發(fā)布時(shí)間：2018-01-14 15:10

  本文關(guān)鍵詞：免固定電化學(xué)核酸傳感分析新方法及性能研究　出處：《青島科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： DNA 電化學(xué)生物傳感平臺(tái) 均相 信號(hào)放大 免固定

【摘要】：本論文構(gòu)建了三種新穎的免固定電化學(xué)DNA生物傳感器,實(shí)現(xiàn)了對(duì)靶標(biāo)分子核酸的高靈敏、高選擇、新穎的檢測(cè),為電化學(xué)DNA生物傳感器的研究開拓了新思路、新方法,有望應(yīng)用于早期臨床疾病的檢測(cè)。1、提出了一個(gè)獨(dú)特的基于外切酶Ⅲ催化釋放亞甲基藍(lán)標(biāo)記的單核苷酸的免固定的電化學(xué)DNA生物傳感策略,并且釋放出的亞甲基藍(lán)標(biāo)記的單核苷酸可以與十二硫醇疏水的烷鏈相互結(jié)合,從而富集亞甲基藍(lán)分子到十二硫醇單分子層上,產(chǎn)生電化學(xué)信號(hào)的響應(yīng)。由于其不需固定并且是在均相中進(jìn)行的生物識(shí)別和放大過程,因此實(shí)現(xiàn)了對(duì)于靶DNA簡(jiǎn)單、快速,高靈敏的檢測(cè),對(duì)于靶DNA的檢測(cè)限低至1pM,并且此方法可以擴(kuò)展到檢測(cè)任何可促使亞甲基藍(lán)信號(hào)分子釋放的靶標(biāo)分子,因此,為免固定電化學(xué)生物傳感器的發(fā)展提供了廣闊的空間。2、提出了一個(gè)基于T7核酸外切酶的輔助循環(huán)放大的免固定的電化學(xué)DNA生物傳感策略,實(shí)現(xiàn)了對(duì)完全互補(bǔ)的靶標(biāo)分子在均相溶液中的循環(huán)放大檢測(cè)。當(dāng)單堿基錯(cuò)配時(shí)無法實(shí)現(xiàn)均相中的反應(yīng),進(jìn)而達(dá)到在電極表面富集情況的區(qū)別,實(shí)現(xiàn)了對(duì)錯(cuò)配堿基的檢測(cè),為基因單堿基突變的檢測(cè)提供了新穎、高效、靈敏的檢測(cè)策略。3、基于等溫?cái)U(kuò)增技術(shù)和hemin/G-quadruplex DNAzyme構(gòu)筑了一種高效、靈敏、方便、免固定檢測(cè)靶標(biāo)DNA的生物傳感器。在靶標(biāo)DNA存在時(shí),基于等溫?cái)U(kuò)增技術(shù)擴(kuò)增出大量的產(chǎn)物鏈,產(chǎn)物鏈包括重要的兩部分,一部分為鏈霉親和素的適體鏈,另一部分為富G堿基鏈。首先通過電極表面的鏈霉親和素和其適體結(jié)合,將產(chǎn)物鏈固定到電極表面,產(chǎn)物鏈上的富G堿基遇到K+和Hemin時(shí)就會(huì)形成G四鏈體,它具有辣根過氧化物酶的活性,其可以催化H2O2,通過測(cè)H2O2的電催化產(chǎn)生的電化學(xué)信號(hào)來實(shí)現(xiàn)對(duì)靶標(biāo)DNA的檢測(cè)。此策略通過聚合內(nèi)切循環(huán)放大反應(yīng)有助于G四鏈體的形成,從而可以對(duì)靶標(biāo)DNA進(jìn)行高靈敏檢測(cè)�？傊�,本論文所構(gòu)建的免固定的電化學(xué)DNA生物傳感器可以擴(kuò)充到其他生物分子的檢測(cè)中,對(duì)今后的研究和診斷具有重大意義。
[Abstract]:In this paper, three novel immobilization free electrochemical DNA biosensors were constructed to detect target molecular nucleic acids with high sensitivity, high selectivity and novel detection. It opens up new ideas and methods for the study of electrochemical DNA biosensor and is expected to be used in the detection of early clinical diseases. 1. A novel immobilized electrochemical DNA biosensor strategy based on the catalytic release of methylene blue labeled single nucleotides was proposed. The released methylene blue labeled single nucleotides can bind to the hydrophobic alkane chain of 12 mercaptan, thus enriching the methylene blue molecule onto the 12 mercaptan monolayer. Because it does not need to be fixed and is carried out in the homogeneous biometric recognition and amplification process, it realizes simple, fast and highly sensitive detection for target DNA. The detection limit for target DNA is as low as 1 pm, and this method can be extended to detect any target molecule that can induce the release of methylene blue signal molecule. In order to provide a wide space for the development of electrochemical biosensor, a novel non-fixed electrochemical DNA biosensor strategy based on T7 nucleic acid exonuclease was proposed. The cyclic amplification detection of completely complementary target molecules in homogeneous solution is realized. When the single base mismatch is not realized, the reaction in homogeneous phase can not be realized, and then the difference of enrichment on the electrode surface is achieved. The detection of mismatch base is realized, which provides a novel, efficient and sensitive detection strategy for the detection of single base mutation of gene. Based on isothermal amplification technique and hemin/G-quadruplex DNAzyme, a kind of high efficiency, sensitivity and convenience was constructed. In the presence of target DNA, a large number of product chains were amplified based on isothermal amplification technique, which included two important parts. One is the aptamer chain of streptavidin and the other is the G-rich base chain. Firstly, the product chain is immobilized to the electrode surface through the binding of the streptavidin on the electrode surface to the aptamer. G-rich bases on the product chain will form G quadruplex when they encounter K and Hemin. It has the activity of horseradish peroxidase and can catalyze H _ 2O _ 2. The detection of target DNA is realized by measuring the electrochemical signal produced by electrocatalysis of H2O2. This strategy is helpful to the formation of G quadruplex by polymeric internal cycle amplification reaction. Therefore, the target DNA can be detected with high sensitivity. In a word, the immobilized electrochemical DNA biosensor can be extended to the detection of other biomolecules. It is of great significance for future research and diagnosis.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TP212.3;O657.1

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本文編號(hào)：1424113