發(fā)布時(shí)間：2018-01-10 23:27

  本文關(guān)鍵詞：基于核酸放大及共反應(yīng)放大策略構(gòu)建的電致化學(xué)發(fā)光生物傳感器的研究　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 電致化學(xué)發(fā)光 信號(hào)放大 共反應(yīng)試劑 共反應(yīng)促進(jìn)劑 核酸擴(kuò)增技術(shù)

【摘要】：電致化學(xué)發(fā)光(ECL)生物傳感器是一類以生物分子如核酸分子、適體、抗體等為分子識(shí)別元件,結(jié)合ECL技術(shù)實(shí)現(xiàn)目標(biāo)物檢測(cè)的傳感器。ECL生物傳感器具有靈敏度高、選擇性好、響應(yīng)速度快、操作簡(jiǎn)便、成本低等優(yōu)點(diǎn),被廣泛應(yīng)用于疾病相關(guān)核酸分子、蛋白質(zhì)、功能小分子等的檢測(cè)。通常,向ECL體系中引入共反應(yīng)試劑可顯著增強(qiáng)ECL信號(hào)強(qiáng)度,從而提高ECL生物傳感器的檢測(cè)靈敏度。然而,一些共反應(yīng)試劑不穩(wěn)定且難以標(biāo)記,從而增加了固載難度。同時(shí),其與發(fā)光體相互作用時(shí),電子傳輸距離長(zhǎng)、能量損失大。鑒于此,本論文主要從提高發(fā)光體和共反應(yīng)試劑之間的相互作用效率著手,通過合成自增強(qiáng)型ECL試劑和引入新型共反應(yīng)促進(jìn)劑放大策略,并結(jié)合核酸擴(kuò)增技術(shù)以及多種納米復(fù)合材料,構(gòu)建了多個(gè)ECL生物傳感器,實(shí)現(xiàn)了多種生物分子如micro RNAs、蛋白等的高靈敏檢測(cè)。本論文主要的研究工作如下:1.基于目標(biāo)物催化發(fā)夾組裝和分子內(nèi)/分子間共反應(yīng)信號(hào)放大策略構(gòu)建的ECL生物傳感器研究目前,將電致化學(xué)發(fā)光技術(shù)和核酸擴(kuò)增技術(shù)結(jié)合起來用于micro RNAs(mi RNAs)的檢測(cè)日漸成熟。然而,大多數(shù)的核酸擴(kuò)增技術(shù)都依賴酶的參與,因此會(huì)受到一些不利因素,如操作步驟繁瑣、操作條件苛刻和測(cè)試成本較高等的制約。目標(biāo)物催化發(fā)夾組裝(T-CHA)技術(shù)能在無酶的條件下實(shí)現(xiàn),其不僅具有靈敏度高、分析時(shí)間短、操作簡(jiǎn)單等特點(diǎn),而且還能同時(shí)實(shí)現(xiàn)目標(biāo)物的循環(huán)擴(kuò)增。另外,在構(gòu)建ECL生物傳感器過程中,引入合適的共反應(yīng)試劑,同時(shí)有效的提高共反應(yīng)試劑和發(fā)光體的作用效率尤為重要。通過共價(jià)交聯(lián)作用,將共反應(yīng)試劑和發(fā)光體合成為一個(gè)分子,制得自增強(qiáng)型釕復(fù)合物發(fā)光試劑(PEI-Ru(II)),縮短了發(fā)光體Ru(II)與共反應(yīng)試劑聚乙烯亞胺(PEI)之間電子傳輸距離,減少了Ru(II)與PEI作用中能量的損失,提高了發(fā)光體與共反應(yīng)試劑的作用效率,進(jìn)而獲得更強(qiáng)且穩(wěn)定的ECL信號(hào)。本研究首次將新型的共反應(yīng)試劑氨基硫脲(TSC)引入PEI-Ru(II)自增強(qiáng)發(fā)光體系中,實(shí)現(xiàn)了分子內(nèi)/分子間雙重共反應(yīng)放大,進(jìn)一步結(jié)合無酶T-CHA構(gòu)建了生物傳感器來高靈敏的檢測(cè)mi R-21,其檢測(cè)范圍為1.0×10-16 mol/L到1.0×10-11 mol/L,檢測(cè)限低至30 amol/L。2.基于傒衍生物作為新型共反應(yīng)促進(jìn)劑信號(hào)放大構(gòu)建的ECL生物傳感器研究量子點(diǎn)(QDs)具有尺寸和發(fā)射波長(zhǎng)可控、發(fā)光產(chǎn)率高和化學(xué)穩(wěn)定性好等優(yōu)點(diǎn),其作為ECL試劑已被廣泛用于生物標(biāo)志物的分析中。值得注意的是,QDs的固載是固態(tài)ECL生物傳感器構(gòu)建過程中的關(guān)鍵步驟。石墨烯憑借優(yōu)良的導(dǎo)電性和大的比表面積等優(yōu)勢(shì),可作為QDs理想的固載基質(zhì)。同時(shí),過硫酸根(S2O82-)作為QDs最常用的共反應(yīng)試劑使QDs產(chǎn)生高效穩(wěn)定的ECL信號(hào),但是由于其難以固載,使其應(yīng)用受到了一定的限制。本研究在S2O82-/QDs體系中引入共反應(yīng)促進(jìn)劑解決了這一難題。其中,L-賴氨酸功能化的傒四甲酸的復(fù)合物(PTC-Lys)作為一種新型的傒衍生物,能夠促進(jìn)S2O82-產(chǎn)生更多的強(qiáng)氧化劑中間體SO4·-,從而增強(qiáng)QDs的ECL信號(hào)。基于PTC-Lys作為新型的共反應(yīng)促進(jìn)劑構(gòu)建了高靈敏的適體傳感器用于檢測(cè)凝血酶(TB),檢測(cè)范圍為1.0×10-16 mol/L~1.0×10-8 mol/L,檢測(cè)限為34 amol/L。3.基于金屬有機(jī)骨架材料/量子點(diǎn)復(fù)合材料作為多功能信號(hào)探針構(gòu)建的ECL生物傳感器研究金屬有機(jī)骨架材料(MOFs)一般是以金屬離子為連接點(diǎn),有機(jī)配體為支撐構(gòu)筑的3D空間網(wǎng)絡(luò)結(jié)構(gòu)材料,具有比表面積大、孔尺寸和孔形狀可調(diào)等優(yōu)點(diǎn),使其成為理想的固載材料。本研究為了固載更多的發(fā)光試劑Cd Te QDs,同時(shí)采用了“內(nèi)腔填充”、“內(nèi)表面修飾”以及“外表面修飾”方法制得異格金屬有機(jī)骨架材料-3(IRMOF-3)內(nèi)外表面均修飾有Cd Te QDs的復(fù)合材料Cd Te@IRMOF-3/Cd Te QDs。值得注意的是,IRMOF-3不僅可作為Cd Te QDs的固載基質(zhì),而且能促進(jìn)S2O82-產(chǎn)生更多的強(qiáng)氧化劑中間體SO4·-發(fā)射更強(qiáng)的ECL信號(hào),即起到了共反應(yīng)促進(jìn)劑的作用。因此,本文基于IRMOF-3和Cd Te QDs的復(fù)合材料作為多功能信號(hào)探針,構(gòu)建了一個(gè)高靈敏的免疫傳感器用以檢測(cè)心肌肌鈣蛋白I(c Tn I),并獲得了較寬的響應(yīng)范圍(1.1 fg/m L~11 ng/m L)和低的檢測(cè)限(0.46 fg/m L)。
[Abstract]:Electrochemiluminescence (ECL) biosensor is a kind of biological molecules such as nucleic acid molecules, such as antibodies, aptamers, molecular recognition element, sensor.ECL biosensor target detection with high sensitivity, good selectivity with ECL technology, fast response speed, simple operation, low cost, is widely used in disease related nucleic acid molecules, proteins, small molecules detection function. Usually, introducing common reagent can significantly enhance the signal intensity of ECL to ECL system, thus improving the detection sensitivity of ECL biosensor. However, some common reagents unstable and difficult to mark, thus increasing the difficulty of immobilization. At the same time, with the light body electronic interaction, long transmission distance, large energy loss. In view of this, this paper mainly from the body and improve the luminous efficiency of interaction between the co reaction reagent to the synthesis of self reinforced ECL test The introduction of a new agent and co reaction accelerator amplification strategy, combined with the nucleic acid amplification technology and a variety of nano composite materials, construction of a number of ECL biological sensors, to achieve a variety of biological molecules such as micro, RNAs, high sensitive protein detection. The main works of this thesis are as follows: 1. based on the target assembly and molecular / catalytic hairpin the intermolecular co response signal amplification of ECL biosensor construction strategy at present, electrochemiluminescence technology and nucleic acid amplification technology together for micro RNAs (MI RNAs) detection is becoming more and more mature. However, most of the nucleic acid amplification techniques rely on enzymes, so it will have some adverse factors, such as complicated steps harsh operating conditions, and the test cost is higher. The restricted object assembly (T-CHA) hairpin catalytic technology can be realized in non enzymatic conditions, it not only has high sensitivity, divided Analysis of short time, simple operation and other characteristics, but also realize the target of cyclic amplification. In addition, in the process of constructing ECL biosensor, introducing common reagent suitable, and effectively improve the co reaction reagent and luminous efficiency of the body is particularly important. Through covalent crosslinking, CO reaction reagent and luminescence the body into a molecule, prepared from ruthenium complexes enhanced luminescence reagent (PEI-Ru (II)), Ru phosphor (II) and shorten the reaction reagent of polyethyleneimine (PEI) between the electronic transmission distance, reduced Ru (II) and PEI in energy loss, improve the luminous body with the total reaction efficiency of reagents, and ECL signals were enhanced and stable. This is the first time of the co reaction reagent Aminothiourea model (TSC) into PEI-Ru (II) from the enhanced chemiluminescence system, the intramolecular / intermolecular co amplification reaction, into a No enzyme T-CHA to construct biosensor to the sensitive detection of MI combined with R-21, the detection range of 1 * 10-16 to 1 * mol/L 10-11 mol/L, the detection limit is low to 30 amol/L.2. based on Xi derivatives as novel co reaction accelerator signal amplification to construct ECL biosensor based on quantum dots (QDs) with the size and emission wavelength the advantages of light controlled, high yield and good chemical stability, as the ECL reagent has been widely used for biomarker analysis. It is worth noting that QDs supported is the key step in the process of solid ECL biosensors. Graphene with excellent electrical conductivity and large specific surface area and other advantages, solid matrix can be used as the ideal QDs. At the same time, persulfate (S2O82-) as the most commonly used QDs co reaction reagent QDs ECL signal is stable and efficient, but because of its difficult immobilization, the application by a certain Limited. This study introduces co reaction accelerator to solve this problem in the S2O82-/QDs system. Among them, compound L- lysine functionalized Xi four formic acid (PTC-Lys) as a new type of Xi derivatives can promote S2O82- produced more strong oxidizing agent intermediate SO4 - ECL, thereby enhancing the signal QDs PTC-Lys. As a new co reaction accelerator built high sensitive aptasensor for thrombin detection based on (TB), the detection range of 1 * 10-16 mol/L~1.0 * 10-8 mol/L, the detection limit is 34 amol/L.3. based on metal organic frameworks / quantum dots composite materials as ECL biosensor based on metal organic framework of multi functional signal probe the construction of the (MOFs) are generally based on metal ions as the connection point, to support the construction of organic ligand 3D space network structure material, has the advantages of large specific surface area, pore size and pore shape adjustable, Make it a solid material ideal. In this study, in order to Cd Te QDs reagent supported more at the same time, the "cavity filling", "modified" and "surface modification" prepared different lattice metal organic frameworks (-3 IRMOF-3) both internal and external surface composite modified Cd Te@IRMOF-3/Cd Te QDs. Cd Te QDs, it is worth noting that IRMOF-3 not only can be used as immobilization matrix Cd Te QDs, but also can promote S2O82- to produce more strong oxidant agent intermediate SO4 - emission stronger ECL signals, which play the role of CO reaction accelerator. Therefore, the composite materials of IRMOF-3 and Cd Te as QDs multifunctional signal probe based on the construction of a highly sensitive immunosensor for the detection of cardiac troponin I (C Tn I), and received a wide range of response (1.1 fg/m L~11 ng/m L) and low detection limit (0.46 fg/m L).

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3;TP212.3

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本文編號(hào)：1407242