纳米材料在电化学生物传感器中的应用研究
摘要
纳米材料由于其特殊的结构而产生了一系列独特的物理、化学性质。将纳米材料作为一种新型的生物传感介质应用于生物化学领域吸引了众多研究者的兴趣。纳米材料修饰的电化学生物传感器的研制是纳米技术与生命科学的交叉,它可以在纳米尺度空间从分子层次上研究目标分子的结构与功能的关系,解决纳米技术在生物医学领域以及环境检测领域中应用的基础问题,发展新技术和新方法。本论文将生物化学、纳米技术和电分析化学理论和方法有机地结合起来,致力于构建新型纳米材料修饰的电化学传感器,本论文共分五个部分。
Ⅰ.合成了一种用于处理废水中重金属的新型硅纳米多孔吸附剂,同时以铜离子(Cu2+)作为废水处理中重金属离子的模板,验证了这种新型材料在废水处理过程中的的有效性。以蔗糖和聚乙二醇为印迹分子,将含有致孔剂、壳聚糖和无机硅烷的混合溶液涂覆于硅胶表面,通过室温下壳聚糖与γ-环氧丙氧丙基三甲氧基硅烷的共价交联、有机-无机杂化制备得到以硅胶为支持物、具有表面多孔结构的壳聚糖基质。电解废水经过柱层析后,Cu2+的浓度得到了很好的修正。制备的印迹吸附材料就有很好的再生利用性,制备方法简单,制备过程中没有使用有机溶剂,成本低,效果好,稳定性强,在生物吸附领域就有很好的应用前景。
Ⅱ.以半胱胺(cysteamine)修饰的金电极为基础电极,以戊二醛(glutaraldehyde)为交联剂,利用希夫碱(Schiff base)反应,将表面经过氨基修饰的纳米二氧化硅粒子(SiO2 nanoparticles)固定在电极表面上,制成DNA纳米生物传感器,用于DNA片段的检测。
Ⅲ.将胶体纳米金和羧基功能化的CdS纳米粒子固定在金电极表面,制成一种新型的电化学生物传感器。胶体纳米金和CdS纳米粒子在电极表面上良好的导电性和生物兼容性,为DNA的固定提供了较大的比表面积和充足的结合位点。在整个的电极组装过程中,电化学循环伏安法(CV)和电化学交流阻抗法(EIS)被用于表征每一步的组装过程。以邻菲咯啉钴[Co(phen)2(Cl)(H2O)]Cl·2H2O作为指示剂,运用微分脉冲伏安技术(DPV)考察了DNA的固定和杂交过程。本文所制备的新型传感器在检测DNA过程中显示了良好的灵敏度、选择性、重现性和稳定性。
Ⅳ.将金纳米粒子(Au NPs)和硫化铅纳米粒子(PbS NPs)固定在修饰磁球的表面上,利用了生物条形码和金纳米粒子信号的放大作用,制备了一种新型灵敏的DNA电化学生物传感器。通过静电作用将聚丙烯胺氢氯化物/聚苯乙烯磺酸钠/聚丙烯胺氢氯化物/聚丙烯酸(PAH/PSS/PAH/PAA)在磁球表面依次进行了修饰,使磁球表面具有更多的自由羧基来固定氨基修饰的捕获DNA,该传感器首先将氨基功能化的捕获DNA探针结合到磁球上,然后再与靶DNA的一端杂交,耙DNA的另一端和标记Au纳米粒子的DNA探针杂交。Au纳米粒子上DNA的固定采用了生物条形码技术,将PbS纳米粒子作为检测靶DNA的一种标记物,通过DNA链固定在Au纳米粒子,来提高该生物传感器的灵敏度。PbS纳米粒子的电化学溶出法测定铅,通过阳极溶出伏安技术(ASV)预富集铅离子的过程进一步提高了传感器的灵敏度。研究结果表明,该方法制备的生物传感器具有很好的选择性和灵敏度。
Ⅴ.利用多壁碳纳米管和室温离子液体(RTIL),N-丁基吡啶-六氯代磷酸盐(BPPF6)的混合纳米材料构建了一种新型微过氧化物酶(MP-11)生物催化的用于检测过氧化氢的电化学生物传感器。传感器电化学信号响应快,灵敏度高,稳定性好,具有很好的生物活性和选择性。
Nanomaterials have special structure, which results in serials of interesting chemical and physical properties. In our work, the nanomaterials are used to construct the electrochemical biosensors by means of the combination of biochemistry and electrochemical methods and we aim to develop new types of biosensors based on nanomaterials for the purpose of improving the long-term stability and the higher sensitivity of biosensors. The details are summarized as follows:
(1) A new porous sorbent for waste water treatment of meta lions was synthesized by covalent grafting of molecularly imprinted organic-inorganic hybrid on silica gel. With sucrose and polyethylene glycol 4000 (PEG 4000) being synergic imprinting molecules, covalent surface coating on silica gel was achieved by using polysaccharide-incorporated sol–gel process starting from the functional biopolymer, chitosan and an inorganic epoxy-precursor, gamma-glycid oxypropyltrimethoxy siloxane (GPTMS) at room temperature. The prepared porous sorbent was characterized by using simultaneous thermogravimetry and differential scanning calorimeter (TG/DSC), scanning electronmicroscopy (SEM), nitrogen adsorption porosimetry measurement and X-ray diffraction (XRD). Copper ion, Cu2+, was chosen as the model metal ion to evaluate the effectiveness of the new biosorbent in wastewater treatment. The influence of epoxy-siloxane dose, buffer pH and co-existed ions on Cu2+ adsorption was assessed through batch experiments. The imprinted composite sorbent offered a fast kinetics for the adsorption of Cu2+. The uptake capacity of the sorbent imprinted by two pore-building components was higher than those imprinted with only a single component. The dynamic adsorption in column underwent a good elimination of Cu2+ in treating electric plating wastewater. The prepared composite sorbent exhibited high reusability. Easy preparation of the described porous composite sorbent, absence of organic solvents, cost-effectiveness and high stability make this approach attractive in biosorption.
(2) A feasible approach modified nanoSiO2 particles on the Au electrode surface to construct a novel DNA biosensor is described. On the basis of Schiff base reaction between the -CHO groups and -NH2 groups, cysteamine and glutaraldehyde was used as covelent attachment cross-linkers. The covalent attachment processes were followed and confirmed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridized dsDNA biosensor was studied by Different Pulse Voltammetry (DPV). From the analysis of voltammetric signals, the linear response range of the biosensor is 6×10-8 M ~ 8×10-10 M, the detection limit is 3×10-10 M. In addition, the sensitivity biosensor is easily manipulated and exhibited good stability and long-term life.
(3) In this article, colloidal gold nanoparticles (Au NPs) and carboxyl group-functionalized CdS Nanoparticles (CdS NPs) were immobilized on the Au electrode surface to fabricate a novel electrochemical DNA biosensor. Both Au NPs and CdS NPs, well known to be good biocompatibile and conductive materials, could provide larger surface area and sufficient amount of binding points for DNA immobilization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments were performed to follow the whole electrode fabrication process. DNA immobilization and hybridization were characterized with differential pulse voltammetry (DPV) by using [Co(phen)2(Cl)(H2O)]Cl?2H2O as an electrochemical hybridization indicator. With this approach, the target DNA could be quantified at a linear range from 2.0×10-10 to 1.0×10-8 M, with a detection limit of 2.0×10-11 M by 3σ. In addition, the biosensor exhibited a good repeatability and stability for the determination of DNA sequences.
(4) A novel and sensitive sandwich electrochemical biosensor based on the amplification of magnetic microbeads and Au nanoparticles (NPs) modified with bio bar code and PbS nanoparticals was constructed in the present work. In this method, the magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The amino-functionalized capture DNA on the surface of magnetic microbeads hybridized with one end of target DNA, the other end of which was hybridized with signal DNA probe labelled with Au NPs on the terminus. The Au NPs was modified with bio bar code and the PbS NPs was used as a marker for identifying the target oligoncleotide. The modification of magnetic microbeads could immobilize more amino-group terminal capture DNA, and the bio bar code could increase the amount of Au NPs that combined with the target DNA. The detection of lead ions performed by anodic stripping voltammetry (ASV) technology further improved the sensitivity of the biosensor. As a result, the present DNA biosensor showed good selectivity and sensitivity by the combined amplification. Under the optimum conditions, the linear relationship with the concentration of the target DNA was ranging from 2.0×10?14 M to 1.0×10?12 M and a detection limit as low as 5.0×10?15 M were obtained.
(5) A novel nanocomposite material of muti-walled carbon nanotubes (MWCNTs) and room-temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF6) was explored and was used to construct a novel Microperoxidase-11 (MP-11) biosensor for the determination of H2O2. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H2O2 could be detected in a linear calibration range of 0.5×10-7 ~ 7.0×10-7 M with a correlation coefficient of 0.9949 (n = 9) and a detection limit of 3.8×10-9 M at 3σ. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, good bioactivity and selectivity.
Ⅰ.合成了一种用于处理废水中重金属的新型硅纳米多孔吸附剂,同时以铜离子(Cu2+)作为废水处理中重金属离子的模板,验证了这种新型材料在废水处理过程中的的有效性。以蔗糖和聚乙二醇为印迹分子,将含有致孔剂、壳聚糖和无机硅烷的混合溶液涂覆于硅胶表面,通过室温下壳聚糖与γ-环氧丙氧丙基三甲氧基硅烷的共价交联、有机-无机杂化制备得到以硅胶为支持物、具有表面多孔结构的壳聚糖基质。电解废水经过柱层析后,Cu2+的浓度得到了很好的修正。制备的印迹吸附材料就有很好的再生利用性,制备方法简单,制备过程中没有使用有机溶剂,成本低,效果好,稳定性强,在生物吸附领域就有很好的应用前景。
Ⅱ.以半胱胺(cysteamine)修饰的金电极为基础电极,以戊二醛(glutaraldehyde)为交联剂,利用希夫碱(Schiff base)反应,将表面经过氨基修饰的纳米二氧化硅粒子(SiO2 nanoparticles)固定在电极表面上,制成DNA纳米生物传感器,用于DNA片段的检测。
Ⅲ.将胶体纳米金和羧基功能化的CdS纳米粒子固定在金电极表面,制成一种新型的电化学生物传感器。胶体纳米金和CdS纳米粒子在电极表面上良好的导电性和生物兼容性,为DNA的固定提供了较大的比表面积和充足的结合位点。在整个的电极组装过程中,电化学循环伏安法(CV)和电化学交流阻抗法(EIS)被用于表征每一步的组装过程。以邻菲咯啉钴[Co(phen)2(Cl)(H2O)]Cl·2H2O作为指示剂,运用微分脉冲伏安技术(DPV)考察了DNA的固定和杂交过程。本文所制备的新型传感器在检测DNA过程中显示了良好的灵敏度、选择性、重现性和稳定性。
Ⅳ.将金纳米粒子(Au NPs)和硫化铅纳米粒子(PbS NPs)固定在修饰磁球的表面上,利用了生物条形码和金纳米粒子信号的放大作用,制备了一种新型灵敏的DNA电化学生物传感器。通过静电作用将聚丙烯胺氢氯化物/聚苯乙烯磺酸钠/聚丙烯胺氢氯化物/聚丙烯酸(PAH/PSS/PAH/PAA)在磁球表面依次进行了修饰,使磁球表面具有更多的自由羧基来固定氨基修饰的捕获DNA,该传感器首先将氨基功能化的捕获DNA探针结合到磁球上,然后再与靶DNA的一端杂交,耙DNA的另一端和标记Au纳米粒子的DNA探针杂交。Au纳米粒子上DNA的固定采用了生物条形码技术,将PbS纳米粒子作为检测靶DNA的一种标记物,通过DNA链固定在Au纳米粒子,来提高该生物传感器的灵敏度。PbS纳米粒子的电化学溶出法测定铅,通过阳极溶出伏安技术(ASV)预富集铅离子的过程进一步提高了传感器的灵敏度。研究结果表明,该方法制备的生物传感器具有很好的选择性和灵敏度。
Ⅴ.利用多壁碳纳米管和室温离子液体(RTIL),N-丁基吡啶-六氯代磷酸盐(BPPF6)的混合纳米材料构建了一种新型微过氧化物酶(MP-11)生物催化的用于检测过氧化氢的电化学生物传感器。传感器电化学信号响应快,灵敏度高,稳定性好,具有很好的生物活性和选择性。
Nanomaterials have special structure, which results in serials of interesting chemical and physical properties. In our work, the nanomaterials are used to construct the electrochemical biosensors by means of the combination of biochemistry and electrochemical methods and we aim to develop new types of biosensors based on nanomaterials for the purpose of improving the long-term stability and the higher sensitivity of biosensors. The details are summarized as follows:
(1) A new porous sorbent for waste water treatment of meta lions was synthesized by covalent grafting of molecularly imprinted organic-inorganic hybrid on silica gel. With sucrose and polyethylene glycol 4000 (PEG 4000) being synergic imprinting molecules, covalent surface coating on silica gel was achieved by using polysaccharide-incorporated sol–gel process starting from the functional biopolymer, chitosan and an inorganic epoxy-precursor, gamma-glycid oxypropyltrimethoxy siloxane (GPTMS) at room temperature. The prepared porous sorbent was characterized by using simultaneous thermogravimetry and differential scanning calorimeter (TG/DSC), scanning electronmicroscopy (SEM), nitrogen adsorption porosimetry measurement and X-ray diffraction (XRD). Copper ion, Cu2+, was chosen as the model metal ion to evaluate the effectiveness of the new biosorbent in wastewater treatment. The influence of epoxy-siloxane dose, buffer pH and co-existed ions on Cu2+ adsorption was assessed through batch experiments. The imprinted composite sorbent offered a fast kinetics for the adsorption of Cu2+. The uptake capacity of the sorbent imprinted by two pore-building components was higher than those imprinted with only a single component. The dynamic adsorption in column underwent a good elimination of Cu2+ in treating electric plating wastewater. The prepared composite sorbent exhibited high reusability. Easy preparation of the described porous composite sorbent, absence of organic solvents, cost-effectiveness and high stability make this approach attractive in biosorption.
(2) A feasible approach modified nanoSiO2 particles on the Au electrode surface to construct a novel DNA biosensor is described. On the basis of Schiff base reaction between the -CHO groups and -NH2 groups, cysteamine and glutaraldehyde was used as covelent attachment cross-linkers. The covalent attachment processes were followed and confirmed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridized dsDNA biosensor was studied by Different Pulse Voltammetry (DPV). From the analysis of voltammetric signals, the linear response range of the biosensor is 6×10-8 M ~ 8×10-10 M, the detection limit is 3×10-10 M. In addition, the sensitivity biosensor is easily manipulated and exhibited good stability and long-term life.
(3) In this article, colloidal gold nanoparticles (Au NPs) and carboxyl group-functionalized CdS Nanoparticles (CdS NPs) were immobilized on the Au electrode surface to fabricate a novel electrochemical DNA biosensor. Both Au NPs and CdS NPs, well known to be good biocompatibile and conductive materials, could provide larger surface area and sufficient amount of binding points for DNA immobilization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments were performed to follow the whole electrode fabrication process. DNA immobilization and hybridization were characterized with differential pulse voltammetry (DPV) by using [Co(phen)2(Cl)(H2O)]Cl?2H2O as an electrochemical hybridization indicator. With this approach, the target DNA could be quantified at a linear range from 2.0×10-10 to 1.0×10-8 M, with a detection limit of 2.0×10-11 M by 3σ. In addition, the biosensor exhibited a good repeatability and stability for the determination of DNA sequences.
(4) A novel and sensitive sandwich electrochemical biosensor based on the amplification of magnetic microbeads and Au nanoparticles (NPs) modified with bio bar code and PbS nanoparticals was constructed in the present work. In this method, the magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The amino-functionalized capture DNA on the surface of magnetic microbeads hybridized with one end of target DNA, the other end of which was hybridized with signal DNA probe labelled with Au NPs on the terminus. The Au NPs was modified with bio bar code and the PbS NPs was used as a marker for identifying the target oligoncleotide. The modification of magnetic microbeads could immobilize more amino-group terminal capture DNA, and the bio bar code could increase the amount of Au NPs that combined with the target DNA. The detection of lead ions performed by anodic stripping voltammetry (ASV) technology further improved the sensitivity of the biosensor. As a result, the present DNA biosensor showed good selectivity and sensitivity by the combined amplification. Under the optimum conditions, the linear relationship with the concentration of the target DNA was ranging from 2.0×10?14 M to 1.0×10?12 M and a detection limit as low as 5.0×10?15 M were obtained.
(5) A novel nanocomposite material of muti-walled carbon nanotubes (MWCNTs) and room-temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF6) was explored and was used to construct a novel Microperoxidase-11 (MP-11) biosensor for the determination of H2O2. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H2O2 could be detected in a linear calibration range of 0.5×10-7 ~ 7.0×10-7 M with a correlation coefficient of 0.9949 (n = 9) and a detection limit of 3.8×10-9 M at 3σ. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, good bioactivity and selectivity.
引文
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