基于纳米复合材料的电化学传感器检测堆肥中酚类物质及木质素功能基因研究
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摘要
堆肥法是目前有机固体废物资源化的一种有效处理方法。在堆肥系统中,各种有机固体废物中含有不少酚类污染物,微生物降解多环芳烃的过程中还会产生对苯二酚和邻苯二酚两种中间产物。木质素是一种堆肥过程中大量存在且难以降解的芳香族化合物,锰过氧化物酶(MnP)和纤维二糖脱氢酶(CDH)在微生物降解木质素的过程中其中起了关键性作用。电化学生物传感器以其操作简单、选响应快速、选择性好、灵敏度高、成本低廉等特点在环境、生物、医药及食品工程等领域受到广泛关注。将纳米材料独特的导电性、催化活性及生物相容性与生物分子高度的催化能力和特异性识别能力结合起来,构建功能化的复合电极界面,为堆肥环境中污染物及其功能基因的分析检测提供了一个新的技术平台,对堆肥过程控制及环境监测具有十分重要的意义。
本文以堆肥环境体系为研究对象,探讨了基于碳纳米管、石墨烯、金、铂等纳米复合材料的制备及功能化方法,合成并表征了多种水溶性纳米材料,研究了它们的电化学性质并将其应用于生物传感界面的修饰。通过酶、核酸探针等生物分子在电极上的固定,构建了一系列基于水溶性纳米复合材料修饰的生物传感器,用于堆肥环境中对苯二酚、邻苯二酚及锰过氧化物酶和纤维二糖脱氢酶功能基因的高灵敏检测。
利用硼氢化钠化学还原及L-半胱氨酸共价结合的方法制备出一种水溶性石墨烯-纳米铂复合材料,构建出一种新型电化学传感器用于堆肥系统中的对苯二酚和邻苯二酚的同时检测。该纳米复合物具有良好的水溶性和生物相容性,可明显增大电极的有效比表面积、导电性能及催化活性。采用差分脉冲伏安法将其用于堆肥浸出液中对苯二酚和邻苯二酚的同时检测,检测下限分别为2.0×10-8M和1.0×10-8M。该传感器特异性好,稳定性强,灵敏度高,且不易受系统中其他物质的干扰,能快速实现酚类污染物的实时在线监测。
制备了一种基于水溶性碳纳米管/L-赖氨酸/纳米金共同修饰的漆酶生物传感器用于对苯二酚和邻苯二酚两种物质的高灵敏测定。将L-赖氨酸通过电沉积的方法聚合在纳米金修饰后的玻碳电极上,滴涂刚果红功能化的碳纳米管水溶液,利用戊二醛交联的方法将漆酶固定。此固定化方法能很好地保持漆酶的生物活性,使得电极的催化能力大大提高,碳纳米管及纳米金均能明显提高电极的导电性、比表面积等电化学特性。该传感器制作简单,响应快速,灵敏度高,选择性好,检测下限达到了1.0×10-8M和1.0×10-9M,可用于各种环境中酚类污染物的实时监测。
采用聚合酶链式反应扩增及限制性内切酶技术与基因传感技术相结合,研制出种基于辣根过氧化物酶和漆酶共同标记的生物传感器,用于锰过氧化物酶和纤维二糖脱氢酶编码基因的同时检测。利用目标链与巯基修饰的捕获探针及双酶标记的响应探针间夹心式杂交将其固定在电极表面,通过双酶信号放大来实现目标基因的快速检测。在最优实验条件下,采用计时电流法得到的还原电流与MnP和CDH两种目标基因浓度的常用对数值成良好的线性相关性,检测下限分别为6.2×10-12M和3.0×10-11。该生物传感器选择性好,精密度高,稳定性和可重复性好,为污染物生物降解过程中微生物功能基因的协同作用机理分析提供了很好的技术支持。
研制了一种基于纳米金/多壁碳纳米管/1,5-萘二胺复合膜修饰的生物传感器用于纤维二糖脱氢酶功能基因的快速检测。1,5-萘二胺导电聚合膜带有大量自由氨基,有利于羧基化碳纳米管在电极表面的固定。经刚果红共价修饰后的碳纳米管导电性能好,水溶性也大大增强,为纳米金提供了一定的结合位点,增强了DNA分子在电极表面的固定量,提高了生物催化反应活性。将复合膜修饰后的电极通过扫描电镜、循环伏安法及交流阻抗法进行了表征。该传感器的灵敏度高,特异性好,操作简便,成本低,检测下限达到了1.2×10-16M,可应用于生命科学、临床医学以及环境污染控制系统中实时在线监测。
开发出一种基于纳米金/多壁碳纳米管/石墨烯共同修饰的基因生物传感器用于锰过氧化物酶功能基因的超灵敏检测。L-半胱氨酸和刚果红分别对石墨烯和碳纳米管进行功能化修饰,得到一种水溶性较好、导电性能优越、生物相容性好的纳米复合材料。为纳米金的电化学沉积提供了良好的载体条件,明显增大了电极的比表面积,同时也提高了DNA分子在电极表面的固载量。通过扫描电镜、透射电镜、红外光谱扫描等方法对材料的形态和结构进行了表征。在最优实验条件下,采用计时电流法得到的还原电流与MnP目标基因浓度的常用对数值呈良好的线性相关性,检测下限达到了2.0×10-17M。该基因传感器的灵敏度极高,选择性和稳定性好,制备方法简单,成本低,可对环境中其他特定核苷酸序列进行快速灵敏检测。
Composting is an effective disposal method for resource utilization of organic solid wastes. Phenol compounds are common pollutants in organic solid wastes composting. Hydroquinone and catechol are the two intermediate products during biodegradation of polycyclic aromatic hydrocarbons. Lignin is one kind of aromatic polymer in composting and is extremely recalcitrant in an aerobic oxidative process. Both manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) play important roles in lignin mineralization. Electrochemical biosensors have received particular attention due to their easy operation, fast response, good selectivity, high sensitivity and low cost in environment, biology, medicine, and food stuff. The incorporation of the highly selective catalytic and recognition properties of biomolecules with the unique conductivity, catalytic activity and biocompatibility of nanomaterials provide a new technical platform for the analysis and measurement of contaminants and functional genes in composting, and are also of great importance for compost process control and environmental monitoring.
This dissertation took the composting system for example and discussed the preparation and functionalization of carbon nanotubes, graphene, gold and platinum nanocomposite. Several water-soluble nanocomposites were synthesised and characterized and their electrochemical properties were also studied. A series of water-soluble nanocomposite modified electrodes were constructed by the immobilization of enzyme and nucleic acid probes for highly sensitive detection of hydroquinone, catechol, MnP and CDH functional genes in composting.
Water soluble graphene-platinum nanocomposite was prepared by the combination of chemical reduction using sodium borohydride and covalent modification with L-cysteine. A new electrochemical sensor based on the nanocomposite was developed for simultaneous detection of hydroquinone and catechol in composting. The good solubility and biocompatibility of nanocomposite could greatly improve the effective specific surface area, conductivity and catalytic activity of the electrode. The concentrations of hydroquinone and catechol in the compost extract were determined by differential pulse voltammetry, which showed that the detection limits of the two phenol compounds were2.0×10-8M and1.0×10-8M, respectively. This sensor exhibited good selectivity, stability, sensitivity and excellent capacity of resisting disturbance and it would be an effective approach for rapid and on-line detection of phenol compounds.
A laccase biosensor was constructed based on the water-soluble carbon nanotubes/L-lysine/gold nanoparticles for highly sensitive detection of hydroquinone and catechol. After modification of gold nanoparticles, the L-lysine was decorated on the glassy carbon electrode by the electrochemical polymerization. Carbon nanotubes was functionalized by Congo red and the solution was cast on the modified electrode. Then the laccase was immobilized on the electrode using covalently crosslinking with glutaraldehyde. The immobilization of laccase showed good biological activity which significantly enhanced the catalytic capability of electrode. The conductivity and surface area was also improved by the synergy of carbon nanotubes and gold nanoparticles. This laccase biosensor was simple and response fast with high sensitivity and selectivity. The detection limits of hydroquinone and catechol were1.0×10-8M and1.0×10-9M, respectively. This sensor can be used for the real-time determination of all kinds of phenol compounds.
An electrochemical DNA sensor based on horseradish peroxidase (HRP) and laccase (LAC) labels was developed by the incorporation of polymerase chain reaction and restriction endonuclease digestion with sensor technology for simultaneous detection of manganese peroxidase and cellobiose dehydrogenase functional genes. The target sequences were immobilized by hybridization with both capture probes modified with thiol and detection probes labeled with HRP and LAC, which were applied for enzyme-amplified amperometric measurement. Under the optimal conditions, the amperometric current responses by chronoamperometry were linearly related to the common logarithm of two target nucleic acids concentrations and the detection limits were6.2×10-12M and3.0×10-11M, respectively. The DNA biosensor exhibited good selectivity, precision, stability and reproducibility. The DNA sensor presented here is a promising step to study the mutual relationship between two functional genes for environmental pollutant biodegradation.
An electrochemical sensor based on gold nanoparticles (GNPs)/multiwalled carbon nanotubes (MWCNTs)/poly (1,5-naphthalenediamine) films modified glassy carbon electrode for rapid detection of cellobiose dehydrogenase gene was fabricated. The monomer of1,5-naphthalenediamine was electropolymerized on the GCE surface with abundant free amino groups which enhanced the stability of MWCNTs modified electrode. Congo red-functionalized MWCNTs possess excellent conductivity as well as high solubility in water. It created a large number of binding sites for electrodeposition of GNPs and improved the DNA immobilization amount and biological catalytic activity. Scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry were used to investigate the film assembly and DNA hybridization processes. The electrochemical biosensor exhibited high sensitivity, selectivity, easy operation and low cost, with the detection limit of1.2×10-16M. It offered a possible method for "on-the-spot" monitoring in life sciences, clinical medicine and environmental pollution control systems.
A sensitive electrochemical DNA sensor based on gold nanoparticles/multiwalled carbon nanotubes/graphene composite films modified glassy carbon electrode was fabricated for the determination of manganese peroxidase gene. After functionalization with Congo red (CR) and L-cysteine (Cys), the MWCNT-CR/GR-Cys nanocomposite exhibited high solubility in water, excellent conductivity as well as good biocompatibility, which created a large number of binding sites for electrodeposition of GNPs, greatly enhanced the surface area and DNA immobilization amount. The characteristics of the modified electrode were investigated by transmission electron microscopy, scanning electron microscopy and fouriertrans-form infrared spectroscopy. Under the optimal conditions, the current improvement catalyzed by HRP was linearly related to the common logarithm of MnP target sequences in the detection limit of2.0×10-17M. In addition, this DNA sensor exhibited high sensitivity, good selectivity and stability. The biosensor was easy to prepare and cost low, which could be further used for the rapid and sensitive determination of other specific ssDNA sequence in environment.
本文以堆肥环境体系为研究对象,探讨了基于碳纳米管、石墨烯、金、铂等纳米复合材料的制备及功能化方法,合成并表征了多种水溶性纳米材料,研究了它们的电化学性质并将其应用于生物传感界面的修饰。通过酶、核酸探针等生物分子在电极上的固定,构建了一系列基于水溶性纳米复合材料修饰的生物传感器,用于堆肥环境中对苯二酚、邻苯二酚及锰过氧化物酶和纤维二糖脱氢酶功能基因的高灵敏检测。
利用硼氢化钠化学还原及L-半胱氨酸共价结合的方法制备出一种水溶性石墨烯-纳米铂复合材料,构建出一种新型电化学传感器用于堆肥系统中的对苯二酚和邻苯二酚的同时检测。该纳米复合物具有良好的水溶性和生物相容性,可明显增大电极的有效比表面积、导电性能及催化活性。采用差分脉冲伏安法将其用于堆肥浸出液中对苯二酚和邻苯二酚的同时检测,检测下限分别为2.0×10-8M和1.0×10-8M。该传感器特异性好,稳定性强,灵敏度高,且不易受系统中其他物质的干扰,能快速实现酚类污染物的实时在线监测。
制备了一种基于水溶性碳纳米管/L-赖氨酸/纳米金共同修饰的漆酶生物传感器用于对苯二酚和邻苯二酚两种物质的高灵敏测定。将L-赖氨酸通过电沉积的方法聚合在纳米金修饰后的玻碳电极上,滴涂刚果红功能化的碳纳米管水溶液,利用戊二醛交联的方法将漆酶固定。此固定化方法能很好地保持漆酶的生物活性,使得电极的催化能力大大提高,碳纳米管及纳米金均能明显提高电极的导电性、比表面积等电化学特性。该传感器制作简单,响应快速,灵敏度高,选择性好,检测下限达到了1.0×10-8M和1.0×10-9M,可用于各种环境中酚类污染物的实时监测。
采用聚合酶链式反应扩增及限制性内切酶技术与基因传感技术相结合,研制出种基于辣根过氧化物酶和漆酶共同标记的生物传感器,用于锰过氧化物酶和纤维二糖脱氢酶编码基因的同时检测。利用目标链与巯基修饰的捕获探针及双酶标记的响应探针间夹心式杂交将其固定在电极表面,通过双酶信号放大来实现目标基因的快速检测。在最优实验条件下,采用计时电流法得到的还原电流与MnP和CDH两种目标基因浓度的常用对数值成良好的线性相关性,检测下限分别为6.2×10-12M和3.0×10-11。该生物传感器选择性好,精密度高,稳定性和可重复性好,为污染物生物降解过程中微生物功能基因的协同作用机理分析提供了很好的技术支持。
研制了一种基于纳米金/多壁碳纳米管/1,5-萘二胺复合膜修饰的生物传感器用于纤维二糖脱氢酶功能基因的快速检测。1,5-萘二胺导电聚合膜带有大量自由氨基,有利于羧基化碳纳米管在电极表面的固定。经刚果红共价修饰后的碳纳米管导电性能好,水溶性也大大增强,为纳米金提供了一定的结合位点,增强了DNA分子在电极表面的固定量,提高了生物催化反应活性。将复合膜修饰后的电极通过扫描电镜、循环伏安法及交流阻抗法进行了表征。该传感器的灵敏度高,特异性好,操作简便,成本低,检测下限达到了1.2×10-16M,可应用于生命科学、临床医学以及环境污染控制系统中实时在线监测。
开发出一种基于纳米金/多壁碳纳米管/石墨烯共同修饰的基因生物传感器用于锰过氧化物酶功能基因的超灵敏检测。L-半胱氨酸和刚果红分别对石墨烯和碳纳米管进行功能化修饰,得到一种水溶性较好、导电性能优越、生物相容性好的纳米复合材料。为纳米金的电化学沉积提供了良好的载体条件,明显增大了电极的比表面积,同时也提高了DNA分子在电极表面的固载量。通过扫描电镜、透射电镜、红外光谱扫描等方法对材料的形态和结构进行了表征。在最优实验条件下,采用计时电流法得到的还原电流与MnP目标基因浓度的常用对数值呈良好的线性相关性,检测下限达到了2.0×10-17M。该基因传感器的灵敏度极高,选择性和稳定性好,制备方法简单,成本低,可对环境中其他特定核苷酸序列进行快速灵敏检测。
Composting is an effective disposal method for resource utilization of organic solid wastes. Phenol compounds are common pollutants in organic solid wastes composting. Hydroquinone and catechol are the two intermediate products during biodegradation of polycyclic aromatic hydrocarbons. Lignin is one kind of aromatic polymer in composting and is extremely recalcitrant in an aerobic oxidative process. Both manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) play important roles in lignin mineralization. Electrochemical biosensors have received particular attention due to their easy operation, fast response, good selectivity, high sensitivity and low cost in environment, biology, medicine, and food stuff. The incorporation of the highly selective catalytic and recognition properties of biomolecules with the unique conductivity, catalytic activity and biocompatibility of nanomaterials provide a new technical platform for the analysis and measurement of contaminants and functional genes in composting, and are also of great importance for compost process control and environmental monitoring.
This dissertation took the composting system for example and discussed the preparation and functionalization of carbon nanotubes, graphene, gold and platinum nanocomposite. Several water-soluble nanocomposites were synthesised and characterized and their electrochemical properties were also studied. A series of water-soluble nanocomposite modified electrodes were constructed by the immobilization of enzyme and nucleic acid probes for highly sensitive detection of hydroquinone, catechol, MnP and CDH functional genes in composting.
Water soluble graphene-platinum nanocomposite was prepared by the combination of chemical reduction using sodium borohydride and covalent modification with L-cysteine. A new electrochemical sensor based on the nanocomposite was developed for simultaneous detection of hydroquinone and catechol in composting. The good solubility and biocompatibility of nanocomposite could greatly improve the effective specific surface area, conductivity and catalytic activity of the electrode. The concentrations of hydroquinone and catechol in the compost extract were determined by differential pulse voltammetry, which showed that the detection limits of the two phenol compounds were2.0×10-8M and1.0×10-8M, respectively. This sensor exhibited good selectivity, stability, sensitivity and excellent capacity of resisting disturbance and it would be an effective approach for rapid and on-line detection of phenol compounds.
A laccase biosensor was constructed based on the water-soluble carbon nanotubes/L-lysine/gold nanoparticles for highly sensitive detection of hydroquinone and catechol. After modification of gold nanoparticles, the L-lysine was decorated on the glassy carbon electrode by the electrochemical polymerization. Carbon nanotubes was functionalized by Congo red and the solution was cast on the modified electrode. Then the laccase was immobilized on the electrode using covalently crosslinking with glutaraldehyde. The immobilization of laccase showed good biological activity which significantly enhanced the catalytic capability of electrode. The conductivity and surface area was also improved by the synergy of carbon nanotubes and gold nanoparticles. This laccase biosensor was simple and response fast with high sensitivity and selectivity. The detection limits of hydroquinone and catechol were1.0×10-8M and1.0×10-9M, respectively. This sensor can be used for the real-time determination of all kinds of phenol compounds.
An electrochemical DNA sensor based on horseradish peroxidase (HRP) and laccase (LAC) labels was developed by the incorporation of polymerase chain reaction and restriction endonuclease digestion with sensor technology for simultaneous detection of manganese peroxidase and cellobiose dehydrogenase functional genes. The target sequences were immobilized by hybridization with both capture probes modified with thiol and detection probes labeled with HRP and LAC, which were applied for enzyme-amplified amperometric measurement. Under the optimal conditions, the amperometric current responses by chronoamperometry were linearly related to the common logarithm of two target nucleic acids concentrations and the detection limits were6.2×10-12M and3.0×10-11M, respectively. The DNA biosensor exhibited good selectivity, precision, stability and reproducibility. The DNA sensor presented here is a promising step to study the mutual relationship between two functional genes for environmental pollutant biodegradation.
An electrochemical sensor based on gold nanoparticles (GNPs)/multiwalled carbon nanotubes (MWCNTs)/poly (1,5-naphthalenediamine) films modified glassy carbon electrode for rapid detection of cellobiose dehydrogenase gene was fabricated. The monomer of1,5-naphthalenediamine was electropolymerized on the GCE surface with abundant free amino groups which enhanced the stability of MWCNTs modified electrode. Congo red-functionalized MWCNTs possess excellent conductivity as well as high solubility in water. It created a large number of binding sites for electrodeposition of GNPs and improved the DNA immobilization amount and biological catalytic activity. Scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry were used to investigate the film assembly and DNA hybridization processes. The electrochemical biosensor exhibited high sensitivity, selectivity, easy operation and low cost, with the detection limit of1.2×10-16M. It offered a possible method for "on-the-spot" monitoring in life sciences, clinical medicine and environmental pollution control systems.
A sensitive electrochemical DNA sensor based on gold nanoparticles/multiwalled carbon nanotubes/graphene composite films modified glassy carbon electrode was fabricated for the determination of manganese peroxidase gene. After functionalization with Congo red (CR) and L-cysteine (Cys), the MWCNT-CR/GR-Cys nanocomposite exhibited high solubility in water, excellent conductivity as well as good biocompatibility, which created a large number of binding sites for electrodeposition of GNPs, greatly enhanced the surface area and DNA immobilization amount. The characteristics of the modified electrode were investigated by transmission electron microscopy, scanning electron microscopy and fouriertrans-form infrared spectroscopy. Under the optimal conditions, the current improvement catalyzed by HRP was linearly related to the common logarithm of MnP target sequences in the detection limit of2.0×10-17M. In addition, this DNA sensor exhibited high sensitivity, good selectivity and stability. The biosensor was easy to prepare and cost low, which could be further used for the rapid and sensitive determination of other specific ssDNA sequence in environment.
引文
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