基于DNA納米技術(shù)和核酸酶的核酸化學(xué)發(fā)光傳感分析
發(fā)布時(shí)間：2018-07-17 19:14
【摘要】：隨著循環(huán)腫瘤DNA、循環(huán)microRNA(miRNA)和融合基因等在臨床疾病發(fā)生和診療中作用的逐步揭示,無細(xì)胞核酸分析在生物學(xué)研究和醫(yī)學(xué)診療中變得日趨重要。基于此,本論文擬將整合臨床檢驗(yàn)診斷學(xué)和生物分析化學(xué)學(xué)科交叉的最新研究成果,以DNA納米自組裝和功能核酸酶為納米級(jí)工具,結(jié)合臨床檢驗(yàn)診斷的需求,構(gòu)建一系列簡單、快速、等溫、特異、靈敏、免標(biāo)記的核酸化學(xué)發(fā)光傳感分析新策略。本研究主要包括以下三個(gè)部分:1.基于核酸酶級(jí)聯(lián)指數(shù)擴(kuò)增的化學(xué)發(fā)光成像用于miRNA傳感分析本研究基于功能核酸酶技術(shù)和級(jí)聯(lián)指數(shù)擴(kuò)增(exponential amplification reaction,EXPAR)分子機(jī)器,構(gòu)建高靈敏、高特異且可視化的化學(xué)發(fā)光生物傳感器,為循環(huán)miRNA提供全新檢測新策略。研究中發(fā)卡開關(guān)能夠特異識(shí)別靶miRNA并形成雜交雙鏈,從而啟動(dòng)擴(kuò)增機(jī)器I,產(chǎn)生引物鏈。引物鏈與模板II探針互補(bǔ)雜交形成雙鏈進(jìn)級(jí)聯(lián)激活指數(shù)擴(kuò)增機(jī)器II,產(chǎn)生大量G四鏈體辣根過氧化物模擬酶,催化發(fā)光底物發(fā)光,實(shí)現(xiàn)信號(hào)的轉(zhuǎn)換輸出。發(fā)卡結(jié)構(gòu)DNA分子探針的設(shè)計(jì)提高檢測的特異性,級(jí)聯(lián)型的指數(shù)擴(kuò)增提高檢測的靈敏度,功能核酸酶免標(biāo)記技術(shù)實(shí)現(xiàn)信號(hào)轉(zhuǎn)導(dǎo)和可視化的miRNA生物傳感分析。本章所構(gòu)建的化學(xué)發(fā)光傳感方法檢測miRNA的線性范圍為10 fM到100 pM,最低檢測限為2.91 fM,且具有單堿基錯(cuò)配分辨能力。同時(shí),該傳感方法具有等溫、均相、免標(biāo)記和可視化的優(yōu)點(diǎn),有望為循環(huán)mirna分析和臨床疾病診斷提供有力的檢測工具。2.基于雙t型dna納米開關(guān)和核酸酶的化學(xué)發(fā)光成像用于bcr/abl融合基因傳感分析本研究基于dna納米技術(shù)和級(jí)聯(lián)型dna擴(kuò)增機(jī)器構(gòu)建bcr/abl融合基因化學(xué)發(fā)光型生物傳感器,為融合基因檢測提供超靈敏、高特異的化學(xué)發(fā)光成像傳感新策略。利用nupack軟件設(shè)計(jì)功能化分子探針,其能夠邏輯性識(shí)在bcr/abl融合基因并自組裝形成bis-3wj納米結(jié)構(gòu)(bis-threewayjunctionnanostructure,bis-3wj)。該結(jié)構(gòu)激活級(jí)聯(lián)擴(kuò)增機(jī)器,產(chǎn)生大量g四鏈體辣根過氧化物模擬酶亞單元。該亞單元能夠自組裝為完整模擬酶,級(jí)聯(lián)催化化學(xué)發(fā)光底物實(shí)現(xiàn)光信號(hào)轉(zhuǎn)換輸出和邏輯門操作。本章所構(gòu)建的化學(xué)發(fā)光成像檢測bcr/abl融合基因的最低檢測限23fm,線性范圍達(dá)7個(gè)數(shù)量級(jí),且具有邏輯識(shí)別能力和單堿基錯(cuò)配分辨能力。該方法在復(fù)雜基質(zhì)中顯示好的回收率,具有等溫、均相、免標(biāo)記的優(yōu)點(diǎn),為慢性粒細(xì)胞白血病診斷提供全新的化學(xué)發(fā)光成像策略。3.基于發(fā)卡開關(guān)和原位非線性雜交鏈反應(yīng)的電致化學(xué)發(fā)光用于bcr/abl融合基因傳感分析本研究基于發(fā)卡探針和原位分支化雜交鏈反應(yīng)(hybridizationchainreaction,hcr)構(gòu)建bcr/abl融合基因的電化學(xué)發(fā)光型生物傳感器,為慢性粒細(xì)胞白血病診斷提供操作簡便、超靈敏、高特異的傳感新策略。首先,將捕獲探針固定在金電極。隨后,將分支化雜交鏈反應(yīng)系統(tǒng)溶液滴加在電極表面,在發(fā)卡開關(guān)上的觸發(fā)鏈DNA觸發(fā)兩種底物鏈和兩種輔助鏈的級(jí)聯(lián)自組裝形成分支狀DNA納米結(jié)構(gòu)。在目標(biāo)分子存在的情況下,其打開發(fā)卡開關(guān)并將分支狀DNA納米結(jié)構(gòu)連接在電極表面。這種原位HCR反應(yīng)形成的DNA納米結(jié)構(gòu)能夠通過其DNA雙鏈凹槽與電致發(fā)光指示劑(Ru(phen)32+)間的相互作用將隨后加入的指示劑引入到電極表面,從而實(shí)現(xiàn)信號(hào)的指數(shù)形式的輸出放大。發(fā)卡探針和原位非線性雜交鏈反應(yīng)的引入到電致發(fā)光傳感器設(shè)計(jì)增強(qiáng)了傳感器的特異性和靈敏度。本章所構(gòu)建的ECL方法檢測BCR/ABL融合基因的最低檢測限為5.49 fM,線性范圍為10 fM至1 nM,且具有與正常BCR基因和正常ABL基因進(jìn)行邏輯區(qū)別的能力。另外,該方法具有無酶,免標(biāo)記,低成本的優(yōu)勢,有望成為白血病臨床診斷中融合基因檢測提供新的診療策略。
[Abstract]:With the gradual discovery of the role of circulating tumor DNA, circulating microRNA (miRNA) and fusion gene in the occurrence and diagnosis of clinical diseases, acellular nucleic acid analysis becomes more and more important in biological research and medical diagnosis and treatment. Based on this, this paper will integrate the latest research results in the interdisciplinary of clinical diagnostics and bioanalytical chemistry. DNA nano self-assembly and functional nuclease are nanoscale tools, combined with the needs of clinical diagnosis. A series of simple, fast, isothermal, specific, sensitive, and non labeled nucleic acid chemiluminescence sensing analysis strategies are constructed. This study mainly includes the following three parts: 1. based chemiluminescence imaging of the nuclease cascade index for miR NA sensing analysis is based on functional nuclease technology and cascade index amplification (exponential amplification reaction, EXPAR) molecular machines to construct highly sensitive, highly specific and visualized chemiluminescence biosensors, which provide new detection strategies for circulating miRNA. In the study, a hairpin switch can specifically identify the target miRNA and form a hybridization. Double strands, thus initiating the amplification machine I, producing the primer chain. The primer chain and the template II probe complement each other to form a double chain into the cascade activation index to amplify the machine II, producing a large number of G four chain body horseradish peroxidase analog enzymes, catalyzing the luminescence of the luminescent substrate, and realizing the conversion of the signal. The design of the DNA molecular probe of the hairpin structure improves the specificity of the detection. Cascaded exponential amplification improves detection sensitivity, functional nuclease free labeling technique for signal transduction and visual miRNA biosensor analysis. The linear range of miRNA is 10 fM to 100 pM in this chapter, with a minimum detection limit of 2.91 fM and a single base mismatch resolution. The sensing method has the advantages of isothermal, homogeneous, non labeling and visualization. It is expected to provide a powerful detection tool for cyclic miRNA analysis and clinical disease diagnosis.2. based on double T type DNA nanoscale switch and nuclease chemiluminescence imaging for bcr/abl fusion gene sensing analysis based on DNA nanotechnology and cascade DNA amplification machines to construct B Cr/abl fusion gene chemiluminescence biosensor provides a super sensitive, high specific chemiluminescence imaging sensor for fusion gene detection. Using nupack software, a functional molecular probe is designed, which can logically identify the bcr/abl fusion gene and form a bis-3wj nanoscale structure (bis-threewayjunctionnanostructure, bis-3w). J). The structure activates the cascade amplification machine to produce a large number of G four chain horseradish peroxidase subunits. This subunit can self assemble as a complete analog enzyme, cascade catalytic chemiluminescence substrates to realize optical signal conversion output and logic gate operation. The minimum detection limit of bcr/abl fusion gene for detection of chemiluminescence imaging in this chapter is 23 FM, which has a linear range of 7 orders of magnitude, and has the ability to distinguish between logical recognition and single base mismatch. The method shows good recovery in the complex matrix and has the advantages of isothermal, homogeneous and non labeling. It provides a new chemiluminescent imaging strategy for the diagnosis of chronic myelocytic leukemia,.3. based on the hairpin switch and in situ nonlinear hybrid chain. Electrochemiluminescence (electrochemiluminescence) for bcr/abl fusion gene sensing analysis, an electrochemiluminescence biosensor based on the hairpin probe and in situ branched hybrid chain reaction (hybridizationchainreaction, HCR) was used to construct a bcr/abl fusion gene, which provides a simple, sensitive and highly specific sensing for the diagnosis of chronic myelocytic leukemia. First, the capture probe is fixed on the gold electrode. Then, the branched hybrid chain reaction system solution is added to the surface of the electrode. The trigger chain DNA on the card switch triggers two substrate chains and two kinds of auxiliary chains to form a branched DNA nanostructure. The branched DNA nanostructure is connected to the surface of the electrode. The DNA nanostructure formed by the in situ HCR reaction can be introduced into the electrode surface by the interaction between the DNA double chain grooves and the electroluminescent indicator (Ru (phen) 32+), thus realizing the output amplification of the exponential form of the signal, the hairpin probe and the in situ. The introduction of the nonlinear hybrid chain reaction to the electroluminescent sensor design enhanced the specificity and sensitivity of the sensor. The minimum detection limit for detection of BCR/ABL fusion gene by the ECL method in this chapter is 5.49 fM, and the linear range is 10 fM to 1 nM, and it has the ability to distinguish the normal BCR gene and the normal ABL gene. The method has the advantages of no enzyme, label free and low cost, and is expected to provide a new strategy for the detection of fusion genes in clinical diagnosis of leukemia.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R440
本文編號(hào)：2130659
[Abstract]:With the gradual discovery of the role of circulating tumor DNA, circulating microRNA (miRNA) and fusion gene in the occurrence and diagnosis of clinical diseases, acellular nucleic acid analysis becomes more and more important in biological research and medical diagnosis and treatment. Based on this, this paper will integrate the latest research results in the interdisciplinary of clinical diagnostics and bioanalytical chemistry. DNA nano self-assembly and functional nuclease are nanoscale tools, combined with the needs of clinical diagnosis. A series of simple, fast, isothermal, specific, sensitive, and non labeled nucleic acid chemiluminescence sensing analysis strategies are constructed. This study mainly includes the following three parts: 1. based chemiluminescence imaging of the nuclease cascade index for miR NA sensing analysis is based on functional nuclease technology and cascade index amplification (exponential amplification reaction, EXPAR) molecular machines to construct highly sensitive, highly specific and visualized chemiluminescence biosensors, which provide new detection strategies for circulating miRNA. In the study, a hairpin switch can specifically identify the target miRNA and form a hybridization. Double strands, thus initiating the amplification machine I, producing the primer chain. The primer chain and the template II probe complement each other to form a double chain into the cascade activation index to amplify the machine II, producing a large number of G four chain body horseradish peroxidase analog enzymes, catalyzing the luminescence of the luminescent substrate, and realizing the conversion of the signal. The design of the DNA molecular probe of the hairpin structure improves the specificity of the detection. Cascaded exponential amplification improves detection sensitivity, functional nuclease free labeling technique for signal transduction and visual miRNA biosensor analysis. The linear range of miRNA is 10 fM to 100 pM in this chapter, with a minimum detection limit of 2.91 fM and a single base mismatch resolution. The sensing method has the advantages of isothermal, homogeneous, non labeling and visualization. It is expected to provide a powerful detection tool for cyclic miRNA analysis and clinical disease diagnosis.2. based on double T type DNA nanoscale switch and nuclease chemiluminescence imaging for bcr/abl fusion gene sensing analysis based on DNA nanotechnology and cascade DNA amplification machines to construct B Cr/abl fusion gene chemiluminescence biosensor provides a super sensitive, high specific chemiluminescence imaging sensor for fusion gene detection. Using nupack software, a functional molecular probe is designed, which can logically identify the bcr/abl fusion gene and form a bis-3wj nanoscale structure (bis-threewayjunctionnanostructure, bis-3w). J). The structure activates the cascade amplification machine to produce a large number of G four chain horseradish peroxidase subunits. This subunit can self assemble as a complete analog enzyme, cascade catalytic chemiluminescence substrates to realize optical signal conversion output and logic gate operation. The minimum detection limit of bcr/abl fusion gene for detection of chemiluminescence imaging in this chapter is 23 FM, which has a linear range of 7 orders of magnitude, and has the ability to distinguish between logical recognition and single base mismatch. The method shows good recovery in the complex matrix and has the advantages of isothermal, homogeneous and non labeling. It provides a new chemiluminescent imaging strategy for the diagnosis of chronic myelocytic leukemia,.3. based on the hairpin switch and in situ nonlinear hybrid chain. Electrochemiluminescence (electrochemiluminescence) for bcr/abl fusion gene sensing analysis, an electrochemiluminescence biosensor based on the hairpin probe and in situ branched hybrid chain reaction (hybridizationchainreaction, HCR) was used to construct a bcr/abl fusion gene, which provides a simple, sensitive and highly specific sensing for the diagnosis of chronic myelocytic leukemia. First, the capture probe is fixed on the gold electrode. Then, the branched hybrid chain reaction system solution is added to the surface of the electrode. The trigger chain DNA on the card switch triggers two substrate chains and two kinds of auxiliary chains to form a branched DNA nanostructure. The branched DNA nanostructure is connected to the surface of the electrode. The DNA nanostructure formed by the in situ HCR reaction can be introduced into the electrode surface by the interaction between the DNA double chain grooves and the electroluminescent indicator (Ru (phen) 32+), thus realizing the output amplification of the exponential form of the signal, the hairpin probe and the in situ. The introduction of the nonlinear hybrid chain reaction to the electroluminescent sensor design enhanced the specificity and sensitivity of the sensor. The minimum detection limit for detection of BCR/ABL fusion gene by the ECL method in this chapter is 5.49 fM, and the linear range is 10 fM to 1 nM, and it has the ability to distinguish the normal BCR gene and the normal ABL gene. The method has the advantages of no enzyme, label free and low cost, and is expected to provide a new strategy for the detection of fusion genes in clinical diagnosis of leukemia.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R440
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