(xiàn)微弱的熒光。隨著H1N1待測DNA濃度的增加,DNA銀納米簇對(duì)與待測DNA共存溶液的熒光強(qiáng)度降低,待測DNA濃度與熒光強(qiáng)度在一定范圍內(nèi)存在良好的線性。而據(jù)此設(shè)計(jì)了檢測H1N1待測DNA的方法,推廣了DNA銀納米簇在DNA檢測方面的應(yīng)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.
【學(xué)位授予單位】：清華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：O657.3;TB383.1

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