本文選題：納米探針 + DNA雜交��； 參考：《江南大學(xué)》2008年碩士論文

【摘要】： DNA雜交分析及免疫分析在生命科學(xué)研究中具有重要的意義,廣泛應(yīng)用于生物學(xué)、醫(yī)學(xué)診斷等方面。已有分析方法的局限性促使研究者們積極的研究普遍適用性好、操作簡(jiǎn)便、靈敏度高的DNA雜交檢測(cè)及免疫檢測(cè)新方法。 本文將納米探針技術(shù)與高靈敏化學(xué)發(fā)光技術(shù)結(jié)合應(yīng)用于DNA雜交檢測(cè)及免疫檢測(cè),對(duì)特定序列的寡聚核苷酸,人IgG及黃曲霉毒素B1進(jìn)行了超微量檢測(cè),取得了令人滿意的結(jié)果。 以CoFe_2O_4/Au核殼復(fù)合納米顆粒標(biāo)記巰基化沙門氏菌特異寡核苷酸序列,用納米金標(biāo)記沙門氏菌另一特異寡核苷酸序列,通過(guò)DNA雜交反應(yīng)與沙門氏菌特異目標(biāo)DNA序列互補(bǔ)形成夾心結(jié)構(gòu)。經(jīng)過(guò)簡(jiǎn)單的磁分離,去除其他沒(méi)有雜交的部分,最后將磁分離得到部分的納米金溶出成為Au~(3+),結(jié)合luminol化學(xué)發(fā)光體系實(shí)現(xiàn)對(duì)目標(biāo)DNA的高靈敏檢測(cè)。結(jié)果表明,發(fā)光強(qiáng)度和目標(biāo)DNA濃度在1-100 pmol·L~(-1)范圍內(nèi)相關(guān)性良好,對(duì)目標(biāo)DNA的檢測(cè)限為0.3 pmol·L~(-1)(3S/N),相對(duì)標(biāo)準(zhǔn)偏差為3.6%(10 pmol·L~(-1), n=7)。 同時(shí)以CoFe2O4/Au核殼復(fù)合納米顆粒為載體與羊抗人IgG構(gòu)建捕獲探針復(fù)合結(jié)構(gòu),首先與目標(biāo)分析物人IgG發(fā)生免疫反應(yīng),捕獲的人IgG再與納米金標(biāo)記的二抗(金標(biāo)羊抗人IgG)發(fā)生免疫反應(yīng),形成三明治夾心結(jié)構(gòu);通過(guò)磁分離去除未結(jié)合物質(zhì)干擾,將分離得到的標(biāo)記納米金溶出成為Au~(3+),結(jié)合Au~(3+)催化luminol化學(xué)發(fā)光分析方法實(shí)現(xiàn)對(duì)目標(biāo)分析物人IgG的高靈敏檢測(cè)。在實(shí)驗(yàn)優(yōu)化條件下,化學(xué)發(fā)光強(qiáng)度與人IgG的濃度在2-100 ng·mL~(-1)范圍內(nèi)呈良好的線性關(guān)系,檢測(cè)限為0.5 ng·mL~(-1)。 黃曲霉毒素B1的快速靈敏檢測(cè)在食品安全檢測(cè)工作中具有重要意義。本文建立了兩種基于銀增強(qiáng)納米金標(biāo)記探針的高靈敏度免疫分析方法。第一種方法用黃曲霉毒素B1(AFB1)抗體與金標(biāo)抗原、待測(cè)抗原進(jìn)行競(jìng)爭(zhēng)免疫反應(yīng),然后加入銀增強(qiáng)溶液,以金為核沉積生長(zhǎng)銀,通過(guò)檢測(cè)吸光度來(lái)確定待測(cè)物中AFB_1的含量,該方法的檢出限可達(dá)到0.01 ng·mL~(-1)。第二種方法在前一種方法的基礎(chǔ)上,將銀化學(xué)溶出,通過(guò)化學(xué)發(fā)光法檢測(cè)沉積的銀的量來(lái)確定待測(cè)物中AFB1的含量,該方法的檢出限可達(dá)到0.002 ng·mL~(-1)。 論文還建立了納米金標(biāo)記-銀增強(qiáng)-化學(xué)發(fā)光聯(lián)用檢測(cè)沙門氏菌的新方法。通過(guò)沙門氏菌捕獲探針、金標(biāo)沙門氏菌顯示探針與沙門氏菌目標(biāo)核酸序列之間的DNA雜交,形成三明治復(fù)合體,然后通過(guò)銀增強(qiáng)在標(biāo)記的納米金表面選擇性沉積銀,實(shí)現(xiàn)第一次信號(hào)放大;隨后結(jié)合溶出化學(xué)發(fā)光檢測(cè)技術(shù),實(shí)現(xiàn)信號(hào)第二次放大。結(jié)果表明,在優(yōu)化條件下,化學(xué)發(fā)光強(qiáng)度和目標(biāo)DNA濃度在1-1000 fmol·L~(-1)范圍內(nèi)相關(guān)性良好,對(duì)目標(biāo)DNA的檢測(cè)限為0.3 fmol·L~(-1)(3S/N),相對(duì)標(biāo)準(zhǔn)偏差為2.2%(10 fmol·L~(-1), n=7)。
[Abstract]:DNA hybridization analysis and immunoassay are of great significance in life science research, and are widely used in biology, medical diagnosis and so on. The limitations of existing analytical methods have prompted researchers to actively study new methods of DNA hybridization and immunoassay with good applicability, simple operation and high sensitivity. In this paper, the nanoprobe technique combined with highly sensitive chemiluminescence technique was applied to the detection of DNA hybridization and immunoassay. The specific sequences of oligonucleotides, human IgG and aflatoxin B1 were detected in ultramicro amounts, and satisfactory results were obtained. CoFe2O4 / au core-shell composite nanoparticles were used to label the specific oligonucleotide sequence of Salmonella mercaptobacter, and another specific oligonucleotide sequence was labeled with gold nanoparticles. The sandwich structure was formed by DNA hybridization with the specific target DNA sequence of Salmonella. After simple magnetic separation, the other parts without hybridization were removed. Finally, the partial gold nanocrystalline from magnetic separation was dissolved into Au3, and the highly sensitive detection of target DNA was realized by combining with the chemiluminescence system of luminol. The results showed that there was a good correlation between the luminescence intensity and the concentration of target DNA in the range of 1-100 pmol L ~ (-1). The detection limit of target DNA was 0.3 pmol L ~ (-1) (3s / N), and the relative standard deviation was 3.6% (10 pmol L ~ (-1), 7). At the same time, CoFe2O4 / au core-shell composite nanoparticles were used as carrier and goat anti-human IgG as carrier to construct the structure of trap probe. Firstly, the captured human IgG reacted with the target analyte human IgG, and then the captured human IgG reacted with gold labeled second antibody (gold-labeled goat anti-human IgG). The sandwich structure was formed, and the labeled gold nanoparticles were dissolved into Au3 by magnetic separation, and the high sensitive detection of human IgG was realized by using Au3 catalytic luminol chemiluminescence analysis method. Under the optimized conditions, there was a good linear relationship between the intensity of chemiluminescence and the concentration of human IgG in the range of 2-100 ng mL ~ (-1). The detection limit was 0.5 ng mL ~ (-1). The rapid and sensitive detection of aflatoxin B1 is of great significance in food safety detection. In this paper, two high sensitivity immunoassay methods based on silver-enhanced gold nanoparticles labeling probe were established. The first method is to use aflatoxin B1 (AFB1) antibody and gold-labeled antigen to carry on competitive immune reaction with the antigen to be tested, then to add the silver enhancement solution, then deposit the growing silver with gold as the nucleus, and determine the content of AFB1 in the object to be tested by measuring absorbance. The detection limit of this method is 0.01 ng mL ~ (-1). The second method is based on the former method, the silver is chemically dissolved, and the content of AFB1 in the sample is determined by chemiluminescence method. The detection limit of the method can reach 0.002 ng mL ~ (-1). A new method for the detection of Salmonella by gold-silver-enhanced chemiluminescence assay was established. The DNA hybridization between the probe and the target nucleic acid sequence of Salmonella was demonstrated by the capture probe of Salmonella aureus to form a sandwich complex, and then the selective deposition of silver on the labeled gold surface was enhanced by silver. The first signal amplification is realized, and then the second signal amplification is realized with the dissolution chemiluminescence detection technology. The results showed that the chemiluminescence intensity was well correlated with the concentration of target DNA in the range of 1-1000 fmol L ~ (-1). The detection limit of target DNA was 0.3 fmol L ~ (-1) (3s / N), and the relative standard deviation was 2.2% (10 fmol L ~ (-1), n ~ (7).
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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