辣根过氧化物酶，漆酶生物传感器的研究及应用
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摘要
目的:利用电化学酶传感器来检测分析环境中有机污染物具有了简便快捷特点。本研究采用顺序注射分析法(SIA)的丝网印刷型酶传感器联用技术,检测辣根过氧化物酶(horseradish peroxidas,HRP)和漆酶的催化电流,反映苯酚类等有机污染物的浓度。为环境污染物快速测定提供了一种准确,灵敏及自动化的检测技术。
方法:实验研究中以单位点HRP,漆酶丝网印刷电极和四位点(HRP,漆酶)丝网印刷电极为基础,利用牛血清白蛋白(BSA)和戊二醛形成的交联大分子将HRP,漆酶或两种酶与锇聚合物(锇-聚乙烯基吡啶,Os(bpy)_2(PVP)_(10)Cl_2,Os-PVP)的混合酶液分别固定于单位点丝网印刷电极和四位点电极的表面,采用顺序注射分析(SIA)与丝网印刷生物传感器联用法对各种酶电极进行检测分析,并对其缓冲液pH值及浓度、泵流速、样品量等实验条件进行优化。
结果:
(1)对单位点,无Os-PVP修饰的HRP电极,游离铁氰化钾修饰的漆酶电极检测分析中,优化得到最佳条件分别为①HRP电极:泵流速153.9μL/min、样品量100μL、0.1 mol/L ,pH 6.5的PBS缓冲液,HRP催化邻苯二胺(OPD)-H_2O_2反应时,H_2O_2浓度变化的电流响应线性方程为y=14.59+114.61x,R~2=0.9845,电流响应灵敏度为110.11 nA/ (mmol/L),OPD浓度变化的电流响应线性方程为y = 27.595x + 17.925,R~2=0.9941,电流响应灵敏度为26.88 nA/ (mmol/L)该酶电极重现性良好,RSD为3.8%,用于实际水样加标回收率测定时,回收率大于60%。②游离铁氰化钾修饰的漆酶电极:0.1 mol/L ,pH 4.5 PBS缓冲液,漆酶催化邻苯二酚与铁氰化钾的电流响应线性方程为y=49.7x+17.582,R~2=0.9827,电流响应灵敏度为50 nA/ (mmol/L),呈现良好的线性关系。
(2)对单位点及Os-PVP修饰的HRP,漆酶电极检测分析,在最适的PBS缓冲液条件0.1mol/L,pH分别为7.5,4.5下,HRP催化OPD-H_2O_2反应的电流响应线性方程为y=300.39x-6.7031,R~2=0.9875,电流响应灵敏度为97.46 nA/ (mmol/L);漆酶催化邻苯二酚反应的电流响应线性方程为y=1.166x+0.078,R~2=0.9801,电流响应灵敏度为1.13μA/ (mmol/L)。HRP-(Os-PVP)电极用于实际水样加标回收率为70 % ~ 93 %。漆酶-(Os-PVP)电极用于检测垃圾滤液,其中含邻苯二酚浓度的平均值为0.028 mmol/L,标准偏差(RSD)为0.34%。
(3)四位点酶电极的分析中按照HRP,漆酶,HRP,漆酶的顺序将酶分别与Os-PVP混合后固定于丝网印刷电极表面,得到在最适PBS缓冲液(0.15 mmol/L,pH 6.0 )条件下,同H_2O_2,邻苯二酚共同反应的电流响应线性方程分别为1位点(HRP):y=1.572x-0.165,R~2=0.9559,电流响应灵敏度为1.44μA/(mmol/L);2位点(漆酶):y=0.5519x-0.023,R~2=0.949,电流响应灵敏度为0.54μA/ (mmol/L);3位点(HRP):y=1.022x+0.01,R~2=0.9604,电流响应灵敏度为0.91μA/ (mmol/L);4位点(漆酶):y=0.349x-0.0042,R~2=0.9641,电流响应灵敏度为0.33μA/ (mmol/L)。对垃圾滤液中含邻苯二酚浓度检测结果浓度分别为1.09 mmol/L,,1.02 mmol/L,1.04 mmol/L,1.09 mmol/L,平均值为1.05 mmol/L,相对偏差(RSD)为3.4%。
结论:建立了一种顺序注射分析法与丝网印刷型酶传感器联用技术及对四位点酶电极的研究。该联用技术提高了传感器分析的精确度和灵敏度及利于在线长期实时的检测环境;四位点酶电极的研究为复杂的环境检测手段扩宽了检测范围及选择性。
Objective: Using enzyme systems of electrochemical biosensor to detect organic pollutants in the analytic environment is easily and quickly. In this study,sequential injection analysis (SIA) was combined with the screen-printed enzyme sensor technology to detect catalytic current of horseradish peroxidase(HRP) and laccase,which can reflect the concentration of phenol and other organic pollutants.It provides an accurate,sensitive and automated detection technology for rapidly detect environmental pollutants.
Methods: This study based on the screen-printed electrode of Single enzyme sites HRP,laccase and four enzyme point(HRP,laccase),using bovine serum albumin(BSA) and glutaraldehyde to immobilize HRP,laccase or mix liquid of two enzymes with osmium polymer([Os(bipyridine)2Cl2]poly(4-vinylpyridine),Os(bpy)2(PVP)10Cl2,Os-PVP) on the screen-printed electrodes and the surface of four point electrode. And then utilizing the technology of SIA coupled with screen-printed biosensor to process detect and analysis on each kind of Enzyme electrode,and optimize the relevant test conditions such as the buffer pH and concentration, pump flow rate, sample volume and etc.
Results:
(1) Analyzed the detection system of single enzyme sites,no Os-PVP modified HRP electrode,got the optimum conditions of the experiment via analysis on detection of free ferricyanide modified laccase electrode.The optimum conditions of the experiment are as follows:
①HRP electrode:153.9μL/min flow rate,100μL sample volume and pH 6.5,0.1 mol/L buffer.The reaction of HRP catalyzing o-phenylenediamine(OPD)-H_2O_2,which the current response linear equationIn of H_2O_2 concentration is y=14.59+114.61x,R~2=0.9845,and Current response sensitivity is 110.11nA/(mmol/L).The current response linear equationIn of OPD concentration is y=27.595x+17.925,R~2=0.9941, and Current response sensitivity is 26.88 nA/(mmol/L).In addition, the biosenser had advantages of repeatability,RSD was 3.8%.The recovery ration for OPD in the real sample was more than 60%.②free ferricyanide modified laccase electrodes:0.1 mol/L,pH 4.5 PBS buffer,the current response linear equationIn of laccase catalyzed catechol and ferricyanide is y=49.7x+17.582,R~2=0.9827,and Current response sensitivity is 50 nA/(mmol/L),they Showed a good linear relationship.
(2)Analyzed the detection system of single enzyme sites of HRP electrode and laccase electrode, which modified by Os-PVP. Under the respectively optimum PBS buffer conditions of 0.1 mol/L, pH 7.5 and 4.5, the current response linear equation ,which HRP catalyzed OPD-H_2O_2, is y=300.39x-6.7031,R~2=0.9875,and Current response sensitivity is 97.46 nA/ (mmol/L).The current response linear equations, which laccase catalyzed catechol, is y=1.166x+0.078,R~2=0.9801,and Current response sensitivity is 1.13μA/ (mmol/L). The recycle rate of HRP-(Os-PVP) electrode used for detecting real water samples varies from 70% to 93%. And the laccase-(Os-PVP) electrode used for detecting the landfill leachate,the average concentration of catechol is 0.028 mmol/L,and the standard deviation(RSD) is 0.34%.
(3) To analyzed the Four-point enzyme electrode, respectively mixed HRP,laccase,HRP,laccase with Os-PVP in sequence on the surface of screen-printed electrodes.The optimum conditons are as follows: 0.15 mmol/L,pH 6.0 of PBS buffer.The first enzyme point(HRP) current response linear equations of a common reaction of H_2O_2 and catechol is y=1.572x-0.165,R~2=0.9559,and current response sensitivity is 1.44μA/(mmol/L).The seconde enzyme point(laccase) current response linear equations of a common reaction of H_2O_2 and catechol is y=0.5519x-0.023,R~2=0.949,and current response sensitivity is 0.54μA/(mmol/L).The third enzyme point(HRP) current response linear equations of a common reaction of H_2O_2 and catechol is y=1.022x+0.01, R~2=0.9604,and current response sensitivity is 0.91μA/(mmol/L).The forth enzyme point(laccase) current response linear equations of a common reaction of H_2O_2 and catechol is y=0.349x-0.004,R~2=0.9641,and current response sensitivity is 0.33μA/(mmol/L).In addition,to detect the respectively catechol concentrition of landfill leachate is 1.09 mmol/L,1.02 mmol/L,1.04 mmol/L,1.09 mmol/L,and average value is 1.05 mmol/L. The relative deviation (RSD) was 3.4%.
Conclusion: Here established a technology of screen-printed enzyme sensor coupled with sequential injection analysis(SIA) and studied on four point enzyme electrode. This technology improved the sensor analytical accuracy and sensitivity, and it will use to testing environment of long-term,real-time online. Enzyme electrode of the four points provided a method for the complex environment detection,which widened the scope of testing and selective.
方法:实验研究中以单位点HRP,漆酶丝网印刷电极和四位点(HRP,漆酶)丝网印刷电极为基础,利用牛血清白蛋白(BSA)和戊二醛形成的交联大分子将HRP,漆酶或两种酶与锇聚合物(锇-聚乙烯基吡啶,Os(bpy)_2(PVP)_(10)Cl_2,Os-PVP)的混合酶液分别固定于单位点丝网印刷电极和四位点电极的表面,采用顺序注射分析(SIA)与丝网印刷生物传感器联用法对各种酶电极进行检测分析,并对其缓冲液pH值及浓度、泵流速、样品量等实验条件进行优化。
结果:
(1)对单位点,无Os-PVP修饰的HRP电极,游离铁氰化钾修饰的漆酶电极检测分析中,优化得到最佳条件分别为①HRP电极:泵流速153.9μL/min、样品量100μL、0.1 mol/L ,pH 6.5的PBS缓冲液,HRP催化邻苯二胺(OPD)-H_2O_2反应时,H_2O_2浓度变化的电流响应线性方程为y=14.59+114.61x,R~2=0.9845,电流响应灵敏度为110.11 nA/ (mmol/L),OPD浓度变化的电流响应线性方程为y = 27.595x + 17.925,R~2=0.9941,电流响应灵敏度为26.88 nA/ (mmol/L)该酶电极重现性良好,RSD为3.8%,用于实际水样加标回收率测定时,回收率大于60%。②游离铁氰化钾修饰的漆酶电极:0.1 mol/L ,pH 4.5 PBS缓冲液,漆酶催化邻苯二酚与铁氰化钾的电流响应线性方程为y=49.7x+17.582,R~2=0.9827,电流响应灵敏度为50 nA/ (mmol/L),呈现良好的线性关系。
(2)对单位点及Os-PVP修饰的HRP,漆酶电极检测分析,在最适的PBS缓冲液条件0.1mol/L,pH分别为7.5,4.5下,HRP催化OPD-H_2O_2反应的电流响应线性方程为y=300.39x-6.7031,R~2=0.9875,电流响应灵敏度为97.46 nA/ (mmol/L);漆酶催化邻苯二酚反应的电流响应线性方程为y=1.166x+0.078,R~2=0.9801,电流响应灵敏度为1.13μA/ (mmol/L)。HRP-(Os-PVP)电极用于实际水样加标回收率为70 % ~ 93 %。漆酶-(Os-PVP)电极用于检测垃圾滤液,其中含邻苯二酚浓度的平均值为0.028 mmol/L,标准偏差(RSD)为0.34%。
(3)四位点酶电极的分析中按照HRP,漆酶,HRP,漆酶的顺序将酶分别与Os-PVP混合后固定于丝网印刷电极表面,得到在最适PBS缓冲液(0.15 mmol/L,pH 6.0 )条件下,同H_2O_2,邻苯二酚共同反应的电流响应线性方程分别为1位点(HRP):y=1.572x-0.165,R~2=0.9559,电流响应灵敏度为1.44μA/(mmol/L);2位点(漆酶):y=0.5519x-0.023,R~2=0.949,电流响应灵敏度为0.54μA/ (mmol/L);3位点(HRP):y=1.022x+0.01,R~2=0.9604,电流响应灵敏度为0.91μA/ (mmol/L);4位点(漆酶):y=0.349x-0.0042,R~2=0.9641,电流响应灵敏度为0.33μA/ (mmol/L)。对垃圾滤液中含邻苯二酚浓度检测结果浓度分别为1.09 mmol/L,,1.02 mmol/L,1.04 mmol/L,1.09 mmol/L,平均值为1.05 mmol/L,相对偏差(RSD)为3.4%。
结论:建立了一种顺序注射分析法与丝网印刷型酶传感器联用技术及对四位点酶电极的研究。该联用技术提高了传感器分析的精确度和灵敏度及利于在线长期实时的检测环境;四位点酶电极的研究为复杂的环境检测手段扩宽了检测范围及选择性。
Objective: Using enzyme systems of electrochemical biosensor to detect organic pollutants in the analytic environment is easily and quickly. In this study,sequential injection analysis (SIA) was combined with the screen-printed enzyme sensor technology to detect catalytic current of horseradish peroxidase(HRP) and laccase,which can reflect the concentration of phenol and other organic pollutants.It provides an accurate,sensitive and automated detection technology for rapidly detect environmental pollutants.
Methods: This study based on the screen-printed electrode of Single enzyme sites HRP,laccase and four enzyme point(HRP,laccase),using bovine serum albumin(BSA) and glutaraldehyde to immobilize HRP,laccase or mix liquid of two enzymes with osmium polymer([Os(bipyridine)2Cl2]poly(4-vinylpyridine),Os(bpy)2(PVP)10Cl2,Os-PVP) on the screen-printed electrodes and the surface of four point electrode. And then utilizing the technology of SIA coupled with screen-printed biosensor to process detect and analysis on each kind of Enzyme electrode,and optimize the relevant test conditions such as the buffer pH and concentration, pump flow rate, sample volume and etc.
Results:
(1) Analyzed the detection system of single enzyme sites,no Os-PVP modified HRP electrode,got the optimum conditions of the experiment via analysis on detection of free ferricyanide modified laccase electrode.The optimum conditions of the experiment are as follows:
①HRP electrode:153.9μL/min flow rate,100μL sample volume and pH 6.5,0.1 mol/L buffer.The reaction of HRP catalyzing o-phenylenediamine(OPD)-H_2O_2,which the current response linear equationIn of H_2O_2 concentration is y=14.59+114.61x,R~2=0.9845,and Current response sensitivity is 110.11nA/(mmol/L).The current response linear equationIn of OPD concentration is y=27.595x+17.925,R~2=0.9941, and Current response sensitivity is 26.88 nA/(mmol/L).In addition, the biosenser had advantages of repeatability,RSD was 3.8%.The recovery ration for OPD in the real sample was more than 60%.②free ferricyanide modified laccase electrodes:0.1 mol/L,pH 4.5 PBS buffer,the current response linear equationIn of laccase catalyzed catechol and ferricyanide is y=49.7x+17.582,R~2=0.9827,and Current response sensitivity is 50 nA/(mmol/L),they Showed a good linear relationship.
(2)Analyzed the detection system of single enzyme sites of HRP electrode and laccase electrode, which modified by Os-PVP. Under the respectively optimum PBS buffer conditions of 0.1 mol/L, pH 7.5 and 4.5, the current response linear equation ,which HRP catalyzed OPD-H_2O_2, is y=300.39x-6.7031,R~2=0.9875,and Current response sensitivity is 97.46 nA/ (mmol/L).The current response linear equations, which laccase catalyzed catechol, is y=1.166x+0.078,R~2=0.9801,and Current response sensitivity is 1.13μA/ (mmol/L). The recycle rate of HRP-(Os-PVP) electrode used for detecting real water samples varies from 70% to 93%. And the laccase-(Os-PVP) electrode used for detecting the landfill leachate,the average concentration of catechol is 0.028 mmol/L,and the standard deviation(RSD) is 0.34%.
(3) To analyzed the Four-point enzyme electrode, respectively mixed HRP,laccase,HRP,laccase with Os-PVP in sequence on the surface of screen-printed electrodes.The optimum conditons are as follows: 0.15 mmol/L,pH 6.0 of PBS buffer.The first enzyme point(HRP) current response linear equations of a common reaction of H_2O_2 and catechol is y=1.572x-0.165,R~2=0.9559,and current response sensitivity is 1.44μA/(mmol/L).The seconde enzyme point(laccase) current response linear equations of a common reaction of H_2O_2 and catechol is y=0.5519x-0.023,R~2=0.949,and current response sensitivity is 0.54μA/(mmol/L).The third enzyme point(HRP) current response linear equations of a common reaction of H_2O_2 and catechol is y=1.022x+0.01, R~2=0.9604,and current response sensitivity is 0.91μA/(mmol/L).The forth enzyme point(laccase) current response linear equations of a common reaction of H_2O_2 and catechol is y=0.349x-0.004,R~2=0.9641,and current response sensitivity is 0.33μA/(mmol/L).In addition,to detect the respectively catechol concentrition of landfill leachate is 1.09 mmol/L,1.02 mmol/L,1.04 mmol/L,1.09 mmol/L,and average value is 1.05 mmol/L. The relative deviation (RSD) was 3.4%.
Conclusion: Here established a technology of screen-printed enzyme sensor coupled with sequential injection analysis(SIA) and studied on four point enzyme electrode. This technology improved the sensor analytical accuracy and sensitivity, and it will use to testing environment of long-term,real-time online. Enzyme electrode of the four points provided a method for the complex environment detection,which widened the scope of testing and selective.
引文
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[27]刘宇,郭晨,王锋等.磁性SiO2纳米粒子的制备及其用于漆酶固定化[J].过程工程学报.2008,8(3):583-588
[28] Xu XH,Guo MQ,Lu P,et al.Development of amperometric laccase biosensor through immobilizing enzyme in copper-containing ordered mesoporous carbon (Cu-OMC)/chitosan matrix[J].Materials Science and Engineering C. 2010,30(5): 722-729
[29]刘强.电化学制备聚合物修饰酶电极及其在酶燃料电池中的应用[D].天津:天津大学化工学院,2007.
[30] Xue CH,Luo FT,Chen J,et al.Synthesis and biosensing application of highly water-soluble and cross-linkable poly(p-phenyleneethynylene) containing osmium(II) complex and aldehyde groups[J].Analytica Chimica Acta 2006,569(1/2):27-34
[31] Ackermann Y,Guschin DA,Eckhard K,et al.Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Os-complex containing redox polymer with entrapped Trametes hirsuta laccase[J].Electrochemistry Communications 2010,12(5):640-643
[32] Kumi Y. Inouea,Kosuke Ino,Hitoshi Shiku,et al.Electrochemical monitoring of hydrogen peroxide released from leucocytes on horseradish peroxidase redox polymer coated electrode chip[J].Biosensors and Bioelectronics.2010,25(7): 1723–1728
[33]张君,王月伶,袁倬斌.可检测有机磷农药残留的丝网印刷酶电极[J].化学学报.2006,64(5):428-434
[34]陈向强,何苗,蔡强等.锇聚合物修饰丝网印刷电极的辣根过氧化物酶传感器[J].清华大学学报-自然科学版.2007,47(9):1473-1476
[35]罗细亮,焦奎,孙伟.聚天青A/辣根过氧化物酶电极的制备及性能[J].青岛科技大学学报.2003,24(1):4-7
[36] Zafar MN,Tasca F,Boland S,et al.Wiring of pyranose dehydrogenase with osmium polymers of different redox potentials[J].Bioelectrochemistry.2010,80(1):38-42
[37] Quan D ,Kim Y,Shin W.Characterization of an amperometric laccase electrode covalently immobilized on platinum surface[J].Journal of Electroanalytical Chemistry.2004,56(1/2):181-189
[1]韦明元,郭良宏.环境污染物的免疫传感检测方法进展[J].化学进展.2009,21(2/3):492-502
[2]蔡强,何苗,施汉昌.电化学免疫传感器在环境污染监测中的研究进展[J].传感技术学报.2004(3):526-530
[3]刘真真,张敏,姚海军等.酶生物传感器的研究进展[J].东莞理工学院学报.2007,14(3):97-101
[4]伍林,曹淑超,易德莲等.酶生物传感器的研究进展[J].传感器技术.2005,24(7):4-6
[5]陈玲.生物传感器的研究进展综述[J].传感器与微系统.2006,25(9):4-7
[6]高志勇.生物传感器研究进展[J].华西医学.2008,23(6):1517
[7] Timur S ,Yigzawa Y,Gorton L.Electrical wiring of pyranose oxidase with osmium redox polymers[J].Sensors and Actuators B.2006,113(2):684-691
[8] Razumiene J,Vilkanauskyte A,Gureviciene V,et al.New bioorganometallic ferrocene derivatives as efficient mediators for glucose and ethanol biosensors based on PQQ-dependent dehydrogenases[J].Journal of Organometallic Chemistry.2003,668(1/2):83-90
[9] Teng YJ ,Zuo SH,Lan MB.Direct electron transfer of Horseradish peroxidase on porous structure of screen-printed electrode[J].Biosensors & Bioelectronics. 2009,24(5):1353-1357
[10] Chikkaveeraiah BV,Liu HY,Mani V,et al.A microfluidic electrochemical device for high sensitivity biosensing:Detection of nanomolar hydrogen peroxide[J]. Electrochemistry Communications.2009,11(4):819-822
[11]郭满栋.漆酶电极的研制及应用[J].分析科学学报.1994,10(4):16-19
[12]陈辉,张剑波,王维敬等.固定化漆酶催化去除水中2,4-二氯苯酚[J].北京大学学报(自然科学版).2006,42(4):484-489
[13]岳兰.基于丝网印刷电极和流动注射分析的电流型生物传感器系统的研究[D].杭州:浙江大学生仪学院,2006.
[14]张君,郭伟,袁倬斌.用丝网印刷技术制备薄膜微电极的方法研究[J].分析化学.2005,33(7):1045-1048
[15]刘永庆.生物传感器电极网印技术[J].丝网印刷.2005(6):25-27
[16] Ruille N Ledru S, Boujtita M One-step screen-printed electrode modified in its bulk with HRP based on direct electron transfer for hydrogen peroxide detection in flow injection mode[J].Biosensors & Bioelectronics.2006,21(8):1591-1598
[17]左少华,张玲帆,滕渊洁.普鲁士蓝修饰丝网印刷电极的葡萄糖生物传感器[J].现代仪器.2008,14(2):13-16
[18] Tonning E,Sapelnikova S,Christensen J,et al.Chemometric exploration of an amperometric biosensor array for fast determination of wastewater quality[J].Biosensors and Bioelectronics.2005,21(4):608-617
[19] Chang SC ,Rawson K,McNeil CJ.Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols[J].Biosensors and Bioelectronics 17.2002,17(11/12):1015-1023
[20] Solna R,Dock E,Christenson A,et al.Amperometric screen-printed biosensor arrays with co-immobilised oxidoreductases and cholinesterases[J].Analytica Chimica Acta.2005,528(1):9-19
[21] Sapelnikova S,Dock E,Solna R,et al.Screen-printed multienzyme arrays for use in amperometric batch and flow systems[J].Anal Bioanal Chem.2003,376(7): 1098-1103
[22] Morrin A,Guzman A,Killard AJ,et al.Characterisation of horseradish peroxidase immobilisation on an electrochemical biosensor by colorimetric and amperometric techniques[J].Biosensors & Bioelectronics.2003,18(5/6):715-720
[23]郭立泉,王红宇.酶的固定化技术的研究进展[J].吉林工商学院学报.2008,24(5):83-87
[24]李晔.酶的固定化及其应用[J].分子催化.2008,22(1):86-96
[25] Szamocki R,Flexer V,Levin L,et al.Oxygen cathode based on a layer-by-layer self-assembled laccase and osmium redox mediator[J].Electrochimica Acta.2009,54(7):1970-1977
[26]李明,陈焕文,郑健等.流动注射分析技术的若干进展[J].分析仪器.2003(3):1-5
[27] Perez-Olmos R,Soto J C,Zarate N,et al.Application of sequential injection analysis (SIA) to food analysis[J].Food Chemistry.2005,90(3):471-490
[28]李青,李义兵.流动注射分析在分析化学中的应用与发展[J].太原师范学院学报.2006,5(1):84-88
[29] Gomes SP,Odlozil?kova M,Almeida MG,et al.Application of lactate amperometric sol-gel biosensor to sequential injection determination of l-lactate[J].Journal of Pharmaceutical and Biomedical Analysis.2007,43(4):1376-1381
[30]陈旭伟,范世华.顺序注射分析的新进展[J].冶金分析.2004,24(2):23-29
[31] Stefan RI,Staden JF,Bala C,et al.On-line assay of the S-enantiomers of enalapril, ramipril and pentopril using a sequential injection analysis/amperometric biosensor system[J].Journal of Pharmaceutical and Biomedical Analysis.2004,36 (4):889-892
[32] Chung JW ,Bernhardt R,Pyun JC.Sequential analysis of multiple analytes using a surface plasmon resonance (SPR) biosensor[J].Journal of Immunological Methods.2006,311(1/2):178-188
[33] Slolna R,Sapelnikova S,Skladal p,et al.Multienzyme electrochemical array sensor for determination of phenols and pesticides[J].Talanta.2005,65(2):349-357
[34] Niculescu M,Erichsen T,Sukharev V,et al.Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation[J].Analytica Chimica Acta.2002,463(1):39-51
[35] Stefan RI,Bokretsion RG,Staden van JF,et al.Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system[J].Biosensors & Bioelectronics.2003,19(3):261-267
[36] Stefan RI,Bokretsion RG,Staden van JF,et al.Simultaneous determination of L- and D-carnitine using a sequential injection analysis/amperometric biosensors system[J].Journal of Pharmaceutical and Biomedical Analysis.2003,33(2): 323-328
[37] Stefan RI ,Staden JF,Aboul-Enein HY.Amperometric biosensors:sequential injection analysis system for simultaneous determination of S- and R-captopril[J]. Biosensors & Bioelectronics.2000,15(1/2):1-5
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[13] Niculescu M,Erichsen T,Sukharev V,et al.Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation[J].Analytica Chimica Acta.2002,463(1):39-51
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[26] Chang SC ,Rawson K,McNeil CJ.Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols[J].Biosensors and Bioelectronics 17.2002,17(11/12):1015-1023
[27]刘宇,郭晨,王锋等.磁性SiO2纳米粒子的制备及其用于漆酶固定化[J].过程工程学报.2008,8(3):583-588
[28] Xu XH,Guo MQ,Lu P,et al.Development of amperometric laccase biosensor through immobilizing enzyme in copper-containing ordered mesoporous carbon (Cu-OMC)/chitosan matrix[J].Materials Science and Engineering C. 2010,30(5): 722-729
[29]刘强.电化学制备聚合物修饰酶电极及其在酶燃料电池中的应用[D].天津:天津大学化工学院,2007.
[30] Xue CH,Luo FT,Chen J,et al.Synthesis and biosensing application of highly water-soluble and cross-linkable poly(p-phenyleneethynylene) containing osmium(II) complex and aldehyde groups[J].Analytica Chimica Acta 2006,569(1/2):27-34
[31] Ackermann Y,Guschin DA,Eckhard K,et al.Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Os-complex containing redox polymer with entrapped Trametes hirsuta laccase[J].Electrochemistry Communications 2010,12(5):640-643
[32] Kumi Y. Inouea,Kosuke Ino,Hitoshi Shiku,et al.Electrochemical monitoring of hydrogen peroxide released from leucocytes on horseradish peroxidase redox polymer coated electrode chip[J].Biosensors and Bioelectronics.2010,25(7): 1723–1728
[33]张君,王月伶,袁倬斌.可检测有机磷农药残留的丝网印刷酶电极[J].化学学报.2006,64(5):428-434
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[37] Quan D ,Kim Y,Shin W.Characterization of an amperometric laccase electrode covalently immobilized on platinum surface[J].Journal of Electroanalytical Chemistry.2004,56(1/2):181-189
[1]韦明元,郭良宏.环境污染物的免疫传感检测方法进展[J].化学进展.2009,21(2/3):492-502
[2]蔡强,何苗,施汉昌.电化学免疫传感器在环境污染监测中的研究进展[J].传感技术学报.2004(3):526-530
[3]刘真真,张敏,姚海军等.酶生物传感器的研究进展[J].东莞理工学院学报.2007,14(3):97-101
[4]伍林,曹淑超,易德莲等.酶生物传感器的研究进展[J].传感器技术.2005,24(7):4-6
[5]陈玲.生物传感器的研究进展综述[J].传感器与微系统.2006,25(9):4-7
[6]高志勇.生物传感器研究进展[J].华西医学.2008,23(6):1517
[7] Timur S ,Yigzawa Y,Gorton L.Electrical wiring of pyranose oxidase with osmium redox polymers[J].Sensors and Actuators B.2006,113(2):684-691
[8] Razumiene J,Vilkanauskyte A,Gureviciene V,et al.New bioorganometallic ferrocene derivatives as efficient mediators for glucose and ethanol biosensors based on PQQ-dependent dehydrogenases[J].Journal of Organometallic Chemistry.2003,668(1/2):83-90
[9] Teng YJ ,Zuo SH,Lan MB.Direct electron transfer of Horseradish peroxidase on porous structure of screen-printed electrode[J].Biosensors & Bioelectronics. 2009,24(5):1353-1357
[10] Chikkaveeraiah BV,Liu HY,Mani V,et al.A microfluidic electrochemical device for high sensitivity biosensing:Detection of nanomolar hydrogen peroxide[J]. Electrochemistry Communications.2009,11(4):819-822
[11]郭满栋.漆酶电极的研制及应用[J].分析科学学报.1994,10(4):16-19
[12]陈辉,张剑波,王维敬等.固定化漆酶催化去除水中2,4-二氯苯酚[J].北京大学学报(自然科学版).2006,42(4):484-489
[13]岳兰.基于丝网印刷电极和流动注射分析的电流型生物传感器系统的研究[D].杭州:浙江大学生仪学院,2006.
[14]张君,郭伟,袁倬斌.用丝网印刷技术制备薄膜微电极的方法研究[J].分析化学.2005,33(7):1045-1048
[15]刘永庆.生物传感器电极网印技术[J].丝网印刷.2005(6):25-27
[16] Ruille N Ledru S, Boujtita M One-step screen-printed electrode modified in its bulk with HRP based on direct electron transfer for hydrogen peroxide detection in flow injection mode[J].Biosensors & Bioelectronics.2006,21(8):1591-1598
[17]左少华,张玲帆,滕渊洁.普鲁士蓝修饰丝网印刷电极的葡萄糖生物传感器[J].现代仪器.2008,14(2):13-16
[18] Tonning E,Sapelnikova S,Christensen J,et al.Chemometric exploration of an amperometric biosensor array for fast determination of wastewater quality[J].Biosensors and Bioelectronics.2005,21(4):608-617
[19] Chang SC ,Rawson K,McNeil CJ.Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols[J].Biosensors and Bioelectronics 17.2002,17(11/12):1015-1023
[20] Solna R,Dock E,Christenson A,et al.Amperometric screen-printed biosensor arrays with co-immobilised oxidoreductases and cholinesterases[J].Analytica Chimica Acta.2005,528(1):9-19
[21] Sapelnikova S,Dock E,Solna R,et al.Screen-printed multienzyme arrays for use in amperometric batch and flow systems[J].Anal Bioanal Chem.2003,376(7): 1098-1103
[22] Morrin A,Guzman A,Killard AJ,et al.Characterisation of horseradish peroxidase immobilisation on an electrochemical biosensor by colorimetric and amperometric techniques[J].Biosensors & Bioelectronics.2003,18(5/6):715-720
[23]郭立泉,王红宇.酶的固定化技术的研究进展[J].吉林工商学院学报.2008,24(5):83-87
[24]李晔.酶的固定化及其应用[J].分子催化.2008,22(1):86-96
[25] Szamocki R,Flexer V,Levin L,et al.Oxygen cathode based on a layer-by-layer self-assembled laccase and osmium redox mediator[J].Electrochimica Acta.2009,54(7):1970-1977
[26]李明,陈焕文,郑健等.流动注射分析技术的若干进展[J].分析仪器.2003(3):1-5
[27] Perez-Olmos R,Soto J C,Zarate N,et al.Application of sequential injection analysis (SIA) to food analysis[J].Food Chemistry.2005,90(3):471-490
[28]李青,李义兵.流动注射分析在分析化学中的应用与发展[J].太原师范学院学报.2006,5(1):84-88
[29] Gomes SP,Odlozil?kova M,Almeida MG,et al.Application of lactate amperometric sol-gel biosensor to sequential injection determination of l-lactate[J].Journal of Pharmaceutical and Biomedical Analysis.2007,43(4):1376-1381
[30]陈旭伟,范世华.顺序注射分析的新进展[J].冶金分析.2004,24(2):23-29
[31] Stefan RI,Staden JF,Bala C,et al.On-line assay of the S-enantiomers of enalapril, ramipril and pentopril using a sequential injection analysis/amperometric biosensor system[J].Journal of Pharmaceutical and Biomedical Analysis.2004,36 (4):889-892
[32] Chung JW ,Bernhardt R,Pyun JC.Sequential analysis of multiple analytes using a surface plasmon resonance (SPR) biosensor[J].Journal of Immunological Methods.2006,311(1/2):178-188
[33] Slolna R,Sapelnikova S,Skladal p,et al.Multienzyme electrochemical array sensor for determination of phenols and pesticides[J].Talanta.2005,65(2):349-357
[34] Niculescu M,Erichsen T,Sukharev V,et al.Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation[J].Analytica Chimica Acta.2002,463(1):39-51
[35] Stefan RI,Bokretsion RG,Staden van JF,et al.Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system[J].Biosensors & Bioelectronics.2003,19(3):261-267
[36] Stefan RI,Bokretsion RG,Staden van JF,et al.Simultaneous determination of L- and D-carnitine using a sequential injection analysis/amperometric biosensors system[J].Journal of Pharmaceutical and Biomedical Analysis.2003,33(2): 323-328
[37] Stefan RI ,Staden JF,Aboul-Enein HY.Amperometric biosensors:sequential injection analysis system for simultaneous determination of S- and R-captopril[J]. Biosensors & Bioelectronics.2000,15(1/2):1-5