【作者】 李文娟；

【导师】 袁若；

【作者基本信息】 西南大学 ， 分析化学， 2011， 博士

【摘要】 电化学生物传感器是一种将电化学分析与生物技术研究相结合而发展起来的一门学科。是将具有分子识别能力的生物活性材料(如酶、抗体等)与物理化学换能器有机组装而成。在生物传感器的研制过程中,生物敏感元件的固定方法和固载材料的选择尤为重要。基于此,本文致力于新型的复合纳米材料用于酶和免疫传感界面的构建以及固酶技术等方面进行了一系列探索性的研究。具体研究工作如下：1.基于氨基端基有机硅纳米球功能化的普鲁士蓝为媒介的葡萄糖生物传感器的研究在介体型酶生物传感器的研制过程中,电子媒介体易从电极表面渗漏到测试底液中,造成电化学信号不稳定,从而影响测定的稳定性和灵敏度,已成为制约其发展的瓶颈。为解决这个问题,电子媒介体-普鲁士蓝(PB),被有机硅纳米球保护起来,形成有机硅纳米球功能化的普鲁士蓝(OSiFPB),通过增加PB的分子量和包埋的方法来防止其渗漏。该材料兼具PB的电化学性质,呈现出良好的氧化还原特性。以此为电化学探针,来跟踪指示传感器制备过程中界面的电化学特征以及葡萄糖催化反应的进程。并结合比表面积大、吸附力强、生物亲和性好等优点的正电荷纳米金(PGNs),构建了PGNs/OSiFPB复合膜修饰葡萄糖氧化酶(GOD)的葡萄糖生物传感器。PGNs的引入不仅使GOD牢固的固定在电极表面,并且还可以很好的保持酶的生物活性。同时也起到了纳米导线的作用,加快OSiFPB在氧化还原过程中电子转移的速率。该传感器具有稳定性佳、灵敏度高的优点,实现了对葡萄糖快速、灵敏的测定。2.基于合金功能化的硅纳米纤维和凝集素-糖蛋白为复合固载基质的拟双酶葡萄糖生物传感器的研究通过化学合成的方法解决媒介体渗漏的方法有效,但是合成步骤较繁琐。为了简化试验程序,设计了一种具有独特的电学和光学特性的有机半导体材料---氨基化的北四甲酸(PTC-NH2),直接覆盖在PB的表面,形成结构镶嵌、稳定性好的有机-无机氧化还原复合膜(PTC-NH2/PB).同时,合成了一种全新的纳米复合物---金铂合金功能化的硅纳米纤维(GP-SNFs)为电极增敏材料,并组装在表面含有丰富氨基官能团的PTC-NH2表面。然后通过GP-SNFs强的表面作用力将半刀豆球蛋白A (Con A)捕获在电极表面,最后,结合凝集素-糖蛋白高的特异性吸附作用将葡萄糖氧化酶(GOD)固定在电极上。该传感器具有稳定性好、抗干扰能力强、线性范围宽、检测限低等特点。该工作具有三个特点：一是PTC-NH2/PB复合膜氧化还原活性强,导电性好,且比表面积大、表面活性位点多等优点,是一种极佳的电极修饰材料；二是由于GP-SNFs大的比表面积,提高了蛋白酶的负载量,同时为酶的固定提供了良好的微环境,保持酶的生物活性,所以提高了生物传感器的响应性能；三是从酶的固定方法考虑。采明了凝集素-糖蛋白特异性识别的方法对GOD进行固载,形成一层定向有序复合膜,这种固定方法更好地维持了酶的三维空间结构,使酶分子较好的保持原有的构型。3.基于铂纳米颗粒负载的碳纳米管和糖蛋白-凝集素特异作用构建葡萄糖生物传感器的研究基于直接电催化的第三代生物传感器。无需引入电子媒介体,故无需考虑其渗漏对样品造成污染的问题。但是由于氧化还原蛋白和酶的反应活性中心深埋在分子内部,难以和电极表面进行直接电子传输。合成了一种导电性优良的纳米铂负载碳纳米管的杂化材料(Ptnano-CNTs),扮演“分子导线”的角色,并结合层层自组装(LBL)技术和凝集素-糖蛋白特异性吸附作用将GOD固定在电极表面,成功地实现了GOD的直接电子传输。研究结果表明,几种物质的协同作用使得蛋白质分子在保持良好生物活性和电化学活性的同时,还提高了氧化还原蛋白质与电极之间的电子交换速度,从而制备具有高灵敏度、低检测限和高稳定性的葡萄糖生物传感器。4.基于葡萄糖氧化酶的直接电子转移为媒介的无试剂电流型CA 15-3免疫传感器的研究在传统的电化学免疫传感器构建中,大多需引用电子媒介体作为组装和检测进程中的跟踪试剂,但是其存在造成步骤繁琐、污染样品和信号不稳定等缺点。基于此,利用GOD自身电子转移产生的氧化还原峰为电化学探针,来实现直接对CA 15-3抗原分子的定量检测,设计了一种真止无试剂无媒介型的免疫传感器。将碳纳米管用有机硅纳米球分散,制得壳聚糖纳米球包覆的功能化碳纳米管复合材料(CNTs-OrgSi@CS)为电极基底。同时,采用铂纳米簇(Pt NCs)来增加界面的导电性,由于其粒径和酶的大小很接近,更能接近酶的氧化还原中心,极大地提高了酶与电极之间的电子转移速率,增加响应的灵敏度。在Pt NCs/CNTs-OrgSi@CS纳米复合膜的协同作用下,GOD产生了一对对称性好、可逆性强、电流值大的氧化还原峰。然后,将CA 15-3抗体通过Pt NCs连接在GOD的表面.我们以这对氧化还原峰为跟踪电信号,来考察不同浓度的抗原捕获上去的电流值的变化,进而米实现对抗原的定量检测。另外,使用GOD为封闭剂,封闭免疫电极上的非特异性吸附位点,并同时利用GOD的生物催化放大作用放大响应电流信号,进一步提高免疫传感器灵敏度的新方法。经实验研究证明,该方法操作简单,切实可行,与常规的方法相比,该免疫生物传感器更灵敏、稳定、清洁、绿色。5.基于"Click”化学和磁性金纳米壳协同作用的超灵敏葡萄糖生物传感器的应用研究致力于酶固定方法的研究。"click"化学,其反应具有较高立体选择效和高控制性,常用于有机或医药合成试验中。本研究中将‘’click"化学作为酶的固载技术,并用于传感器的制备过程。为了进一步提高传感器的灵敏度,合成了磁性金纳米壳(Au@Fe3O4)为修饰电极的增敏材料,并嫁接在新型的碳材料石墨烯表面,然后和离子液体-壳聚糖溶液混合(ILs-CS-Gra-Au@Fe3O4)。借助“click"化学,在Cu(Ⅰ)的催化作用下,将叠氮化的ILs-CS-Gra-Au@Fe3O4和炔基化的GOD进行"click"反应,构建了高灵敏的葡萄糖生物传感器。通过“click”化学,实现了对酶蛋白的成功固载。此方法反应条件温和,能够很好的保持酶的生物活性并增加酶的固载量和牢固性,成功的实现了酶自身的直接电子转移过程。较之传统的固定方法而言,该传感器的稳定性、灵敏度和寿命有了显著的提高,因此,通过这种方法所构建的传感器在生物技术领域中具有潜在的应用优势。更多还原

【Abstract】 Electrochemically biosensor is a inter-discipline based on electrochemical analysis and biological technology, which consist of a biological recognition element (such as enzyme, antibody, etc) in intimate contact with a suitable transducer. In the fabricated process of biosensors, it is very important to choice the immobilized methods and materials of biomolecules. Therefore, a series of research focuses on the preparation of multi-functionalized nanomaterials, the construction of the electrochemical sensing interface and biomolecules immobilization. The detaij contents are as follows:1. Amine-terminated organosilica nanosphere functionalized prussian blue for the electrochemical detection of glucose.The mediated-enzyme biosensors have attracted much attention. However, there are still several challenges concerning immobilization of electron-shuttling mediators on the electrode surface since low molecular weight soluble mediators can easily diffuse away from the electrode surface into the bulk solution when the biosensor is used continuously, which would lead to significant signal loss and greatly affect the performance and lifetime of the biosensor. To circumvent this problem, the monomeric mediators are linked directly with the organic polymer or proteins system. First of all, we adopted a facile and bottom-up method to synthesize organosilica nanospheres. which contain rich amino-group. Then prussian blue was prepared in organosilica nanospheres suspension, which is donated as amine-terminated organosilica nanosphere functionalized prussian blue (OSiFPB). The OSiFPB compound could not only effectively prevent the leakage of the PB mediator during measurements, but also easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore. with the negatively charged surface of OSiFPB. this film could be used as an interface to adsorb the positively charged gold nanoparticles (PGNs). which were prepared in organic solvents at relatively high concentrations with improved monodispersity compared to those prepared in aqueous solution. The presence of PGNs provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance. Finally, with glucose oxidase as a model biomolecular, the proposed sensor showed rapid and highly sensitive amperometric response to glucose and this immobilization approach effectively improved the stability of the electron transfer mediator. This work would be promising for construction of biosensor and bioelectronic devices.2. Determination of glucose using pseud o-bienzyme channeling based on sugar-lectin biospecific interactions in a novel organic-inorganic composite matrix.It is effective method to prevent the leakage of the mediate through chemical linking, but the step is complexity. A porous organic conducting polymer containing abundant amino groups (PTC-NH2) was coated on the surface of PB film, which could not only avoid the leakage of the PB efficiently, but also provide an interface of abundant amino-groups to further modifiability. At the same time, gold/platinum hybrid nanostructure supported on silica nanofibers (GP-SNFs) were synthesize to enhance the sensitivity. Finally, the glucose oxidase (GOD) was attached on the electrode surface through the strong biological affinity links between Con A and sugar chains intrinsically. The proposed biosensor possessed high sensitivity and stability for the detection of glucose. Several advantages of the obtained sensors should be highlighted. Firstly, it was found that this resulting nanomaterial exhibited a high electrocatalytic activity toward glucose, and a necessary pathway of electron transfer. Secondly, with the use of porous PTC-NH2, overcame the defect of mediator leakage successfully, which enhanced the stability and sensitivity of the bioassay. Thirdly, the pseudo-bienzymatic sensors could be operated at low working potentials, and interferences from other electroactive compounds are minimal in biological samples. The proposed method can be extended to a large group of enzymes to provide great promise platforms for biosensor and bioelectronics applications.3. Study of the biosensor based on Pt nanoparticles supported on carbon nanotubes and sugar-lectin biospecific interactions for the determination of glucose.The third generation biosensor is based on the direct electrochemistry of the enzyme without the introduction of the redox-mediator, so it is unnecessary to consider that its leakage caused pollution problem of the sample. However, the redox center of the majority of the enzymes is buried inside the protein matrix, which results in a slow process of direct electron transfer between the electrode and enzyme. To facilitate superior electron transport, the nanomaterial may be selectively placed in an optimum position between the redox center and the enzyme periphery. In this concept, Pt nanoparticles supported on carbon nanotubes (Ptnano-CNTs) were prepared in the presence of carbon nanotubes (CNTs), as "molecular wires" to enhance the electronic transfer. Thereafter, concanavalin A (Con A) was adsorbed onto the precursor film by the electrostatic force between positively charged chitosan and the negatively charged Con A. Finally, the multilayers of Con A/GOD films were prepared based on biospecific affinity of Con A and GOD via layer-by-layer (LBL) self-assembly technique. The results shows the proposed biosensor provides a remarkable synergistic action of the response current toward glucose. It also exhibits high sensitivity, low detection limit, excellent reproducibility. good and anti-interfering capability. Thus, the electrode has good application potential in glucose detection.4. Reagentless amperometric cancer antigen 15-3 immunosensor based on enzyme-mediated direct electrochemistry.A novel strategy was proposed for the construction of reagentless and mediatorless immunosensors based on the direct electrochemistry of glucose oxidase (GOD). Firstly, a composite material containing carbon nanotubes (CNTs) and core-shell organosilica@chitosan nanospheres was successfully prepared and cast on the glassy carbon electrode surface directly. Then. Pt nanoclusters (Pt NCs) as an electron relay were deposited on it to form the interface of biocompatibility and huge surface free energy for the adsorption of the first GOD layer. Subsequently, the second Pt NCs layer was deposited on the surface of GOD to capture CA 15-3 antibodies (anti-CA 15-3). Finally. GOD. as a blocking reagent instead of bovine serum albumin, was employed to block the possible remaining active sites of the Pt NCs and avoid the nonspecific adsorption. The immunosensor with the double layer GOD membranes as tracer performed excellent biocompatible and avoided the pollution of mediator molecules, as well as amplify the response of the antigen-antibody reaction was proposed. Such a detection of immunointeraction provided a new promising platform for clinical immunoassay.5. A new immobilized glucose oxidase approach on magnetic gold nanoshell surface via click chemistry and its direct electrochemistry.Immobilizing biomolecular on the sensing electrode surface is one of the most important points to be considered in the construction of amperometric biosensors. The reported methods have exhibited their own advantage and disadvantage. In this concept, we devoted to the method study of enzyme immobilization. In this study, a novel glucose biosensor was developed based on click reaction. Firstly, alkynyl-terminated multifunctional magnetic gold nanoparticles (alkynyl-Mag-GNSs) consisting of gold nanoshells, carboxymethylated chitosan inner layer, and magnetic core were synthesized, then were grafted on the synergistic beneficial characteristics interface of carboxyl-modified graphene oxide, ionic liquids and chitosan modified electrodes. Finally, azido-glucose oxidase (azido-GOD) was covalent immobilized onto the formed films by click reaction. Compared to the conventional methods, the biosensor exhibited more sensitivity, a lower detection limit, and a wider linear range to glucose detection. Overall, this work provided a new avenue for electrochemical investigation of enzyme immobilization.更多还原