基于次血红素六肽的过氧化氢和葡萄糖灵敏比色方法
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摘要
采用次血红素六肽(DhHP-6)催化H_2O_2氧化4-氨基安替吡啉-氯代苯酚显色体系,建立了测定H_2O_2和葡萄糖的方法。研究了pH值、底物浓度和DhHP-6浓度对实验的影响,检测了比色方法的反应线性、稳定性、相关性和回收率。在最佳反应条件下,DhHP-6在不同时间及温度条件下,活性要优于过氧化物酶(POD);DhHP-6催化H_2O_2的米氏常数(K_m)和最大反应速率(v_(max))分别为0. 171 mmo/L和4. 22×10~(-6)mol/s; H_2O_2响应的线性范围为0. 39～25. 0 mmol/L;高、中、低3水平测定的变异系数(CV)和加标回收率分别在1. 29%～2. 16%和94. 5%～101. 1%之间;与葡萄糖商品试剂盒比较相关系数R~2=0. 9946; 36例血液样品中的葡萄糖浓度在4. 26～17. 48 mmol/L之间。与葡萄糖检测商品试剂盒之间的两组数据经统计差异不显著(P> 0. 05)。该方法是一种简单、廉价、方便、灵敏的比色测定方法。
A simple colorimetric method for determination of H_2O_2 and glucose was established based on the peroxidase-like activity of deuterohemin-Ala His Thr Val Glu Lys( DhHP-6) to catalyze the oxidation of4-aminoantipyrine( 4-APP)-chlorophenol color reaction system in the presence of H_2O_2. The effects of p H,substrate concentration and DhHP-6 concentration were observed, and the reaction linearity, stability,correlation and recovery rate of the colorimetric method were detected. Under optimal reaction conditions,the activity of DhHP-6 is superior to that of peroxidase( POD) under different temperatures and times; the Michaelis constant( K_m) and maximum reaction rate( v_(max)) of DhHP-6 catalyzing H_2O_2 are 0. 171 mmol/L and 4. 22 × 10~(-6) mol/s,respectively; the linearity range of H_2O_2 response is 0. 39 ～ 25. 0 mmol/L,the coefficient of variation( CV) and spiked recovery of the high,medium and low levels are between 1. 29% ～ 2. 16% and 94. 5% ～ 101. 1%, respectively; and correlation coefficient with the commercial kit is 0. 9946; the concentration of glucose in 36 blood samples are 4. 26 ～ 17. 48 mmol/L. There is no significant difference between the two sets of data for glucose assay kit( P > 0. 05). Therefore,this is a simple,cheap,convenient and sensitive colorimetric determination method.
A simple colorimetric method for determination of H_2O_2 and glucose was established based on the peroxidase-like activity of deuterohemin-Ala His Thr Val Glu Lys( DhHP-6) to catalyze the oxidation of4-aminoantipyrine( 4-APP)-chlorophenol color reaction system in the presence of H_2O_2. The effects of p H,substrate concentration and DhHP-6 concentration were observed, and the reaction linearity, stability,correlation and recovery rate of the colorimetric method were detected. Under optimal reaction conditions,the activity of DhHP-6 is superior to that of peroxidase( POD) under different temperatures and times; the Michaelis constant( K_m) and maximum reaction rate( v_(max)) of DhHP-6 catalyzing H_2O_2 are 0. 171 mmol/L and 4. 22 × 10~(-6) mol/s,respectively; the linearity range of H_2O_2 response is 0. 39 ～ 25. 0 mmol/L,the coefficient of variation( CV) and spiked recovery of the high,medium and low levels are between 1. 29% ～ 2. 16% and 94. 5% ～ 101. 1%, respectively; and correlation coefficient with the commercial kit is 0. 9946; the concentration of glucose in 36 blood samples are 4. 26 ～ 17. 48 mmol/L. There is no significant difference between the two sets of data for glucose assay kit( P > 0. 05). Therefore,this is a simple,cheap,convenient and sensitive colorimetric determination method.
引文
[1]Jv Y,Li B X,Cao R. Positively-charged Gold Nanoparticles as Peroxidiase Mimic and Their Application in Hydrogen Peroxide and Glucose Detection[J]. Chem Commun,2010,46:8017-8019.
[2]Marinho H S,Real C,Cyrne L,et al. Hydrogen Peroxide Sensing,Signaling and Regulation of Transcription Factors[J].Redox Biol,2014,2:535-562.
[3]Doskey C M,Buranasudja V,Wagner B A,et al. Tumor Cells Have Decreased Ability to Metabolize H2O2:Implications for Pharmacological Ascorbate in Cancer Therapy[J]. Redox Biol,2016,10(C):274-284.
[4]Sasaki A,Horiuchi N. Studies on Normal Blood Glucose Level-Statistical Approach to Interpretation of Glucose Tolerance Test[J]. J Chronic Dis,1976,29(2):129-140.
[5]Chang Q,Tang H Q. Optical Determination of Glucose and Hydrogen Peroxide Using a Nanocomposite Prepared from Glucose Oxidase and Magnetite Nanoparticles Immobilized on Graphene Oxide[J]. Microchim Acta,2014,181:527-534.
[6]Lan D,Li B X,Zhang Z J. Chemiluminescence Flow Biosensor for Glucose Based on Gold Nanoparticle-Enhanced Activities of Glucose Oxidase and Horseradish Peroxidase[J]. Biosens Bioelectron,2008,24:934-938.
[7]Chang Q,Zhu L,Jiang G,et al. Sensitive Fluorescent Probes for Determination of Hydrogen Peroxide and Glucose Based on Enzyme-Immobilized Magnetite/Silica Nanoparticles[J]. Anal Bioanal Chem,2009,395(7):2377-2385.
[8]Zhao K,Gu W,Zheng S S,et al. SDS-MoS2Nanoparticles as Highly-Efficient Peroxidase Mimetics for Colorimetric Detection of H2O2and Glucose[J]. Talanta,2015,141:47-52.
[9]Rauf S,Hayat Nawaz M A,Badea M,et al. Nano-Engineered Biomimetic Optical Sensors for Glucose Monitoring in Diabetes[J]. Sensors(Basel),2016,16(11):e1931.
[10]Huang W,Lin T Y,Cao Y,et al. Hierarchical Ni Co2O4Hollow Sphere as a Peroxidase Mimetic for Colorimetric Detection of H2O2and Glucose[J]. Sensors(Basel),2017,17(1):e217.
[11]Zhang W,Ma D,Du J. Prussian Blue Nanoparticles as Peroxidase Mimetics for Sensitive Colorimetric Detection of Hydrogen Peroxide and Glucose[J]. Talanta,2014,120:362-367.
[12]Lin J M,Arakawa H,Yamada M. Flow Injection Chemiluminescent Determination of Trace Amounts of Hydrogen Peroxide in Snow-Water Using KIO4K2CO3System[J]. Anal Chim Acta,1998,371:171-176.
[13]CAI Zijun,KUANG Yongqing,PAN Dan,et al. Sythesis and Characterization of a Novel ELF-97-based Fluorescent Probe for Hydrogen Peroxide Detection[J]. Chinese J Anal Chem,2015,43(11):1671-1675(in Chinese).蔡子君,匡永清,潘丹,等.基于ELF-97的新型H2O2荧光探针的合成与性能研究[J].分析化学,2015,43(11):1671-1675.
[14]Xu J R,Chen Z M. Determination of Peroxides in Environmental Samples by High Performance Liquid Chromatography[J]. Chinese J Chromatogr,2005,23:366-369.
[15]LI Li,LU Hongmei,DENG Liu. H2O2Electrochemistry Biosensor Based on Graphene and Gold Nanorods Composites[J].Chinese J Anal Chem,2013,41(5):719-724(in Chinese).李理,卢红梅,邓留.基于石墨烯和金纳米棒复合物的过氧化氢电化学传感器[J].分析化学,2013,41(5):719-724.
[16]Travasso R D M,Sampaio Dos Aidos F,Bayani A,et al. Localized Redox Relays as a Privileged Mode of Cytoplasmic Hydrogen Peroxide Signaling[J]. Redox Biol,2017,12:233-245.
[17]Shoji E,Freund M S J. Potentiometric Sensors Based on the Inductive Effect on the p K(a)of Poly(aniline):A Nonenzymatic Glucose Sensor[J]. J Am Chem Soc,2001,123:3383-3384.
[18]Wei H,Wang E K. Nanomaterials with Enzyme-Like Characteristics(Nanozymes):Next-Generation Artificial Enzymes[J].Chem Soc Rev,2013,42:6060-6093.
[19]Daltrop O,Allen J W A,Willis A C. In Vitro Studies on Thioether Bond Formation Between Hydrogenobacter thermophilus Apocytochrome c(552)with Metalloprotoporphyrin Derivatives[J]. J Biol Chem,2004,279(44):45347-45353.
[20]HUANG Yingping,CAI Ruxiu. Studies on the Enzyme Catalytic Color Reaction System Using Hemoglobin as Enzyme Mimetic of Peroxidase and Its Application[J]. Chinese J Anal Chem,2001,29(4):378-382(in Chinese).黄应平,蔡汝秀.血红蛋白作为过氧化物模拟酶催化显色体系的研究与应用[J].分析化学,2001,49(4):378-382.
[21]XI Yongqing,QIU Haiou,YANG Ming,et al. Determination of Glucose by Catulltic Flurimetry with Hemoglobin Mimetic Peroxidase[J]. Chinese J Anal Chem,2008,36(10):1343-1348(in Chinese).席永清,邱海鸥,杨明,等.血红蛋白模拟酶催化荧光法测定葡萄糖[J].分析化学,2008,36(10):1343-1348??
[22]CHEN Qiuying,LI Donghui,ZHENG Hong,et al. Investigation on the Potential Use of the Mimetic Peroxidase-Catalyzed Reaction of Hydrogen Peroxide and o-Hydroxyphenylfluorone in Fluorescence Analysis[J]. Chinese J Anal Chem,1999,27(9):997-999(in Chinese).陈秋影,李东辉,郑洪,等.四磺基铁酞菁做为过氧化物模拟酶在过氧化氢及葡萄糖测定中的应用[J].分析化学,1999,27(9):997-999.
[23]Gao L Z,Zhuang J,Nie L,et al. Intrinsic Peroxidase-like Activity of Ferromagnetic Nanoparticles[J]. Nat Nanotechnol,2007,2:577-583.
[24]Zhang W,Ma D,Dun J. Prussian Blue Nanoparticles as Peroxidase Mimetics for Sensitive Colorimetric Detection of Hydrogen Peroxide and Glucose[J]. Talanta,2014,120:362-367.
[25]Wang X X,Wu Q,Shan Z,et al. BSA-Stabilized Au Clusters as Peroxidase Mimetics for Use in Xanthine Detection[J].Biosens Bioelectron,2011,26(8):3614-3619.
[26]Shi W,Zhang X D,He S H,et al. Co Fe2O4Magnetic Nanoparticles as a Peroxidase Mimic Mediated Chemiluminescence for Hydrogen Peroxide and Glucose[J]. Chem Commun(Camb),2011,47(38):10785-10787.
[27]Chen W,Chen J,Liu A L,et al. Peroxidase-Like Activity of Cupric Oxide Nanoparticle[J]. Chem Catal Chem,2013,3:1151-1154.
[28]Sun J H,Li C Y,Qi Y F,et al. Optimizing Colorimetric Assay Based on V2O5Nanozymes for Sensitive Detection of H2O2and Glucose[J]. Sensors(Basel),2016,16(4). pii:E584. doi:10. 3390/s16040584.
[29]Cai S F,Xiao W,Duan H H,et al. Single-Layer Rh Nanosheets with Ultrahigh Peroxidase-Like Activity for Colorimetric Biosensing[J]. Nano Res,2018,11:6304-6315.
[30]Xie J X,Zhang X D,Wang H,et al. Analytical and Environmental Applications of Nanoparticles as Enzyme Mimetics[J].Trends Anal Chem,2012,39:114-129.
[31]WANG Liping,LIU Yali,YANG Hui,et al. Synthesis and Anti-cataract Activity of a Novel Peroxidase Mimetic[J]. Chem J Chinese Univ,2004,25(11):2171-2173(in Chinese).王丽萍,刘亚丽,杨卉,等.一个新的过氧化物酶模拟物的合成及抗白内障活性研究[J].高等学校化学学报,2004,25(11):2171-2173.
[32]XIU Zhiming,LIN lin,ZHOU Peng,et al. Preparation of Deuterohemin by Recrystallization Method[J]. J Jilin Univ(Sci Ed),2013,51(2),331-334(in Chinese).修志明,林琳,周朋,等.重结晶法制备次氯血红素[J].吉林大学学报(理学版),2013,51(2),331-334.
[33]GUO Changrun,GUAN Shuwen,HUANG Lei,et al. Dh HP-6 Protection of Caenorhabditis Elegans from Heat Shock Injury Induced by Au Nanoparticles[J]. J Jilin Univ(Sci Ed),2012,50(1):153-156(in Chinese).郭常闰,关树文,黄磊,等.次血红素六肽对纳米金诱导线虫热激损伤的保护作用[J].吉林大学学报:理学版,2012,50(1):153-156.
[34]PENG Yan,LIU Yali,SUN Xiaoli,et al. Protective Effect of Heme Hexapeptide on Myocardial Mitochondrial Antioxidative Damage[J]. Chinese J Lab Diagn,2013,17(11):1952-1955(in Chinese).彭燕,刘亚丽,孙晓丽,等.次血红素六肽保护心肌线粒体抗氧化损伤作用研究[J].中国实验诊断学,2013,17(11):1952-1955
[35]侯玥,董占梅,马野,等. Dh HP-6替代过氧化物酶在葡萄糖检测中的应用[J].长春理工大学学报(自然科学版),2016,39(3):139-142.
[2]Marinho H S,Real C,Cyrne L,et al. Hydrogen Peroxide Sensing,Signaling and Regulation of Transcription Factors[J].Redox Biol,2014,2:535-562.
[3]Doskey C M,Buranasudja V,Wagner B A,et al. Tumor Cells Have Decreased Ability to Metabolize H2O2:Implications for Pharmacological Ascorbate in Cancer Therapy[J]. Redox Biol,2016,10(C):274-284.
[4]Sasaki A,Horiuchi N. Studies on Normal Blood Glucose Level-Statistical Approach to Interpretation of Glucose Tolerance Test[J]. J Chronic Dis,1976,29(2):129-140.
[5]Chang Q,Tang H Q. Optical Determination of Glucose and Hydrogen Peroxide Using a Nanocomposite Prepared from Glucose Oxidase and Magnetite Nanoparticles Immobilized on Graphene Oxide[J]. Microchim Acta,2014,181:527-534.
[6]Lan D,Li B X,Zhang Z J. Chemiluminescence Flow Biosensor for Glucose Based on Gold Nanoparticle-Enhanced Activities of Glucose Oxidase and Horseradish Peroxidase[J]. Biosens Bioelectron,2008,24:934-938.
[7]Chang Q,Zhu L,Jiang G,et al. Sensitive Fluorescent Probes for Determination of Hydrogen Peroxide and Glucose Based on Enzyme-Immobilized Magnetite/Silica Nanoparticles[J]. Anal Bioanal Chem,2009,395(7):2377-2385.
[8]Zhao K,Gu W,Zheng S S,et al. SDS-MoS2Nanoparticles as Highly-Efficient Peroxidase Mimetics for Colorimetric Detection of H2O2and Glucose[J]. Talanta,2015,141:47-52.
[9]Rauf S,Hayat Nawaz M A,Badea M,et al. Nano-Engineered Biomimetic Optical Sensors for Glucose Monitoring in Diabetes[J]. Sensors(Basel),2016,16(11):e1931.
[10]Huang W,Lin T Y,Cao Y,et al. Hierarchical Ni Co2O4Hollow Sphere as a Peroxidase Mimetic for Colorimetric Detection of H2O2and Glucose[J]. Sensors(Basel),2017,17(1):e217.
[11]Zhang W,Ma D,Du J. Prussian Blue Nanoparticles as Peroxidase Mimetics for Sensitive Colorimetric Detection of Hydrogen Peroxide and Glucose[J]. Talanta,2014,120:362-367.
[12]Lin J M,Arakawa H,Yamada M. Flow Injection Chemiluminescent Determination of Trace Amounts of Hydrogen Peroxide in Snow-Water Using KIO4K2CO3System[J]. Anal Chim Acta,1998,371:171-176.
[13]CAI Zijun,KUANG Yongqing,PAN Dan,et al. Sythesis and Characterization of a Novel ELF-97-based Fluorescent Probe for Hydrogen Peroxide Detection[J]. Chinese J Anal Chem,2015,43(11):1671-1675(in Chinese).蔡子君,匡永清,潘丹,等.基于ELF-97的新型H2O2荧光探针的合成与性能研究[J].分析化学,2015,43(11):1671-1675.
[14]Xu J R,Chen Z M. Determination of Peroxides in Environmental Samples by High Performance Liquid Chromatography[J]. Chinese J Chromatogr,2005,23:366-369.
[15]LI Li,LU Hongmei,DENG Liu. H2O2Electrochemistry Biosensor Based on Graphene and Gold Nanorods Composites[J].Chinese J Anal Chem,2013,41(5):719-724(in Chinese).李理,卢红梅,邓留.基于石墨烯和金纳米棒复合物的过氧化氢电化学传感器[J].分析化学,2013,41(5):719-724.
[16]Travasso R D M,Sampaio Dos Aidos F,Bayani A,et al. Localized Redox Relays as a Privileged Mode of Cytoplasmic Hydrogen Peroxide Signaling[J]. Redox Biol,2017,12:233-245.
[17]Shoji E,Freund M S J. Potentiometric Sensors Based on the Inductive Effect on the p K(a)of Poly(aniline):A Nonenzymatic Glucose Sensor[J]. J Am Chem Soc,2001,123:3383-3384.
[18]Wei H,Wang E K. Nanomaterials with Enzyme-Like Characteristics(Nanozymes):Next-Generation Artificial Enzymes[J].Chem Soc Rev,2013,42:6060-6093.
[19]Daltrop O,Allen J W A,Willis A C. In Vitro Studies on Thioether Bond Formation Between Hydrogenobacter thermophilus Apocytochrome c(552)with Metalloprotoporphyrin Derivatives[J]. J Biol Chem,2004,279(44):45347-45353.
[20]HUANG Yingping,CAI Ruxiu. Studies on the Enzyme Catalytic Color Reaction System Using Hemoglobin as Enzyme Mimetic of Peroxidase and Its Application[J]. Chinese J Anal Chem,2001,29(4):378-382(in Chinese).黄应平,蔡汝秀.血红蛋白作为过氧化物模拟酶催化显色体系的研究与应用[J].分析化学,2001,49(4):378-382.
[21]XI Yongqing,QIU Haiou,YANG Ming,et al. Determination of Glucose by Catulltic Flurimetry with Hemoglobin Mimetic Peroxidase[J]. Chinese J Anal Chem,2008,36(10):1343-1348(in Chinese).席永清,邱海鸥,杨明,等.血红蛋白模拟酶催化荧光法测定葡萄糖[J].分析化学,2008,36(10):1343-1348??
[22]CHEN Qiuying,LI Donghui,ZHENG Hong,et al. Investigation on the Potential Use of the Mimetic Peroxidase-Catalyzed Reaction of Hydrogen Peroxide and o-Hydroxyphenylfluorone in Fluorescence Analysis[J]. Chinese J Anal Chem,1999,27(9):997-999(in Chinese).陈秋影,李东辉,郑洪,等.四磺基铁酞菁做为过氧化物模拟酶在过氧化氢及葡萄糖测定中的应用[J].分析化学,1999,27(9):997-999.
[23]Gao L Z,Zhuang J,Nie L,et al. Intrinsic Peroxidase-like Activity of Ferromagnetic Nanoparticles[J]. Nat Nanotechnol,2007,2:577-583.
[24]Zhang W,Ma D,Dun J. Prussian Blue Nanoparticles as Peroxidase Mimetics for Sensitive Colorimetric Detection of Hydrogen Peroxide and Glucose[J]. Talanta,2014,120:362-367.
[25]Wang X X,Wu Q,Shan Z,et al. BSA-Stabilized Au Clusters as Peroxidase Mimetics for Use in Xanthine Detection[J].Biosens Bioelectron,2011,26(8):3614-3619.
[26]Shi W,Zhang X D,He S H,et al. Co Fe2O4Magnetic Nanoparticles as a Peroxidase Mimic Mediated Chemiluminescence for Hydrogen Peroxide and Glucose[J]. Chem Commun(Camb),2011,47(38):10785-10787.
[27]Chen W,Chen J,Liu A L,et al. Peroxidase-Like Activity of Cupric Oxide Nanoparticle[J]. Chem Catal Chem,2013,3:1151-1154.
[28]Sun J H,Li C Y,Qi Y F,et al. Optimizing Colorimetric Assay Based on V2O5Nanozymes for Sensitive Detection of H2O2and Glucose[J]. Sensors(Basel),2016,16(4). pii:E584. doi:10. 3390/s16040584.
[29]Cai S F,Xiao W,Duan H H,et al. Single-Layer Rh Nanosheets with Ultrahigh Peroxidase-Like Activity for Colorimetric Biosensing[J]. Nano Res,2018,11:6304-6315.
[30]Xie J X,Zhang X D,Wang H,et al. Analytical and Environmental Applications of Nanoparticles as Enzyme Mimetics[J].Trends Anal Chem,2012,39:114-129.
[31]WANG Liping,LIU Yali,YANG Hui,et al. Synthesis and Anti-cataract Activity of a Novel Peroxidase Mimetic[J]. Chem J Chinese Univ,2004,25(11):2171-2173(in Chinese).王丽萍,刘亚丽,杨卉,等.一个新的过氧化物酶模拟物的合成及抗白内障活性研究[J].高等学校化学学报,2004,25(11):2171-2173.
[32]XIU Zhiming,LIN lin,ZHOU Peng,et al. Preparation of Deuterohemin by Recrystallization Method[J]. J Jilin Univ(Sci Ed),2013,51(2),331-334(in Chinese).修志明,林琳,周朋,等.重结晶法制备次氯血红素[J].吉林大学学报(理学版),2013,51(2),331-334.
[33]GUO Changrun,GUAN Shuwen,HUANG Lei,et al. Dh HP-6 Protection of Caenorhabditis Elegans from Heat Shock Injury Induced by Au Nanoparticles[J]. J Jilin Univ(Sci Ed),2012,50(1):153-156(in Chinese).郭常闰,关树文,黄磊,等.次血红素六肽对纳米金诱导线虫热激损伤的保护作用[J].吉林大学学报:理学版,2012,50(1):153-156.
[34]PENG Yan,LIU Yali,SUN Xiaoli,et al. Protective Effect of Heme Hexapeptide on Myocardial Mitochondrial Antioxidative Damage[J]. Chinese J Lab Diagn,2013,17(11):1952-1955(in Chinese).彭燕,刘亚丽,孙晓丽,等.次血红素六肽保护心肌线粒体抗氧化损伤作用研究[J].中国实验诊断学,2013,17(11):1952-1955
[35]侯玥,董占梅,马野,等. Dh HP-6替代过氧化物酶在葡萄糖检测中的应用[J].长春理工大学学报(自然科学版),2016,39(3):139-142.