抗污染傳感界面的構(gòu)建及其在生物分子檢測(cè)中的應(yīng)用
發(fā)布時(shí)間：2019-07-09 10:46
【摘要】：制備能夠用于實(shí)際生物樣品中低豐度腫瘤標(biāo)志物的高靈敏度,高選擇性檢測(cè)的電化學(xué)生物傳感器存在很大的挑戰(zhàn)。主要存在問題是實(shí)際生物樣品中蛋白質(zhì),多糖,脂質(zhì)等生物大分子對(duì)傳感界面的非特異性吸附。因此構(gòu)建具有優(yōu)良抗污染性能的傳感界面對(duì)于傳感器的研制是十分必要的。本論文以電化學(xué)方法制備了幾種納米材料,以表面共價(jià)修飾的方法構(gòu)建抗污染傳感界面,系統(tǒng)地研究了傳感器的抗污染傳感性能。本論文的主要研究?jī)?nèi)容如下:(1)成功的制備了一種新穎簡(jiǎn)單的超靈敏且具有抗污染能力的電化學(xué)DNA傳感器。首先通過電化學(xué)氧化玻碳電極的方式得到修飾有羧基官能團(tuán)的界面,再通過共價(jià)鏈接的方法將抗污染材料PEG以及固定探針依次修飾到電極表面,從而制備了這種具有抗污染能力的電化學(xué)DNA傳感器。傳感器的檢測(cè)線性范圍為1.0 fM to 100.0 nM,最低檢測(cè)限為0.3 fM。并且制備的生物傳感器在實(shí)際生物樣品檢測(cè)中具有良好的表現(xiàn),有望在實(shí)際臨床檢測(cè)中得到應(yīng)用。(2)非特異性蛋白質(zhì)吸附是免疫測(cè)定中面臨的關(guān)鍵問題,構(gòu)建可以在復(fù)雜生物介質(zhì)中顯著抵抗蛋白質(zhì)非特異性吸附的界面是非常重要的。本研究,是基于摻雜有透明質(zhì)酸(HA)的聚3,4-乙烯二氧噻吩(PEDOT)新型復(fù)合材料,制備了用于腫瘤標(biāo)志物-癌胚抗原(CEA)檢測(cè)的抗污染電化學(xué)免疫傳感器。電沉積的PEDOT/HA復(fù)合材料顯示出多孔微結(jié)構(gòu)和很強(qiáng)的親水性,并且在其表面上還具有許多羧基官能團(tuán),可以用于固定CEA抗體�；赼nti-CEA/PEDOT/HA的免疫傳感器在從1 pg/mL至0.1μg/m L的寬線性濃度范圍內(nèi)對(duì)CEA表現(xiàn)出高的檢測(cè)靈敏度,檢測(cè)限是0.3 pg/m L。此外,所開發(fā)的免疫傳感器具有高度特異性和抗污染性能,可以用于在真實(shí)的人血清樣品中測(cè)定CEA,避免由于非特異性蛋白吸附而導(dǎo)致的信號(hào)干擾。(3)通過共價(jià)鏈接的方法將MB-rep/probe(亞甲基藍(lán)信號(hào)探針/固定探針)雙鏈固定到AuNCs(金納米簇)/HA(透明質(zhì)酸)/Popd(聚鄰苯二胺)/CoO(氧化鈷)核/殼復(fù)合材料表面,得到一種新型的能夠快速檢測(cè)microRNA(miRNA)的電化學(xué)生物傳感器。在檢測(cè)目標(biāo)物時(shí),形成probe/target雙鏈體(電極表面)和MB-rep發(fā)夾結(jié)構(gòu)(溶液中),其迅速從生物傳感器置換MB-rep,導(dǎo)致SWV(方波脈沖伏安法)信號(hào)的迅速變化。基于生物傳感器的Au/HA/Popd/CoO復(fù)合材料提供更多的電活性表面積和優(yōu)異的抗污染性能,其可以用于生物復(fù)雜介質(zhì)中的miRNA的超靈敏檢測(cè)。生物傳感器顯示出優(yōu)異的特異性和檢測(cè)靈敏性,具有從100 fM至1μM的寬線性范圍,為臨床生物應(yīng)用提供了重要支撐。
[Abstract]:The preparation of an electrochemical biosensor capable of being used for high sensitivity and high-selectivity detection of low-abundance tumor markers in an actual biological sample has a great challenge. The main problems are the non-specific adsorption of the biological macromolecules such as proteins, polysaccharides and lipids in the actual biological sample to the sensing interface. Therefore, it is necessary to construct a sensing interface with excellent anti-pollution performance. In this paper, several nano-materials were prepared by electrochemical method, and the anti-pollution sensing interface was constructed by the method of surface covalent modification, and the anti-pollution sensing performance of the sensor was systematically studied. The main contents of this thesis are as follows: (1) A novel, simple, ultra-sensitive and anti-pollution electrochemical DNA sensor was successfully prepared. Firstly, the interface with the anti-pollution function is obtained through the method of electrochemical oxidation of the glassy carbon electrode, and the anti-pollution material PEG and the fixed probe are sequentially modified to the surface of the electrode through a covalent linkage method, so that the electrochemical DNA sensor with the anti-pollution capability is prepared. The sensor has a linear range of 1.0 fM to 100.0 nM and a minimum detection limit of 0.3 fM. And the prepared biosensor has good performance in actual biological sample detection and is expected to be applied in practical clinical detection. (2) Non-specific protein adsorption is a key problem in immunoassays, and it is very important to construct an interface that can significantly resist the non-specific adsorption of proteins in complex biological media. The present study was based on the novel composite of poly (3,4-ethylene) dioxolane (PEDOT) doped with hyaluronic acid (HA), and prepared the anti-pollution electrochemical immunosensor for tumor marker-carcinoembryonic antigen (CEA) detection. Electrodeposited PEDOT/ HA composites exhibit a porous microstructure and very strong hydrophilicity, and also have a number of base-based functional groups on their surface, which can be used to fix the CEA antibody. The immune sensor based on the anti-CEA/ PEDOT/ HA exhibited high detection sensitivity to CEA in a wide linear concentration range from 1 pg/ mL to 0.1. m u.g/ m L, with a detection limit of 0.3 pg/ m L. In addition, the developed immunosensor has a high specificity and an anti-contamination performance, CEA can be used in a real human serum sample to avoid signal interference due to non-specific protein adsorption. (3) the MB-rep/ probe (methylene blue signal probe/ fixed probe) double-chain is fixed to the surface of the AuNCs (gold nanocluster)/ HA (hyaluronic acid)/ Popd (poly (o-phenylenediamine)/ CoO (cobalt oxide) core/ shell composite material by a covalent link method, A novel electrochemical biosensor capable of rapidly detecting microRNA (miRNA) is obtained. When the target is detected, the probe/ target duplex (electrode surface) and the MB-rep hairpin structure (in solution) are formed, which quickly displaces the MB-rep from the biosensor, resulting in a rapid change in the SWV (square wave pulse voltammetry) signal. The Au/ HA/ Popd/ CoO composite based on the biosensor provides more electroactive surface area and excellent anti-pollution performance, which can be used for ultra-sensitive detection of miRNAs in a biological complex medium. Biosensors exhibit excellent specificity and detection sensitivity, with a wide linear range from 100 fM to 1. m u.M, providing important support for clinical biological applications.
【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)】：O657.1
本文編號(hào)：2512090
[Abstract]:The preparation of an electrochemical biosensor capable of being used for high sensitivity and high-selectivity detection of low-abundance tumor markers in an actual biological sample has a great challenge. The main problems are the non-specific adsorption of the biological macromolecules such as proteins, polysaccharides and lipids in the actual biological sample to the sensing interface. Therefore, it is necessary to construct a sensing interface with excellent anti-pollution performance. In this paper, several nano-materials were prepared by electrochemical method, and the anti-pollution sensing interface was constructed by the method of surface covalent modification, and the anti-pollution sensing performance of the sensor was systematically studied. The main contents of this thesis are as follows: (1) A novel, simple, ultra-sensitive and anti-pollution electrochemical DNA sensor was successfully prepared. Firstly, the interface with the anti-pollution function is obtained through the method of electrochemical oxidation of the glassy carbon electrode, and the anti-pollution material PEG and the fixed probe are sequentially modified to the surface of the electrode through a covalent linkage method, so that the electrochemical DNA sensor with the anti-pollution capability is prepared. The sensor has a linear range of 1.0 fM to 100.0 nM and a minimum detection limit of 0.3 fM. And the prepared biosensor has good performance in actual biological sample detection and is expected to be applied in practical clinical detection. (2) Non-specific protein adsorption is a key problem in immunoassays, and it is very important to construct an interface that can significantly resist the non-specific adsorption of proteins in complex biological media. The present study was based on the novel composite of poly (3,4-ethylene) dioxolane (PEDOT) doped with hyaluronic acid (HA), and prepared the anti-pollution electrochemical immunosensor for tumor marker-carcinoembryonic antigen (CEA) detection. Electrodeposited PEDOT/ HA composites exhibit a porous microstructure and very strong hydrophilicity, and also have a number of base-based functional groups on their surface, which can be used to fix the CEA antibody. The immune sensor based on the anti-CEA/ PEDOT/ HA exhibited high detection sensitivity to CEA in a wide linear concentration range from 1 pg/ mL to 0.1. m u.g/ m L, with a detection limit of 0.3 pg/ m L. In addition, the developed immunosensor has a high specificity and an anti-contamination performance, CEA can be used in a real human serum sample to avoid signal interference due to non-specific protein adsorption. (3) the MB-rep/ probe (methylene blue signal probe/ fixed probe) double-chain is fixed to the surface of the AuNCs (gold nanocluster)/ HA (hyaluronic acid)/ Popd (poly (o-phenylenediamine)/ CoO (cobalt oxide) core/ shell composite material by a covalent link method, A novel electrochemical biosensor capable of rapidly detecting microRNA (miRNA) is obtained. When the target is detected, the probe/ target duplex (electrode surface) and the MB-rep hairpin structure (in solution) are formed, which quickly displaces the MB-rep from the biosensor, resulting in a rapid change in the SWV (square wave pulse voltammetry) signal. The Au/ HA/ Popd/ CoO composite based on the biosensor provides more electroactive surface area and excellent anti-pollution performance, which can be used for ultra-sensitive detection of miRNAs in a biological complex medium. Biosensors exhibit excellent specificity and detection sensitivity, with a wide linear range from 100 fM to 1. m u.M, providing important support for clinical biological applications.
【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)】：O657.1
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