基于碳纳米复合材料与信号放大技术构建电化学生物传感器的研究
发布时间：2019-06-08 12:15
【摘要】：电化学生物传感器是一种将电化学分析方法与生物学技术相结合而发展起来的具有响应快速、灵敏度高、选择性好、操作简单、成本低等优点的生物传感器。碳纳米材料(如石墨烯、富勒烯)因具有比表面积大、表面活性位点高及生物相容性好等优点被广泛的应用到生物传感器领域。近年来,基于新型纳米材料催化、酶催化以及生物学放大技术用于蛋白质检测的电化学生物传感器的研究颇受关注。本文的研究目的是将碳纳米复合材料、生物以及化学等多种放大技术相结合,实现高灵敏的检测。围绕本研究目的主要从功能化复合纳米材料的制备、敏感界面的构建以及新型信号放大技术的应用等进行了探索和研究。研究工作分为以下几个部分：1.电化学催化放大技术用于神经元特异性烯醇化酶的检测本文研制了基于金-石墨烯复合膜／铁氰化镍纳米粒子／纳米金修饰的电流型免疫传感器用于神经元特异性烯醇化酶(NSE)的检测。值得注意的是：1)基于铁氰化镍纳米粒子(NiHCFNPs)固有的电化学活性,NiHCFNPs修饰电极呈现出良好的氧化还原活性,可以用来指示免疫反应发生的进程,构建了无试剂型电化学免疫传感器。2)NiHCFNPs能够有效的催化DA,显著增强信号,避免了使用生物酶在标记过程中易失活这一缺点。3)金和石墨烯复合纳米材料(Au-Gra)具有比表面积大、吸附力强、生物相容性好等优点,大大提高抗体分子的固载量。该方法基于简单的直接法进行,不需要在测试溶液中加入其他的电活性物质,只需将NiHCFNPs固载到电极表面即可,具有操作简单、响应快速的优点。与传统的直接法相比,该传感器有较高的灵敏度,线性范围为0.001～100 ng mL-1,检测下限为0.3 pg mL-1(S/N=3)。2.基于多功能化洋葱状石墨烯层和双重催化放大构建电化学免疫传感器用于两种肿瘤标志物同时检测为了提高传感器的检测通量,本研究以功能化洋葱状石墨烯层结合双重催化放大技术构建了一种夹心型电化学免疫传感器,实现了基于同一敏感界面对于游离前列腺特异性抗原(fPSA)及前列腺特异性抗原(PSA)的同时检测。采用洋葱状石墨烯层为纳米载体,通过静电吸附作用在其表面修饰不同的电活性纳米材料,随后进一步固载亲和素(SA)和生物素标记的碱性磷酸酯酶(bio-AP),形成多重标记的洋葱状石墨烯纳米复合材料。当测试液中存在抗坏血酸酯(AA-P)时,bio-AP首先能够催化AA-P水解生成抗坏血酸(AA),接着,生成的AA进一步被电活性纳米材料(普鲁士蓝纳米粒子：PBNPs或铁氰化镍纳米粒子：NiNPs)催化产生DHA,实现双重信号放大。实验结果表明PBNPs和NiNPs具有良好的氧化还原可逆性且氧化还原峰电位相互分离,结合双重催化信号放大策略,完成了同时对两种目标蛋白质高特异和高灵敏的检测。该免疫传感器对fPSA和PSA的检测限分别达到6.7 pg mL-1和3.4pgmL-1。3.基于磁性石墨烯杂化微球构建电化学免疫传感器用于甲状腺疾病标志物的检测生物酶在标记过程中可能会影响蛋白的特异性位点,致使生物活性丧失。本研究基于杂交链式反应(HCR)为模板固载双酶(细胞色素c氧化酶和葡萄糖氧化酶)能够有效提高酶的固载量和很好的保持酶的生物活性。本文首先利用层层自组装方法制备以Si02为模板的磁性石墨烯杂化微球,该纳米材料集电化学氧化还原活性、磁性于一体,构建可再生的电化学免疫传感器。将制备的磁性石墨烯杂化微球作为纳米载体通过化学键合作用固载信标抗体和引物链S1,通过生物催化放大技术和双酶逐级催化有效的放大响应信号。基于夹心免疫反应,将该免疫传感器用于检测甲状腺疾病标志物,线性范围为0.05 pg mL-1～5 ng mL-1,检测限达15 fgmL-1。经实验研究证明该方法切实可行,为传感器灵敏度的提高提供了新的思路。4.基于多功能化的C60纳米复合材料作为信号标签构建电化学适体传感器随着对碳材料性质研究的进一步深入,C60作为一种生物传感材料开始被应用于电化学传感器领域。C60易溶于苯、甲苯和二硫化碳等非极性有机溶剂,但不溶于水,而且导电性能不高。为了改善C60的水溶性,我们用带有NH2活性端基的傒四甲酸(PTC-NH2)功能化nano-C60,基于超分子化学得到了水溶性好的C60纳米材料(FC60NPs)。接着通过化学键合作用在其表面修饰纳米金包裹的普鲁士蓝纳米粒子(Au@PBNPs),继而得到多功能化的C60纳米复合材料(Au@PB/FC60)。为了提高传感器的灵敏度,将碱性磷酸酯酶(AP)标记到Au@PB/FC60表面,在底物抗坏血酸酯(AA-P)存在下,AP首先能够催化AA-P水解生成抗坏血酸(AA),接着,生成的AA进一步被Au@PB/FC60催化产生DHA,实现双重信号放大。将该适体传感器用于检测血小板源性生长因子(PDGF),线性范围为0.002～40 nmolmL-1,检测限达0.6 pmol mL-1。实验表明,该适体传感器具有选择性好、灵敏度高,有望应用于临床检测中。5.C60纳米材料作为氧化还原纳米探针构建电化学免疫传感器用于兴奋剂的检测碳纳米材料因具有比表面积大、导电性及生物相容性好等特点通常作为纳米载体被广泛的应用到生物传感器领域,却很少被用作氧化还原纳米探针。C60除了具有上述碳纳米材料的优点外,还具有内在的氧化还原特性,如强的接受电子能力容易形成相应的阴离子。本文首先用聚酰胺-胺(PAMAM)功能化C60纳米颗粒(PAMAM-C60NPs),然后利用PAMAM-C60NPs表面大量的氨基可以吸附纳米金,得到了C60氧化还原纳米探针(Au-PAMAM-C60NPs)并用于标记信标抗体构建了夹心型免疫传感器。值得注意的是,当修饰好的免疫电极表面孵育四辛基溴化铵(TOAB)后,Au-PAMAM-C60NPs内在的氧化还原活性被唤醒,在-0.45～0.3 V的电位范围内得到一对可逆的氧化还原峰。基于夹心免疫反应,将该传感器用于检测兴奋剂(EPO),有较宽的线性范围和较低的检测下限,将其用于临床样品检测,得到满意的结果。此外,该研究工作为将碳材料作为氧化还原纳米探针用于电化学生物传感器的构建提供了新方法。
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1;O613.71
本文编号：2495276
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1;O613.71
【参考文献】
相关期刊论文 前6条
1 王丽江;陈松月;刘清君;王平;;纳米技术在生物传感器及检测中的应用[J];传感技术学报;2006年03期
2 陈慧连;陈伟锐;;电化学免疫传感器的发展概述[J];广州化工;2013年11期
3 王广凤;朱艳红;陈玲;王伦;;功能性纳米材料在电化学免疫传感器中的应用[J];分析化学;2013年04期
4 张艳丽;朱静;周敬良;蒋健晖;沈国励;俞汝勤;;适体电化学传感器的研究进展[J];化学传感器;2008年03期
5 杨海朋;陈仕国;李春辉;陈东成;戈早川;;纳米电化学生物传感器[J];化学进展;2009年01期
6 吴波;顾国龙;;神经元特异性烯醇化酶的临床研究进展[J];医学综述;2010年19期
，本文编号：2495276
本文链接：https://www.wllwen.com/kejilunwen/cailiaohuaxuelunwen/2495276.html
