生物燃料电池中酶和蛋白质的直接电化学研究
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摘要
随着21世纪经济的飞速发展,环境污染和能源危机已经成为世界各国面临的两大问题。传统能源的日渐枯竭已满足不了国民经济持续发展对能源的需求,因此,寻找新能源已经成为十分紧迫的课题。
生物燃料电池是近年来开发出来的一种新型绿色环保能源。它作为小功率电源在一些特殊领域的应用十分具有吸引力,然而输出功率密度低是限制生物燃料电池发展的主要问题。解决这一问题的有效途径就是探索合适的催化剂(酶或氧化还原蛋白质)的固定载体和固定方法,使催化剂与电极实现直接快速的电子转移。本论文主要探索能实现酶或氧化还原蛋白质与电极之间直接电化学的新型载体材料以及合适的固定方法,制备酶生物燃料电池中的阳极催化剂和阴极催化剂。论文主要研究工作包括以下几个方面:
1.炭黑(CB)因具有良好的导电性、大的比表面、价格低廉等特点近年来被广泛应用于各个领域。本论文用CB做葡萄糖氧化酶(GOx)、血红蛋白(Hb)的固定载体,用吸附法将GOx、Hb固定到炭黑表面,以Nafion加固,制备了相应的修饰电极,研究了电极的直接电化学性质并考察了对β-D(+)葡萄糖及H_2O_2的电催化性能。此外,还对电极的稳定性作了研究。红外光谱和交流阻抗图谱表明用简单的吸附方法可将葡萄糖氧化酶GOx吸附在CB表面,循环伏安结果表明固定后的GOx可与电极表面可进行有效的直接电子转移,其式量电位E~(0')为-0.436 V,在40-150 mV·s~(-1)范围内,不随扫描速率而变化。电化学反应速率常数ks为0.800s~(-1),比文献报道的大30多倍。这可能归因于炭黑表面的羧基、羟基等含氧功能团,为酶的固定提供了合适的取向和适宜的微环境。固定在CB上的GOx能保持其对β-D(+)葡萄糖氧化的生物电催化活性。即使电极在保存两周后,其电催化活性仅下降5%,说明电极有很好的稳定性。红外、紫外、XPS光谱结果表明用简单的吸附法可将血红蛋白(Hb)吸附在CB表面。吸附在CB表面的Hb能进行准可逆的直接电化学反应,其速率常数是1.02 s~(-1),而且固定化的Hb保持了对H_2O_2的良好的电催化活性,表明CB能促进Hb直接电化学反应。以上结果表明炭黑作为酶的固定载体有望在生物燃料领域有广泛的应用前景,而且电极制备方法简单、有效,可以用来制备生物燃料电池的其他催化剂。
2.为了实现催化剂(酶和蛋白质)与电极之间有效的直接电子转移,制备了具有发散结构的胡须状碳复合物(MCWC)。用吸附的方法将Hb固定在MCWC表面。光谱实验表明固定后的Hb保持了原来二级结构的构象。循环伏安结果表明Hb在电极表面实现了直接电子转移并且表现出良好的电催化活性和稳定性,其电子转移速率常数为2.07 s~(-1),大于文献报道的其他的一些载体;其吸附量为4.56×10~(-13)mol/cm~2,大于在单位比表面的炭黑上的吸附量1.32×10~(-13)mol/cm~2。这可能归功于碳复合物特殊的碳刺结构可以增大Hb吸附的比表面,而且碳刺所形成大量的V-型孔有利于物质在电化学反应时迅速扩散;其次,在碳复合物表面有大量的羧基、羟基等含氧功能团,也为酶的固定提供了适宜的微环境。用同样的方法,我们研究了漆酶(Lac)在MCWC上的直接电化学,也得到了比较好的结果。Lac的电子转移速率常数为0.77 s~(-1),在电极表面吸附量为2.73×10~(-12)mol/cm~2。上述结果表明这种发散的胡须状碳复合物作为一种新型载体有望在生物燃料电池电极制备中得到广泛的应用。
3.生物燃料电池中酶和蛋白质直接电化学除了使用碳材料作载体外,还可以使用硅基介孔分子筛作为载体。三维笼状二氧化硅介孔分子筛(FDU-12)具有大的笼径、与辣根过氧化物酶(HRP)分子大小相匹配的窗口以及良好的生物相溶性,可以作为HRP的固定载体。光谱实验表明用简单的吸附的方法可将HRP固定在FDU-12中,固定后的HRP保持了原有构象。循环伏安结果表明固定化的HRP实现了直接电化学,表现出良好的催化活性和稳定性,其式量电位E~(0')为-0.325 V,电子转移速率常数为1.20 s~(-1),显示了较快的电子传递速率。其吸附量为5.44×10~(-11)mol/cm~2,这个值比HRP在电极表面的最大理论单层覆盖浓度(2×10~(-11)mol/cm~2)高出2倍多。这可能归因于笼状介孔分子筛FDU-12具有良好的生物相容性,特殊的笼状结构为HRP构筑了良好的微环境,使HRP在电极上获得了合适的取向,从而促进了HRP在电极表面的直接电子转移,而且FDU-12所具有的三维介观孔道也有利于物质的传输和扩散。此外HRP-FDU-12/GC电极的稳定性较好。大孔笼状二氧化硅介孔分子筛FDU-12有望为进一步研究氧化还原蛋白质的直接电化学和研制开发新型生物燃料电池、生物传感器酶电极提供新的思路。
As the fast growth of economy in the 21st century,environment pollution and energy crisis have been the two problems all the world must face.The exhausting conventional energy could not meet the need of continuous country economy growth. So,exploring new energy has been a particularly urgent task.
Biofuel cell is a really new green environmental-protecting power source developed in the recent years.As a small power source,the application of biofue cell in some fields has been particularly attractive.However,the low output power density is the main problem which confines the further development of biofuel cell.One of the effective method to solve this problem is to explore appropriate carrier materials and immobilization methods of biocatalyst and realize the direct fast electron -transfer between the biocatalyst and the electrodes.In this thesis,the anodic catalyst and the cathodic catalyst in the biofuel cells have been fabricated on the basis of exploring the new carrier materials and enzyme immobilization methods.The main results and conclusions obtained are summarized as follows:
1.Carbon black(CB) was widely used in many fields due to its good conductivity, large surface area and low cost etc.Spectrometric technique indicated that the oxygen-contained groups for example C=O and C-OH were present on the surface of CB powders.These groups can supply the favorable microenvironment for the immobilization and favor the electron-transfer of enzyme.In this work,CB was used as the carrier of glucose oxidase(GOx)、hemoglobin(Hb).The adsorption method was used to immobilize GOx、Hb on the surface of carbon black powders and the Nafion was used to fix the catalyst.The direct electrochemistry and the electrocatalytic activity towards theβ-D(+)-glucose and H_2O_2 was also investigated.The stability of the electrode was studied.The FTIR spectroscopic and electrochemical measurements demonstrated that GOx could be immobilized on the surface of CB using a simple adsorptionmethod.The electrochemical measurements indicated that GOx immobilized on CB could undergo the quasi-reversible and direct electrochemical reaction and keep the bioelectrocatalytic activity for the glucose oxidation.Its formal potential,E~(0'),is -0.436 V and the electron transfer rate constant k_s value was estimated to be 0.800 s~(-1),which is thirty times larger than those obtained for GOx repoted previously.This maybe be attributed to the oxygen-contained groups on the CB,such as -COOH and-OH groups.These groups can supply the favorable microenvironment for the immobilization of enzyme.Even after conservation for two weeks,its electrocatalytic activity decreased only 5%,illustrating the good stability of GOx immobilized on CB.The FTIR spectrum,UV-Vis spectroscopy and XPS spectra showed that secondary structure of Hb immobilized on the surface of CB was not destroyed and Hb retained its biological activity.The CV experiment results demonstrated that immobilized Hb could undergo a direct quasi-reversible electrochemical reaction.Its formal potential,E~(0'),is -0.330 V in phosphate buffer solution(pH 6.9) at a scan rate of 100 mV/s and is almost independent of the scan rate in the range of 40-200 mV/s.The electron transfer rate constant k_s value was estimated to be 1.02 s~(-1),which is larger than those obtained for Hb immobilized on Au-colloid-cysteamine-modified gold electrode(0.49 s~(-1)),Hb modified CNT powder microelectrodes electrodes(0.062 s~(-1)),indicating a reasonably fast electron transfer between the immobilized Hb and the underlying electrode.The surface concentration (F) of Hb was 3.55×10~(-9) mol/cm~2.The above results showed that as the carrier of enzyme,CB is promised to have wide application in the future.The method presented here is simple and effective and can be easily extended to immobilize and obtain the direct electrochemistry of other enzymes or proteins.
2.In order to realize the effective realize the direct electron -transfer of biocatalyst (enzyme and proteins) and electrode,a whisker-like carbon composite(MCWC) was fabricated.The adsorption method was used to immobilize Hb on the surface of MCWC.Spectrometric technique indicated that immobilized Hb retained its native structure.The cyclic voltammetric results showed that immobilized Hb could undergo a direct quasi-reversible electrochemical reaction and has good bioelectrocatalytic activity and good stability for the reduction of H_2O_2.The electron transfer rate constant,ks,is 2.07 s~(-1),which is larger than that on some other carriers reported previously.The surface concentration of Hb is 4.56×10~(-13) mol/cm~2,which is lager than 1.32×10~(-13) mol/cm~2 on carbon black.This maybe because that the special carbon thorns structure can increase the specific area of adsorption,moreover,the numerous "V-type" channels formed among the thorns facilitates the fast diffusion of products. In addition,the oxygen-contained groups,such as -COOH groups and-OH groups can supply the favorable microenvironment for the immobilization of enzyme.Using the same method,the direct electrochemistry of Laccase on the MCWC was also studied.The electron transfer rate constant k_s value was estimated to be 0.77 s~(-1) and the surface concentration(Γ) of Lac was 2.73×10~(-12) mol/cm~2.The above results showed that as the new carrier of enzyme,MCWC is promised to be used widely in the fabrication of the biocatalyst of biofuel cell.
3.Besides the carbon material,silica mesoporous sieves also could be used as carriers of biocatalyst in biofuel cell.3D large cage-like mesoporous silica sieves FDU-12 can be used as the carrier of horseradish peroxidase(HRP) due to its large cage size, appropriate entrance size and good biocompatibility.Spectrometric technique indicated that immobilized HRP retained its native structure.The cyclic voltammetric results showed that immobilized HRP could undergo a direct quasi-reversible electrochemical reaction and has good bioelectrocatalytic activity and good stability for the reduction of H_2O_2.The electron transfer rate constant k_s value was estimated to be 1.20 s~(-1),indicating fast electron transfer between the immobilized HRP and the electrode.The surface concentration(Γ) of immobilized HRP was 5.44×10~(-11) mol/cm~2, which was twice larger than the single layer adsorption concentration of HRP(2×10~(-11) mol/cm~2).This maybe be attributed to the 3D uniform pore structure of FDU-12 which facilitated the fast diffusion of products and provided favorable microenvironment for HRP.Large cage-like mesoporous silica sieves FDU-12 was promised to provide new idea for the direct electrochemistry of enzyme and development of new biofuel cell and biosensors
生物燃料电池是近年来开发出来的一种新型绿色环保能源。它作为小功率电源在一些特殊领域的应用十分具有吸引力,然而输出功率密度低是限制生物燃料电池发展的主要问题。解决这一问题的有效途径就是探索合适的催化剂(酶或氧化还原蛋白质)的固定载体和固定方法,使催化剂与电极实现直接快速的电子转移。本论文主要探索能实现酶或氧化还原蛋白质与电极之间直接电化学的新型载体材料以及合适的固定方法,制备酶生物燃料电池中的阳极催化剂和阴极催化剂。论文主要研究工作包括以下几个方面:
1.炭黑(CB)因具有良好的导电性、大的比表面、价格低廉等特点近年来被广泛应用于各个领域。本论文用CB做葡萄糖氧化酶(GOx)、血红蛋白(Hb)的固定载体,用吸附法将GOx、Hb固定到炭黑表面,以Nafion加固,制备了相应的修饰电极,研究了电极的直接电化学性质并考察了对β-D(+)葡萄糖及H_2O_2的电催化性能。此外,还对电极的稳定性作了研究。红外光谱和交流阻抗图谱表明用简单的吸附方法可将葡萄糖氧化酶GOx吸附在CB表面,循环伏安结果表明固定后的GOx可与电极表面可进行有效的直接电子转移,其式量电位E~(0')为-0.436 V,在40-150 mV·s~(-1)范围内,不随扫描速率而变化。电化学反应速率常数ks为0.800s~(-1),比文献报道的大30多倍。这可能归因于炭黑表面的羧基、羟基等含氧功能团,为酶的固定提供了合适的取向和适宜的微环境。固定在CB上的GOx能保持其对β-D(+)葡萄糖氧化的生物电催化活性。即使电极在保存两周后,其电催化活性仅下降5%,说明电极有很好的稳定性。红外、紫外、XPS光谱结果表明用简单的吸附法可将血红蛋白(Hb)吸附在CB表面。吸附在CB表面的Hb能进行准可逆的直接电化学反应,其速率常数是1.02 s~(-1),而且固定化的Hb保持了对H_2O_2的良好的电催化活性,表明CB能促进Hb直接电化学反应。以上结果表明炭黑作为酶的固定载体有望在生物燃料领域有广泛的应用前景,而且电极制备方法简单、有效,可以用来制备生物燃料电池的其他催化剂。
2.为了实现催化剂(酶和蛋白质)与电极之间有效的直接电子转移,制备了具有发散结构的胡须状碳复合物(MCWC)。用吸附的方法将Hb固定在MCWC表面。光谱实验表明固定后的Hb保持了原来二级结构的构象。循环伏安结果表明Hb在电极表面实现了直接电子转移并且表现出良好的电催化活性和稳定性,其电子转移速率常数为2.07 s~(-1),大于文献报道的其他的一些载体;其吸附量为4.56×10~(-13)mol/cm~2,大于在单位比表面的炭黑上的吸附量1.32×10~(-13)mol/cm~2。这可能归功于碳复合物特殊的碳刺结构可以增大Hb吸附的比表面,而且碳刺所形成大量的V-型孔有利于物质在电化学反应时迅速扩散;其次,在碳复合物表面有大量的羧基、羟基等含氧功能团,也为酶的固定提供了适宜的微环境。用同样的方法,我们研究了漆酶(Lac)在MCWC上的直接电化学,也得到了比较好的结果。Lac的电子转移速率常数为0.77 s~(-1),在电极表面吸附量为2.73×10~(-12)mol/cm~2。上述结果表明这种发散的胡须状碳复合物作为一种新型载体有望在生物燃料电池电极制备中得到广泛的应用。
3.生物燃料电池中酶和蛋白质直接电化学除了使用碳材料作载体外,还可以使用硅基介孔分子筛作为载体。三维笼状二氧化硅介孔分子筛(FDU-12)具有大的笼径、与辣根过氧化物酶(HRP)分子大小相匹配的窗口以及良好的生物相溶性,可以作为HRP的固定载体。光谱实验表明用简单的吸附的方法可将HRP固定在FDU-12中,固定后的HRP保持了原有构象。循环伏安结果表明固定化的HRP实现了直接电化学,表现出良好的催化活性和稳定性,其式量电位E~(0')为-0.325 V,电子转移速率常数为1.20 s~(-1),显示了较快的电子传递速率。其吸附量为5.44×10~(-11)mol/cm~2,这个值比HRP在电极表面的最大理论单层覆盖浓度(2×10~(-11)mol/cm~2)高出2倍多。这可能归因于笼状介孔分子筛FDU-12具有良好的生物相容性,特殊的笼状结构为HRP构筑了良好的微环境,使HRP在电极上获得了合适的取向,从而促进了HRP在电极表面的直接电子转移,而且FDU-12所具有的三维介观孔道也有利于物质的传输和扩散。此外HRP-FDU-12/GC电极的稳定性较好。大孔笼状二氧化硅介孔分子筛FDU-12有望为进一步研究氧化还原蛋白质的直接电化学和研制开发新型生物燃料电池、生物传感器酶电极提供新的思路。
As the fast growth of economy in the 21st century,environment pollution and energy crisis have been the two problems all the world must face.The exhausting conventional energy could not meet the need of continuous country economy growth. So,exploring new energy has been a particularly urgent task.
Biofuel cell is a really new green environmental-protecting power source developed in the recent years.As a small power source,the application of biofue cell in some fields has been particularly attractive.However,the low output power density is the main problem which confines the further development of biofuel cell.One of the effective method to solve this problem is to explore appropriate carrier materials and immobilization methods of biocatalyst and realize the direct fast electron -transfer between the biocatalyst and the electrodes.In this thesis,the anodic catalyst and the cathodic catalyst in the biofuel cells have been fabricated on the basis of exploring the new carrier materials and enzyme immobilization methods.The main results and conclusions obtained are summarized as follows:
1.Carbon black(CB) was widely used in many fields due to its good conductivity, large surface area and low cost etc.Spectrometric technique indicated that the oxygen-contained groups for example C=O and C-OH were present on the surface of CB powders.These groups can supply the favorable microenvironment for the immobilization and favor the electron-transfer of enzyme.In this work,CB was used as the carrier of glucose oxidase(GOx)、hemoglobin(Hb).The adsorption method was used to immobilize GOx、Hb on the surface of carbon black powders and the Nafion was used to fix the catalyst.The direct electrochemistry and the electrocatalytic activity towards theβ-D(+)-glucose and H_2O_2 was also investigated.The stability of the electrode was studied.The FTIR spectroscopic and electrochemical measurements demonstrated that GOx could be immobilized on the surface of CB using a simple adsorptionmethod.The electrochemical measurements indicated that GOx immobilized on CB could undergo the quasi-reversible and direct electrochemical reaction and keep the bioelectrocatalytic activity for the glucose oxidation.Its formal potential,E~(0'),is -0.436 V and the electron transfer rate constant k_s value was estimated to be 0.800 s~(-1),which is thirty times larger than those obtained for GOx repoted previously.This maybe be attributed to the oxygen-contained groups on the CB,such as -COOH and-OH groups.These groups can supply the favorable microenvironment for the immobilization of enzyme.Even after conservation for two weeks,its electrocatalytic activity decreased only 5%,illustrating the good stability of GOx immobilized on CB.The FTIR spectrum,UV-Vis spectroscopy and XPS spectra showed that secondary structure of Hb immobilized on the surface of CB was not destroyed and Hb retained its biological activity.The CV experiment results demonstrated that immobilized Hb could undergo a direct quasi-reversible electrochemical reaction.Its formal potential,E~(0'),is -0.330 V in phosphate buffer solution(pH 6.9) at a scan rate of 100 mV/s and is almost independent of the scan rate in the range of 40-200 mV/s.The electron transfer rate constant k_s value was estimated to be 1.02 s~(-1),which is larger than those obtained for Hb immobilized on Au-colloid-cysteamine-modified gold electrode(0.49 s~(-1)),Hb modified CNT powder microelectrodes electrodes(0.062 s~(-1)),indicating a reasonably fast electron transfer between the immobilized Hb and the underlying electrode.The surface concentration (F) of Hb was 3.55×10~(-9) mol/cm~2.The above results showed that as the carrier of enzyme,CB is promised to have wide application in the future.The method presented here is simple and effective and can be easily extended to immobilize and obtain the direct electrochemistry of other enzymes or proteins.
2.In order to realize the effective realize the direct electron -transfer of biocatalyst (enzyme and proteins) and electrode,a whisker-like carbon composite(MCWC) was fabricated.The adsorption method was used to immobilize Hb on the surface of MCWC.Spectrometric technique indicated that immobilized Hb retained its native structure.The cyclic voltammetric results showed that immobilized Hb could undergo a direct quasi-reversible electrochemical reaction and has good bioelectrocatalytic activity and good stability for the reduction of H_2O_2.The electron transfer rate constant,ks,is 2.07 s~(-1),which is larger than that on some other carriers reported previously.The surface concentration of Hb is 4.56×10~(-13) mol/cm~2,which is lager than 1.32×10~(-13) mol/cm~2 on carbon black.This maybe because that the special carbon thorns structure can increase the specific area of adsorption,moreover,the numerous "V-type" channels formed among the thorns facilitates the fast diffusion of products. In addition,the oxygen-contained groups,such as -COOH groups and-OH groups can supply the favorable microenvironment for the immobilization of enzyme.Using the same method,the direct electrochemistry of Laccase on the MCWC was also studied.The electron transfer rate constant k_s value was estimated to be 0.77 s~(-1) and the surface concentration(Γ) of Lac was 2.73×10~(-12) mol/cm~2.The above results showed that as the new carrier of enzyme,MCWC is promised to be used widely in the fabrication of the biocatalyst of biofuel cell.
3.Besides the carbon material,silica mesoporous sieves also could be used as carriers of biocatalyst in biofuel cell.3D large cage-like mesoporous silica sieves FDU-12 can be used as the carrier of horseradish peroxidase(HRP) due to its large cage size, appropriate entrance size and good biocompatibility.Spectrometric technique indicated that immobilized HRP retained its native structure.The cyclic voltammetric results showed that immobilized HRP could undergo a direct quasi-reversible electrochemical reaction and has good bioelectrocatalytic activity and good stability for the reduction of H_2O_2.The electron transfer rate constant k_s value was estimated to be 1.20 s~(-1),indicating fast electron transfer between the immobilized HRP and the electrode.The surface concentration(Γ) of immobilized HRP was 5.44×10~(-11) mol/cm~2, which was twice larger than the single layer adsorption concentration of HRP(2×10~(-11) mol/cm~2).This maybe be attributed to the 3D uniform pore structure of FDU-12 which facilitated the fast diffusion of products and provided favorable microenvironment for HRP.Large cage-like mesoporous silica sieves FDU-12 was promised to provide new idea for the direct electrochemistry of enzyme and development of new biofuel cell and biosensors
引文
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