【摘要】：電化學(xué)生物傳感器是一種將電化學(xué)分析方法與生物學(xué)技術(shù)相結(jié)合而發(fā)展起來(lái)的具有響應(yīng)快速、靈敏度高、選擇性好、操作簡(jiǎn)單、成本低等優(yōu)點(diǎn)的生物傳感器。碳納米材料(如石墨烯、富勒烯)因具有比表面積大、表面活性位點(diǎn)高及生物相容性好等優(yōu)點(diǎn)被廣泛的應(yīng)用到生物傳感器領(lǐng)域。近年來(lái),基于新型納米材料催化、酶催化以及生物學(xué)放大技術(shù)用于蛋白質(zhì)檢測(cè)的電化學(xué)生物傳感器的研究頗受關(guān)注。本文的研究目的是將碳納米復(fù)合材料、生物以及化學(xué)等多種放大技術(shù)相結(jié)合,實(shí)現(xiàn)高靈敏的檢測(cè)。圍繞本研究目的主要從功能化復(fù)合納米材料的制備、敏感界面的構(gòu)建以及新型信號(hào)放大技術(shù)的應(yīng)用等進(jìn)行了探索和研究。研究工作分為以下幾個(gè)部分：1.電化學(xué)催化放大技術(shù)用于神經(jīng)元特異性烯醇化酶的檢測(cè)本文研制了基于金-石墨烯復(fù)合膜／鐵氰化鎳納米粒子／納米金修飾的電流型免疫傳感器用于神經(jīng)元特異性烯醇化酶(NSE)的檢測(cè)。值得注意的是：1)基于鐵氰化鎳納米粒子(NiHCFNPs)固有的電化學(xué)活性,NiHCFNPs修飾電極呈現(xiàn)出良好的氧化還原活性,可以用來(lái)指示免疫反應(yīng)發(fā)生的進(jìn)程,構(gòu)建了無(wú)試劑型電化學(xué)免疫傳感器。2)NiHCFNPs能夠有效的催化DA,顯著增強(qiáng)信號(hào),避免了使用生物酶在標(biāo)記過程中易失活這一缺點(diǎn)。3)金和石墨烯復(fù)合納米材料(Au-Gra)具有比表面積大、吸附力強(qiáng)、生物相容性好等優(yōu)點(diǎn),大大提高抗體分子的固載量。該方法基于簡(jiǎn)單的直接法進(jìn)行,不需要在測(cè)試溶液中加入其他的電活性物質(zhì),只需將NiHCFNPs固載到電極表面即可,具有操作簡(jiǎn)單、響應(yīng)快速的優(yōu)點(diǎn)。與傳統(tǒng)的直接法相比,該傳感器有較高的靈敏度,線性范圍為0.001～100 ng mL-1,檢測(cè)下限為0.3 pg mL-1(S/N=3)。2.基于多功能化洋蔥狀石墨烯層和雙重催化放大構(gòu)建電化學(xué)免疫傳感器用于兩種腫瘤標(biāo)志物同時(shí)檢測(cè)為了提高傳感器的檢測(cè)通量,本研究以功能化洋蔥狀石墨烯層結(jié)合雙重催化放大技術(shù)構(gòu)建了一種夾心型電化學(xué)免疫傳感器,實(shí)現(xiàn)了基于同一敏感界面對(duì)于游離前列腺特異性抗原(fPSA)及前列腺特異性抗原(PSA)的同時(shí)檢測(cè)。采用洋蔥狀石墨烯層為納米載體,通過靜電吸附作用在其表面修飾不同的電活性納米材料,隨后進(jìn)一步固載親和素(SA)和生物素標(biāo)記的堿性磷酸酯酶(bio-AP),形成多重標(biāo)記的洋蔥狀石墨烯納米復(fù)合材料。當(dāng)測(cè)試液中存在抗壞血酸酯(AA-P)時(shí),bio-AP首先能夠催化AA-P水解生成抗壞血酸(AA),接著,生成的AA進(jìn)一步被電活性納米材料(普魯士藍(lán)納米粒子：PBNPs或鐵氰化鎳納米粒子：NiNPs)催化產(chǎn)生DHA,實(shí)現(xiàn)雙重信號(hào)放大。實(shí)驗(yàn)結(jié)果表明PBNPs和NiNPs具有良好的氧化還原可逆性且氧化還原峰電位相互分離,結(jié)合雙重催化信號(hào)放大策略,完成了同時(shí)對(duì)兩種目標(biāo)蛋白質(zhì)高特異和高靈敏的檢測(cè)。該免疫傳感器對(duì)fPSA和PSA的檢測(cè)限分別達(dá)到6.7 pg mL-1和3.4pgmL-1。3.基于磁性石墨烯雜化微球構(gòu)建電化學(xué)免疫傳感器用于甲狀腺疾病標(biāo)志物的檢測(cè)生物酶在標(biāo)記過程中可能會(huì)影響蛋白的特異性位點(diǎn),致使生物活性喪失。本研究基于雜交鏈?zhǔn)椒磻?yīng)(HCR)為模板固載雙酶(細(xì)胞色素c氧化酶和葡萄糖氧化酶)能夠有效提高酶的固載量和很好的保持酶的生物活性。本文首先利用層層自組裝方法制備以Si02為模板的磁性石墨烯雜化微球,該納米材料集電化學(xué)氧化還原活性、磁性于一體,構(gòu)建可再生的電化學(xué)免疫傳感器。將制備的磁性石墨烯雜化微球作為納米載體通過化學(xué)鍵合作用固載信標(biāo)抗體和引物鏈S1,通過生物催化放大技術(shù)和雙酶逐級(jí)催化有效的放大響應(yīng)信號(hào)�；趭A心免疫反應(yīng),將該免疫傳感器用于檢測(cè)甲狀腺疾病標(biāo)志物,線性范圍為0.05 pg mL-1～5 ng mL-1,檢測(cè)限達(dá)15 fgmL-1。經(jīng)實(shí)驗(yàn)研究證明該方法切實(shí)可行,為傳感器靈敏度的提高提供了新的思路。4.基于多功能化的C60納米復(fù)合材料作為信號(hào)標(biāo)簽構(gòu)建電化學(xué)適體傳感器隨著對(duì)碳材料性質(zhì)研究的進(jìn)一步深入,C60作為一種生物傳感材料開始被應(yīng)用于電化學(xué)傳感器領(lǐng)域。C60易溶于苯、甲苯和二硫化碳等非極性有機(jī)溶劑,但不溶于水,而且導(dǎo)電性能不高。為了改善C60的水溶性,我們用帶有NH2活性端基的傒四甲酸(PTC-NH2)功能化nano-C60,基于超分子化學(xué)得到了水溶性好的C60納米材料(FC60NPs)。接著通過化學(xué)鍵合作用在其表面修飾納米金包裹的普魯士藍(lán)納米粒子(Au@PBNPs),繼而得到多功能化的C60納米復(fù)合材料(Au@PB/FC60)。為了提高傳感器的靈敏度,將堿性磷酸酯酶(AP)標(biāo)記到Au@PB/FC60表面,在底物抗壞血酸酯(AA-P)存在下,AP首先能夠催化AA-P水解生成抗壞血酸(AA),接著,生成的AA進(jìn)一步被Au@PB/FC60催化產(chǎn)生DHA,實(shí)現(xiàn)雙重信號(hào)放大。將該適體傳感器用于檢測(cè)血小板源性生長(zhǎng)因子(PDGF),線性范圍為0.002～40 nmolmL-1,檢測(cè)限達(dá)0.6 pmol mL-1。實(shí)驗(yàn)表明,該適體傳感器具有選擇性好、靈敏度高,有望應(yīng)用于臨床檢測(cè)中。5.C60納米材料作為氧化還原納米探針構(gòu)建電化學(xué)免疫傳感器用于興奮劑的檢測(cè)碳納米材料因具有比表面積大、導(dǎo)電性及生物相容性好等特點(diǎn)通常作為納米載體被廣泛的應(yīng)用到生物傳感器領(lǐng)域,卻很少被用作氧化還原納米探針。C60除了具有上述碳納米材料的優(yōu)點(diǎn)外,還具有內(nèi)在的氧化還原特性,如強(qiáng)的接受電子能力容易形成相應(yīng)的陰離子。本文首先用聚酰胺-胺(PAMAM)功能化C60納米顆粒(PAMAM-C60NPs),然后利用PAMAM-C60NPs表面大量的氨基可以吸附納米金,得到了C60氧化還原納米探針(Au-PAMAM-C60NPs)并用于標(biāo)記信標(biāo)抗體構(gòu)建了夾心型免疫傳感器。值得注意的是,當(dāng)修飾好的免疫電極表面孵育四辛基溴化銨(TOAB)后,Au-PAMAM-C60NPs內(nèi)在的氧化還原活性被喚醒,在-0.45～0.3 V的電位范圍內(nèi)得到一對(duì)可逆的氧化還原峰�；趭A心免疫反應(yīng),將該傳感器用于檢測(cè)興奮劑(EPO),有較寬的線性范圍和較低的檢測(cè)下限,將其用于臨床樣品檢測(cè),得到滿意的結(jié)果。此外,該研究工作為將碳材料作為氧化還原納米探針用于電化學(xué)生物傳感器的構(gòu)建提供了新方法。
[Abstract]:The electrochemical biosensor is a biosensor with the advantages of rapid response, high sensitivity, good selectivity, simple operation, low cost and the like, which is developed by combining the electrochemical analysis method with the biological technology. The carbon nano material (such as graphene, fullerene) is widely used in the field of biological sensors due to the advantages of large specific surface area, high surface activity site, good biocompatibility and the like. In recent years, the research of electrochemical biosensor based on novel nano-material catalysis, enzyme catalysis and biological amplification technology for protein detection is of great concern. The purpose of this paper is to combine the carbon nano-composite material, biological and chemical amplification technology to realize high-sensitivity detection. The purpose of this study is to explore and study the preparation of functional composite nano-materials, the construction of sensitive interface and the application of new signal amplification technology. The research work is divided into the following parts:1. In this paper, the detection of neuron-specific enolase (NSE) was developed based on the gold-graphene composite membrane/ nickel-nickel-nickel nano-particle/ nano-gold-modified current-type immunosensor. It is worth noting that:1) NiHCFNPs modified electrode exhibits good redox activity based on the inherent electrochemical activity of nickel-nickel-nickel nano-particles (NiHCFNPs), which can be used to indicate the progress of the immune response, The non-reagent type electrochemical immunosensor is constructed.2) NiHCFNPs can effectively catalyze the DA and obviously enhance the signal, so that the defect that the biological enzyme is easy to be inactivated during the labeling process is avoided; and 3) the gold and the graphene composite nano-material (Au-Gra) has the advantages of large specific surface area and strong adsorption force, Has the advantages of good biocompatibility and the like, and greatly improves the solid loading of the antibody molecules. The method is based on a simple direct method, and does not need to add other electroactive substances in the test solution, so that only the NiHCFNPs can be fixed on the surface of the electrode, and the method has the advantages of simple operation and quick response. Compared with the traditional direct method, the sensor has a high sensitivity, the linear range is from 0.001 to 100 ng mL-1, and the detection limit is 0.3 pg mL-1 (S/ N = 3). The invention provides an electrochemical immunosensor based on a multifunctional onion-shaped graphene layer and a dual-catalytic amplification, wherein the electrochemical immunosensor is used for simultaneously detecting two tumor markers in order to improve the detection flux of the sensor, In this study, a sandwich type electrochemical immunosensor was constructed by using the functionalized onion-like graphene layer in combination with the double catalytic amplification technology, and the simultaneous detection of the free prostate specific antigen (fPSA) and the prostate specific antigen (PSA) based on the same sensitive interface was realized. The onion-shaped graphene layer is adopted as a nano carrier, and different electro-active nano-materials are modified on the surface of the nano-carrier by the electrostatic adsorption, and then the avidin (SA) and the biotin-labeled alkaline phosphatase (bio-AP) are further immobilized to form the multi-labeled onion-shaped graphene nano composite material. when ascorbic acid ester (AA-P) is present in the test solution, the bio-AP can first catalyze the hydrolysis of AA-P to generate ascorbic acid (AA), and then the generated AA is further electroactive nano-material (Prussian blue nanoparticles: PBNPs or ferrocyanide nickel nanoparticles: NiNPs) to catalyze the production of DHA, And the double-signal amplification is realized. The results show that PBNPs and NiNPs have good redox reversibility and the oxidation-reduction peak potentials are separated from each other, and combined with the double catalytic signal amplification strategy, the high-specific and high-sensitivity detection of the two target proteins is completed. The detection limits of the immunosensor to fPSA and PSA reached 6.7 pg mL-1 and 3.4 pgmL-1.3, respectively. The use of the magnetic graphene hybrid microspheres to construct the electrochemical immunosensor for detecting the thyroid disease marker can affect the specific site of the protein in the labeling process, so that the biological activity is lost. Based on the hybrid chain reaction (HCR), the two enzymes (cytochrome c oxidase and glucose oxidase), which are used as the template, can effectively improve the solid loading of the enzyme and the bioactivity of the enzyme. In this paper, the magnetic graphene hybrid microspheres with Si02 as the template are prepared by a layer-by-layer self-assembly method, and the nano-material set has the functions of electrochemical oxidation reduction activity and magnetism, and can be used for constructing a reproducible electrochemical immunosensor. The prepared magnetic graphene hybrid microspheres are used as the nano carrier to carry out chemical bond cooperation on the immobilized beacon antibody and the primer chain S1, and the amplified response signals are effectively amplified by the biological catalytic amplification technology and the double-enzyme step-by-step catalysis. Based on the sandwich immune response, the immunosensor was used to detect thyroid disease markers with a linear range of 0.05 pg mL-1 to 5 ng mL-1 and a detection limit of 15 fgmL-1. The experimental results show that the method is feasible and provides a new way for improving the sensitivity of the sensor. The application of C60 as a kind of bio-sensing material is applied to the field of electrochemical sensor as a kind of bio-sensing material, based on the multi-functional C60 nano-composite as the signal label to construct the electrochemical aptamer sensor. C60 is soluble in non-polar organic solvent such as benzene, toluene and carbon disulfide, but is not soluble in water, and has high conductivity. In order to improve the water solubility of the C60, the nano-C60 is functionalized with a tetraformic acid (PTC-NH2) with an NH2 active end group, and a water-soluble C60 nano-material (FC60NPs) is obtained based on the supramolecular chemistry. Then the Prussian blue nano-particles (Au@PBNPs) coated with the nano-gold are modified by chemical bonding, and then the multifunctional C60 nano composite material (Au@PB/ FC60) is obtained. In order to improve the sensitivity of the sensor, an alkaline phosphatase (AP) is labeled to the surface of the Au@PB/ FC60, and in the presence of a substrate ascorbic acid ester (AA-P), the AP can first catalyze the hydrolysis of AA-P to produce ascorbic acid (AA), and then the generated AA is further catalyzed by the Au@PB/ F60 to produce DHA, And the double-signal amplification is realized. The aptamer sensor was used to detect platelet-derived growth factor (PDGF), a linear range of 0.002 to 40 nm-1, and a detection limit of 0.6 pmol mL-1. The experiment shows that the aptamer sensor has the advantages of good selectivity and high sensitivity, and is expected to be applied to clinical detection. The characteristics of electrical conductivity and biocompatibility are generally used as nano-carriers in the field of biosensors, but are rarely used as redox nano-probes. In addition to the advantages of the above-mentioned carbon nano-materials, the C60 also has the intrinsic redox properties, such as strong acceptance of the electron ability, and the corresponding anions can be easily formed. In this paper, the functional C60 nanoparticles (PAMAM-C60NPs) were functionalized with polyfluoroamine-amine (PAMAM), and then the nano-gold was adsorbed by a large amount of amino groups on the surface of the PAMAM-C60NPs, and the nano-probe (Au-PAMAM-C60NPs) was obtained by using the surface of the PAMAM-C60NPs, and the sandwich-type immunosensor was constructed by labeling the beacon antibody. It is to be noted that the redox activity of the Au-PAMAM-C60NPs is awakened and a pair of reversible redox peaks are obtained in the range of-0.45 to 0.3 V when the modified immune electrode surface is incubated with tetraoctyl bromide (TOB). Based on the sandwich immune response, the sensor is used to detect a stimulant (EPO), has a wide linear range and a lower detection limit, and is used for clinical sample detection to obtain a satisfactory result. In addition, that research work to provide a new method for the construction of an electrochemical biosensor using a carbon material as an oxidation-reduction nanoprobe.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：TB383.1;O613.71

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 王麗江;陳松月;劉清君;王平;;納米技術(shù)在生物傳感器及檢測(cè)中的應(yīng)用[J];傳感技術(shù)學(xué)報(bào);2006年03期

2 陳慧連;陳偉銳;;電化學(xué)免疫傳感器的發(fā)展概述[J];廣州化工;2013年11期

3 王廣鳳;朱艷紅;陳玲;王倫;;功能性納米材料在電化學(xué)免疫傳感器中的應(yīng)用[J];分析化學(xué);2013年04期

4 張艷麗;朱靜;周敬良;蔣健暉;沈國(guó)勵(lì);俞汝勤;;適體電化學(xué)傳感器的研究進(jìn)展[J];化學(xué)傳感器;2008年03期

5 楊海朋;陳仕國(guó);李春輝;陳東成;戈早川;;納米電化學(xué)生物傳感器[J];化學(xué)進(jìn)展;2009年01期

6 吳波;顧國(guó)龍;;神經(jīng)元特異性烯醇化酶的臨床研究進(jìn)展[J];醫(yī)學(xué)綜述;2010年19期

，

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