超氧化物歧化酶催化-电化学调控的原子转移自由基聚合方法制备分子印迹聚合物
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摘要
病理学中对含金属蛋白质的敏感检测极其重要。本文以超氧化物歧化酶(SOD)作为金属蛋白,SOD既作为模板分子又作为催化剂进行电化学调控的原子转移自由基聚合(e ATRP)反应制备蛋白质印迹聚合物(PIPs),用于SOD电化学生物传感器。该方法不需要过渡金属离子,具有制备简单、节约试剂、保护环境等优点。我们选用L-半胱氨酸和纳米金修饰的金电极(Au/L-cys/nano Au)作为工作电极将氧化型SOD催化还原为还原型SOD,利用还原型SOD的Cu (Ⅰ)粒子,在引发剂4-硫苯基-2-溴-2-甲基丙酸酯(4-mercaptophenyl2-bromo-2-methylpropanoate,4-HTP-Br)修饰的金电极上调控丙烯酰胺、N,N-亚甲基双丙烯酰胺的e ATRP聚合制备SOD PIPs。利用循环伏安法(CV)和X射线光电子能谱(XPS)方法对其进行了表征。通过微分脉冲伏安法(DPV),在最优的条件下利用此修饰电极对溶液中的SOD进行检测,线性响应范围为1. 0×10-7～1. 0×102mg/L,检测限为6. 8×10-8mg/L(S/N=3),相关系数为0. 995。与其它检测SOD的方法相比,该方法具有更宽的线性范围和较低的检测限。本研究对于制备PIPs,用蛋白质催化的e ATRP和含金属蛋白的敏感检测均有重要意义。
Sensitive detection of metal-containing proteins is extremely important in pathology. Superoxide dismutase(SOD) was represented as metal-containing protein to prepare protein imprinted polymers(PIPs)and used for electrochemical biosensor. SOD was used as a template molecule and as a catalyst for electrochemically mediated atom transfer radical polymerization(eATRP) reaction to prepare SOD PIPs. This method does not require transition metal ions and owns the advantages of simple preparation,reagent saving,environmental protection,etc. In this experiment,we selected L-cysteine(L-cys) and nano gold modified gold electrode(Au/L-cys/nanoAu) as one working electrode for catalytic reduction of SOD, and initiator(4-mercaptophenyl 2-bromo-2-methylpropanoate,4-HTP-Br) modified Au electrode as another working electrode for SOD PIPs modification. The electrode modified with PIPs was characterized by cyclic voltammetry(CV) and X ray photoelectron spectroscopy(XPS). Finally,the PIPs-modified electrode was used as a biosensor for the determination of SOD by differential pulse voltammetry(DPV) measurement. The linear range is 1. 0 × 10-7 to 100 mg/L with the detection limit of 6. 8 × 10-8 mg/L(S/N = 3),the correlation coefficient is 0. 995. Compared with other methods for detecting SOD,this method has a wider linear range and lower detection limits. This work is significant for the preparation of PIPs,eATRP catalyzed by proteins and the sensitive detection of metal-containing proteins.
Sensitive detection of metal-containing proteins is extremely important in pathology. Superoxide dismutase(SOD) was represented as metal-containing protein to prepare protein imprinted polymers(PIPs)and used for electrochemical biosensor. SOD was used as a template molecule and as a catalyst for electrochemically mediated atom transfer radical polymerization(eATRP) reaction to prepare SOD PIPs. This method does not require transition metal ions and owns the advantages of simple preparation,reagent saving,environmental protection,etc. In this experiment,we selected L-cysteine(L-cys) and nano gold modified gold electrode(Au/L-cys/nanoAu) as one working electrode for catalytic reduction of SOD, and initiator(4-mercaptophenyl 2-bromo-2-methylpropanoate,4-HTP-Br) modified Au electrode as another working electrode for SOD PIPs modification. The electrode modified with PIPs was characterized by cyclic voltammetry(CV) and X ray photoelectron spectroscopy(XPS). Finally,the PIPs-modified electrode was used as a biosensor for the determination of SOD by differential pulse voltammetry(DPV) measurement. The linear range is 1. 0 × 10-7 to 100 mg/L with the detection limit of 6. 8 × 10-8 mg/L(S/N = 3),the correlation coefficient is 0. 995. Compared with other methods for detecting SOD,this method has a wider linear range and lower detection limits. This work is significant for the preparation of PIPs,eATRP catalyzed by proteins and the sensitive detection of metal-containing proteins.
引文
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[11]Wang X,Dong S,Bai Q. Preparation of Lysozyme Molecularly Imprinted Polymers and Purification of Lysozyme from Egg White[J]. Biomed Chromatogr,2014,28(6):907-912.
[12]Chen L,Wang X,Lu W,et al. Molecular Imprinting:Perspectives and Applications[J]. Chem Soc Rev,2016,45(8):2137-2211.
[13]Yang L,Zhi L,Song C J. Evaluation the Separation Efficiency of Galangin Molecular Imprinted Polymers by HPLC[J]. J Chengde Med College,2015,132(3):190-192.
[14]Yang Y,Wang X,Fang G,et al. Electrochemiluminescence Analysis Based on Molecular Imprinting Technique[J]. Prog Chem,2016,28(9):1351-1362.
[15]Dan R,Wang Y,Du L,et al. The Synthesis of Molecular Imprinted Chitosan-Gels Copolymerized with Multiform Functional Monomers at Three Different Temperatures and the Recognition for the Template Ovalbumin[J]. Analyst,2013,138(12):3433-3443.
[16]Erdogan-Haug B,Kavanagh M A,Aloshyna L M,et al. Crosslinkable Syrup Copolymers with Aminoalkyl(meth)Acryloyl Solvent Monomers:US,EP2556128. B1[P]. 2014-06-04.
[17]JIANG Jigang. Research and Application of Molecular Imprinting Technology in Drug Analysis[J]. Shandong Normal Univ,2003(in Chinese).姜吉刚.分子印迹技术在药物分析中的研究与应用[J].山东师范大学,2003.
[18]Wu W T,Shen J,Li Y X,et al. Specific Glucose-to-SPR Signal Transduction at Physiological p H by Molecularly Imprinted Responsive Hybrid Microgels[J]. Biomaterials,2012,33(29):7115-7125.
[19]Turner N W,Liu X,Piletsky S A,et al. Recognition of Conformational Changes in Lactoglobulin by Molecularly Imprinted Thin Films[J]. Biomacromolecules,2007,8(9):2781-2787.
[20]Li H,Wang L. Highly Selective Detection of Polycyclic Aromatic Hydrocarbonsusing Multifunctional Magnetic-Luminescent Molecularly Imprinted Polymers[J]. ACS Appl Mater Interfaces,2013,5(21):10502-10509.
[21]Wulff G,Liu J Q. Design of Biomimetic Catalysts by Molecular Imprinting in Synthetic Polymers:The Role of Transition State Stabilization[J]. Acc Chem Res,2012,45(2):239-247.
[22]Panagiotopoulou M,Beyazit S,Nestora S,et al. Initiator-Free Synthesis of Molecularly Imprinted Polymers by Polymerization of Self-initiated Monomers[J]. Polymer,2015,66:43-51.
[23]Tan L,Kang C C,Xu S Y,et al. Selective Room Temperature Phosphorescence Sensing of Target Protein Using Mn-Doped Zn S QDs-Embedded Molecularly Imprinted Polymer[J]. Biosens Bioelectron,2013,48(19):216-223.
[24]Zhang R,Xu S,Luo J,et al. Molecularly Imprinted Photo-sensitive Polyglutamic Acid Nanoparticles for Electrochemical Sensing of Hemoglobin[J]. Microchim Acta,2015,182(1/2):175-183.
[25]MAO Cheng,WEI Zhaoshui,DU Juntao. Research Progress of Acrylamide Polymer Radical Polymerization Initiation System[J]. Guangdong Chem Ind,2013,40(14):126-127(in Chinese).毛程,韦兆水,杜俊涛.丙烯酰胺聚合物自由基聚合引发体系研究进展[J].广东化工,2013,40(14):126-127.
[26]Qiu X,Wu Y,Chen D,et al. Fabrication of Novel Stir Bar Sorptive Extraction Coating Based on Magnetic Molecularly Imprinted Polymer Through Atom Transfer Radical Polymerization for Trace Analysis of Estrogens in Milk[J]. J Nanosci Nanotechnol,2016,16(12):12374-12381.
[27]Sigg S J,Seidi F,Renggli K,et al. Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization[J].Macromol Rapid Commun,2011,32(21):1710-1715.
[28]Shanmugam S,Boyer C. Polymer Synthesis:Organic Photocatalysts for Cleaner Polymer Synthesis[J]. Science,2016,352(6289):1053-1054.
[29]Magenau A J,Strandwitz N C,Gennaro A,et al. Electrochemically Mediated Atom Transfer Radical Polymerization[J].Science,2011,332(6025):81-84.
[30]Silva T B,Spulber M,Kocik M K,et al. Hemoglobin and Red Blood Cells Catalyze Atom Transfer Radical Polymerization[J]. Biomacromol,2013,14(8):2703-2712.
[31] Fodor C,Gajewska B,Rifaie-Graham O,et al. Laccase-catalyzed Controlled Radical Polymerization of N-Vinylimidazole[J]. Polym Chem,2016,7(43):6617-6625.
[32]SUN Yue,DU Hongying,LAN Yuting,et al. Preparation of Hemoglobin Imprinted Polymer by Autocatalytic ATRP:CN,201510410654. 6[P]. 2015-11-04(in Chinese).孙越,杜洪莹,兰玉廷,等.自催化ATRP制备血红蛋白印迹聚合物的方法:中国,201510410654. 6[P]. 2015-11-04.
[33]Sun Y,Du H,Lan Y,et al. Preparation of Hemoglobin(Hb)Imprinted Polymer by Hb Catalyzed e ATRP and Its Application in Biosensor[J]. Biosens Bioelectron,2016,77:894-900.
[34]Sun Y,Du H Y,Deng Y,et al. Preparation of Polyacrylamide via Surface-Initiated Electrochemical-Mediated Atom Transfer Radical Polymerization(SI-eATRP)for Pb2+Sensing[J]. J Solid State Electrochem,2016,20(1):105-113.
[35]Wang C L,Fen M A,Zheng H Y,et al. Synthesis of Salicylic Acid Molecularly Imprinted Polymer Using Acrylamide as a Functional Monomer[J]. Fine Chem,2007,24(8):729-728.
[36]Chen F F,Xie X Y,Shi Y P. Preparation of Magnetic Molecularly Imprinted Polymer for Selective Recognition of Resveratrol in Wine[J]. J Chromatogr A,2013,1300(14):112-118.
[37]LIU Fen,ZHAO Zhijuan,QIU Limei. XPS Photoelectron Peak and Auger Electron Peak Position Table[J]. Anal Test Technol Instrum,2009,15(1):1-17(in Chinese).刘芬,赵志娟,邱丽美. XPS光电子峰和俄歇电子峰峰位表[J].分析测试技术与仪器,2009,15(1):1-17.
[38]HE Xiaoying,WEI Wei,SONG Tao,et al. Electrochemical Behavior of Superoxide Dismutase on the Gold Nanoparticles/L-Cysteine/Au Modified Electrode[J]. Chem Res Appl,2012,24(8):1192-1196(in Chinese).何晓英,魏胤,宋桃,等.超氧化物歧化酶在纳米金/L-半胱氨酸修饰金电极上的电化学行为[J].化学研究与应用,2012,24(8):1192-1196.
[39]Zhidkova T V,Proskurnina E V,Parfenov E A,et al. Determination of Superoxide Dismutase and SOD-Mimetic Activities by a Chemical System:Co2/H2O2/Lucigenin[J]. Anal Bioanal Chem,2011,401(1):381-386.
[40] YANG Tangbin,LIU Zhenxiu,WAN Yumin. Enzyme Immunoassay for Cuprozinc-Superoxide Dismutase in Whole Blood and Urine During Head down Bed Rest[J]. Space Med Med Eng,1998,(3):162-166(in Chinese).杨唐斌,刘振秀,万玉民.头低位-60卧床中血、尿中Cu,Zn-SOD的酶免疫法测定[J].航天医学与医学工程,1998,(3):162-166.
[41]WEI Wei. Research and Application of Optical Probes Interaction with Copper-Zinc Superoxide Dismutase and Amine Small Molecules[D]. Ji'nan:Shandong Normal University,2007(in Chinese).魏薇.光学探针与铜锌超氧化物歧化酶、胺类小分子相互作用的研究及应用[D].济南:山东师范大学,2007.
[2]Olson K R,Gao Y,Arif F,et al. Metabolism of Hydrogen Sulfide(H2S)and Production of Reactive Sulfur Species(RSS)by Superoxide Dismutase[J]. Redox Biol,2018,(15):74-85.
[3]Healy E F,Cervantes L. An in Silico Study of the Effect of SOD1 Electrostatic Loop Dynamics Amyloid-Like Filament Formation[J]. Eur Biophys J,2016,45(8):1-7.
[4]Zhang L,Er J C,Jiang H,et al. A Highly Selective Fluorogenic Probe for the Detection and In vivo Imaging of Cu/Zn Superoxide Dismutase[J]. Chem Commun,2016,52(58):9093-9096.
[5]Perry J J,Shin D S,Getzoff E D,et al. The Structural Biochemistry of the Superoxide Dismutases[J]. Biochim Biophys Acta,2010,1804(2):245-262.
[6]Liu H,Liu L,Qin C,et al. Mitochondrial Damage and Changes of Malondidehyde and Superoxide Dismutase in Acute Alcoholic Liver[J]. J Hubei Eng Univ,2013,33(3):53-55.
[7]Corbisier P,Houbion A,Remacle J. A New Technique for Highly Sensitive Detection of Superoxide Dismutase Activity by Chemiluminescence[J]. Anal Biochem,1987,164(1):240-247.
[8]Marcocci L,Mavelli I,Giulio AD,et al. Room Temperature Electron Spin Resonance of Superoxide Dismutase-Loaded Liposomes and Erythrocytes. A Direct Approach to the Interaction of O2-with Cells[J]. Biochim Biophys Acta,1989,979(1):99-104.
[9]Ukeda H,Kawana D,Maeda S,et al. Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-Soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase[J]. J Agric Chem Soc Jpn,1999,63(3):485-488.
[10] XU Daguang,YIN Zhigang,HOU Xiaoqiang,et al. Application and Progress of Ultraviolet-Visible Spectrophotometry in Drug Analysis[J]. Chinese J Med Phys,2006,23(6):432-433(in Chinese).徐大光,阴志刚,侯晓强,等.紫外-可见分光光度法在药物分析中的应用及进展[J].中国医学物理学杂志,2006,23(6):432-433.
[11]Wang X,Dong S,Bai Q. Preparation of Lysozyme Molecularly Imprinted Polymers and Purification of Lysozyme from Egg White[J]. Biomed Chromatogr,2014,28(6):907-912.
[12]Chen L,Wang X,Lu W,et al. Molecular Imprinting:Perspectives and Applications[J]. Chem Soc Rev,2016,45(8):2137-2211.
[13]Yang L,Zhi L,Song C J. Evaluation the Separation Efficiency of Galangin Molecular Imprinted Polymers by HPLC[J]. J Chengde Med College,2015,132(3):190-192.
[14]Yang Y,Wang X,Fang G,et al. Electrochemiluminescence Analysis Based on Molecular Imprinting Technique[J]. Prog Chem,2016,28(9):1351-1362.
[15]Dan R,Wang Y,Du L,et al. The Synthesis of Molecular Imprinted Chitosan-Gels Copolymerized with Multiform Functional Monomers at Three Different Temperatures and the Recognition for the Template Ovalbumin[J]. Analyst,2013,138(12):3433-3443.
[16]Erdogan-Haug B,Kavanagh M A,Aloshyna L M,et al. Crosslinkable Syrup Copolymers with Aminoalkyl(meth)Acryloyl Solvent Monomers:US,EP2556128. B1[P]. 2014-06-04.
[17]JIANG Jigang. Research and Application of Molecular Imprinting Technology in Drug Analysis[J]. Shandong Normal Univ,2003(in Chinese).姜吉刚.分子印迹技术在药物分析中的研究与应用[J].山东师范大学,2003.
[18]Wu W T,Shen J,Li Y X,et al. Specific Glucose-to-SPR Signal Transduction at Physiological p H by Molecularly Imprinted Responsive Hybrid Microgels[J]. Biomaterials,2012,33(29):7115-7125.
[19]Turner N W,Liu X,Piletsky S A,et al. Recognition of Conformational Changes in Lactoglobulin by Molecularly Imprinted Thin Films[J]. Biomacromolecules,2007,8(9):2781-2787.
[20]Li H,Wang L. Highly Selective Detection of Polycyclic Aromatic Hydrocarbonsusing Multifunctional Magnetic-Luminescent Molecularly Imprinted Polymers[J]. ACS Appl Mater Interfaces,2013,5(21):10502-10509.
[21]Wulff G,Liu J Q. Design of Biomimetic Catalysts by Molecular Imprinting in Synthetic Polymers:The Role of Transition State Stabilization[J]. Acc Chem Res,2012,45(2):239-247.
[22]Panagiotopoulou M,Beyazit S,Nestora S,et al. Initiator-Free Synthesis of Molecularly Imprinted Polymers by Polymerization of Self-initiated Monomers[J]. Polymer,2015,66:43-51.
[23]Tan L,Kang C C,Xu S Y,et al. Selective Room Temperature Phosphorescence Sensing of Target Protein Using Mn-Doped Zn S QDs-Embedded Molecularly Imprinted Polymer[J]. Biosens Bioelectron,2013,48(19):216-223.
[24]Zhang R,Xu S,Luo J,et al. Molecularly Imprinted Photo-sensitive Polyglutamic Acid Nanoparticles for Electrochemical Sensing of Hemoglobin[J]. Microchim Acta,2015,182(1/2):175-183.
[25]MAO Cheng,WEI Zhaoshui,DU Juntao. Research Progress of Acrylamide Polymer Radical Polymerization Initiation System[J]. Guangdong Chem Ind,2013,40(14):126-127(in Chinese).毛程,韦兆水,杜俊涛.丙烯酰胺聚合物自由基聚合引发体系研究进展[J].广东化工,2013,40(14):126-127.
[26]Qiu X,Wu Y,Chen D,et al. Fabrication of Novel Stir Bar Sorptive Extraction Coating Based on Magnetic Molecularly Imprinted Polymer Through Atom Transfer Radical Polymerization for Trace Analysis of Estrogens in Milk[J]. J Nanosci Nanotechnol,2016,16(12):12374-12381.
[27]Sigg S J,Seidi F,Renggli K,et al. Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization[J].Macromol Rapid Commun,2011,32(21):1710-1715.
[28]Shanmugam S,Boyer C. Polymer Synthesis:Organic Photocatalysts for Cleaner Polymer Synthesis[J]. Science,2016,352(6289):1053-1054.
[29]Magenau A J,Strandwitz N C,Gennaro A,et al. Electrochemically Mediated Atom Transfer Radical Polymerization[J].Science,2011,332(6025):81-84.
[30]Silva T B,Spulber M,Kocik M K,et al. Hemoglobin and Red Blood Cells Catalyze Atom Transfer Radical Polymerization[J]. Biomacromol,2013,14(8):2703-2712.
[31] Fodor C,Gajewska B,Rifaie-Graham O,et al. Laccase-catalyzed Controlled Radical Polymerization of N-Vinylimidazole[J]. Polym Chem,2016,7(43):6617-6625.
[32]SUN Yue,DU Hongying,LAN Yuting,et al. Preparation of Hemoglobin Imprinted Polymer by Autocatalytic ATRP:CN,201510410654. 6[P]. 2015-11-04(in Chinese).孙越,杜洪莹,兰玉廷,等.自催化ATRP制备血红蛋白印迹聚合物的方法:中国,201510410654. 6[P]. 2015-11-04.
[33]Sun Y,Du H,Lan Y,et al. Preparation of Hemoglobin(Hb)Imprinted Polymer by Hb Catalyzed e ATRP and Its Application in Biosensor[J]. Biosens Bioelectron,2016,77:894-900.
[34]Sun Y,Du H Y,Deng Y,et al. Preparation of Polyacrylamide via Surface-Initiated Electrochemical-Mediated Atom Transfer Radical Polymerization(SI-eATRP)for Pb2+Sensing[J]. J Solid State Electrochem,2016,20(1):105-113.
[35]Wang C L,Fen M A,Zheng H Y,et al. Synthesis of Salicylic Acid Molecularly Imprinted Polymer Using Acrylamide as a Functional Monomer[J]. Fine Chem,2007,24(8):729-728.
[36]Chen F F,Xie X Y,Shi Y P. Preparation of Magnetic Molecularly Imprinted Polymer for Selective Recognition of Resveratrol in Wine[J]. J Chromatogr A,2013,1300(14):112-118.
[37]LIU Fen,ZHAO Zhijuan,QIU Limei. XPS Photoelectron Peak and Auger Electron Peak Position Table[J]. Anal Test Technol Instrum,2009,15(1):1-17(in Chinese).刘芬,赵志娟,邱丽美. XPS光电子峰和俄歇电子峰峰位表[J].分析测试技术与仪器,2009,15(1):1-17.
[38]HE Xiaoying,WEI Wei,SONG Tao,et al. Electrochemical Behavior of Superoxide Dismutase on the Gold Nanoparticles/L-Cysteine/Au Modified Electrode[J]. Chem Res Appl,2012,24(8):1192-1196(in Chinese).何晓英,魏胤,宋桃,等.超氧化物歧化酶在纳米金/L-半胱氨酸修饰金电极上的电化学行为[J].化学研究与应用,2012,24(8):1192-1196.
[39]Zhidkova T V,Proskurnina E V,Parfenov E A,et al. Determination of Superoxide Dismutase and SOD-Mimetic Activities by a Chemical System:Co2/H2O2/Lucigenin[J]. Anal Bioanal Chem,2011,401(1):381-386.
[40] YANG Tangbin,LIU Zhenxiu,WAN Yumin. Enzyme Immunoassay for Cuprozinc-Superoxide Dismutase in Whole Blood and Urine During Head down Bed Rest[J]. Space Med Med Eng,1998,(3):162-166(in Chinese).杨唐斌,刘振秀,万玉民.头低位-60卧床中血、尿中Cu,Zn-SOD的酶免疫法测定[J].航天医学与医学工程,1998,(3):162-166.
[41]WEI Wei. Research and Application of Optical Probes Interaction with Copper-Zinc Superoxide Dismutase and Amine Small Molecules[D]. Ji'nan:Shandong Normal University,2007(in Chinese).魏薇.光学探针与铜锌超氧化物歧化酶、胺类小分子相互作用的研究及应用[D].济南:山东师范大学,2007.
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