单个铁蛋白分子的机械性能研究
摘要
纳米生物材料研究是目前材料科学研究的一个热点,其相应发展起来的纳米生物技术也将成为21世纪最具有市场应用潜力的新兴科学技术,正在日益受到全世界的关注。纳米生物技术属于纳米技术和生物技术的交叉,在化学与生物纳米结构的构建与应用中具有重要作用,与传统的结构过程相比,这些新颖的过程具有高精度、高度灵活性和低成本等突出优点,因而将广泛应用于生物医学、电子学、材料学等领域。
铁蛋白(ferritin)是一种参与和维持生物体内铁代谢平衡的重要功能蛋白,它是一类广泛存在于动植物及微生物细胞中、含铁量较高的蛋白质。铁蛋白除了重要的生物学功能外,其独特的结构使得它具有特殊的物理化学性质,如磁性、电化学性能等,这使得它除了具有重要的生物学上的功能外,在材料学领域也得到了广泛的应用。例如,铁蛋白可以作为碳纳米导管生长过程中的催化剂,可以作为合成具有磁性或导电性的半导体晶体材料的模版。最近,Shin等人将功能化的铁蛋白纳米颗粒掺入到聚合物中,显著增强了聚合物纳米纤维的机械性能;Bhattacharyya等人也发现,掺入铁蛋白的多层碳纳米管(MWCNTs)和聚合物薄膜间的相互作用可以增强多层碳纳米管合成薄膜的机械性能。
本文阐述了铁蛋白的独特结构和铁蛋白在生物体内是如何参与和维持铁代谢平衡的;介绍了研究的主要工具---原子力显微镜(AFM)的基本原理等相关知识。研究了铁蛋白在不同溶液pH值下,在APS修饰的云母表面的吸附情况,结果表明铁蛋白在APS修饰的云母表面上的吸附量会随着溶液pH值的增大而逐渐增多。本文重点测量了单个铁蛋白分子的弹性模量:先利用AFM的轻敲模式,制备出分散较好的铁蛋白样品;然后在AFM接触模式下测得铁蛋白的力-体积数据,从而提取出力曲线;通过现有的Hertz模型公式可以计算出单个铁蛋白的弹性模量;最后又和相同条件下计算的胶体金颗粒的弹性模量进行了比较。
The nano-biological material is a focus in material science. The nano-biological technique will be the potentialest technique in the 21st century. The nano-biological technique is a joint class by nano meter technique and biological technique. The nano-biological technique is very important in chemistry and biological nano-structure. The new process is high precision, high agility and low cost, so it will be widely used in biological, medicine, electronics and material.
The iron-storage proteins ferritins broadly exist in animals, plants and human bodies. Besides its important biological function, ferritin has been used widely in materials science due to its unique physical and chemical properties, such as magnetic and electrochemical properties. For example, ferritin has been used as a catalyst for carbon nanotube growth and as a template for synthesis of magnetic and photonic nanostructures. Recently, Shin et al. reported that poly(vinyl alcohol) (PVA) nanofibers containing bimolecular ferritin nanoparticles exhibited the enhancement of elastic modulus as compared to pure PVA nanofibers due to chemical interactions between the ferritin and the PVA matrix. Bhattacharyya found that the novel composite films containing ferritin-functionalized multiwall carbon nanotubes (MWCNTs) were reinforced by hydrogen bonding formed between the ferritin functionalized MWCNTs and the polymeric films.
The particular structure of ferritin and how to participate in and how to keep the iron metabolize balance in the body were clarified in this work. The correlative information about Atomic Force Microscopy (AFM) were introduced. We investigated the pH-dependent adsorption of ferritin on mica modifisd by APS. The result was the surface density of ferritin was controlled by a variatin in the solution pH (from 3 to 7). The mechanical property of individual ferritin was measured with force-volume mapping (FV) under contact mode of AFM in this work. The elastic modulus of individual ferritin was estimated by the Hertz mode. In addition, the elastic modulus of individual ferritin was compared with that of the colloid gold nanoparticle.
铁蛋白(ferritin)是一种参与和维持生物体内铁代谢平衡的重要功能蛋白,它是一类广泛存在于动植物及微生物细胞中、含铁量较高的蛋白质。铁蛋白除了重要的生物学功能外,其独特的结构使得它具有特殊的物理化学性质,如磁性、电化学性能等,这使得它除了具有重要的生物学上的功能外,在材料学领域也得到了广泛的应用。例如,铁蛋白可以作为碳纳米导管生长过程中的催化剂,可以作为合成具有磁性或导电性的半导体晶体材料的模版。最近,Shin等人将功能化的铁蛋白纳米颗粒掺入到聚合物中,显著增强了聚合物纳米纤维的机械性能;Bhattacharyya等人也发现,掺入铁蛋白的多层碳纳米管(MWCNTs)和聚合物薄膜间的相互作用可以增强多层碳纳米管合成薄膜的机械性能。
本文阐述了铁蛋白的独特结构和铁蛋白在生物体内是如何参与和维持铁代谢平衡的;介绍了研究的主要工具---原子力显微镜(AFM)的基本原理等相关知识。研究了铁蛋白在不同溶液pH值下,在APS修饰的云母表面的吸附情况,结果表明铁蛋白在APS修饰的云母表面上的吸附量会随着溶液pH值的增大而逐渐增多。本文重点测量了单个铁蛋白分子的弹性模量:先利用AFM的轻敲模式,制备出分散较好的铁蛋白样品;然后在AFM接触模式下测得铁蛋白的力-体积数据,从而提取出力曲线;通过现有的Hertz模型公式可以计算出单个铁蛋白的弹性模量;最后又和相同条件下计算的胶体金颗粒的弹性模量进行了比较。
The nano-biological material is a focus in material science. The nano-biological technique will be the potentialest technique in the 21st century. The nano-biological technique is a joint class by nano meter technique and biological technique. The nano-biological technique is very important in chemistry and biological nano-structure. The new process is high precision, high agility and low cost, so it will be widely used in biological, medicine, electronics and material.
The iron-storage proteins ferritins broadly exist in animals, plants and human bodies. Besides its important biological function, ferritin has been used widely in materials science due to its unique physical and chemical properties, such as magnetic and electrochemical properties. For example, ferritin has been used as a catalyst for carbon nanotube growth and as a template for synthesis of magnetic and photonic nanostructures. Recently, Shin et al. reported that poly(vinyl alcohol) (PVA) nanofibers containing bimolecular ferritin nanoparticles exhibited the enhancement of elastic modulus as compared to pure PVA nanofibers due to chemical interactions between the ferritin and the PVA matrix. Bhattacharyya found that the novel composite films containing ferritin-functionalized multiwall carbon nanotubes (MWCNTs) were reinforced by hydrogen bonding formed between the ferritin functionalized MWCNTs and the polymeric films.
The particular structure of ferritin and how to participate in and how to keep the iron metabolize balance in the body were clarified in this work. The correlative information about Atomic Force Microscopy (AFM) were introduced. We investigated the pH-dependent adsorption of ferritin on mica modifisd by APS. The result was the surface density of ferritin was controlled by a variatin in the solution pH (from 3 to 7). The mechanical property of individual ferritin was measured with force-volume mapping (FV) under contact mode of AFM in this work. The elastic modulus of individual ferritin was estimated by the Hertz mode. In addition, the elastic modulus of individual ferritin was compared with that of the colloid gold nanoparticle.
引文
[1] Ovemey R M,Meyer E,Frommer J,et al.A comparative atomic force microscopic study of liquid crystal films.Langmuir,1993,9:341—346
[2] Song S N,Yi X J,Zheng J Q,Dimensional crossover of Shubnikov-de Haas oscillations in thin films of gray tin.Phys Rev Lett,1990,65:227—210
[3] M F Yu,T Kowalewski, R S Ruof.Investigation of under Controlled the Radial Deformability of Individual Carbon Nanotubes Force,Phys Rev Let.2000,85(7):145
[4] Thomas A M,Rogers J F,Leedman P J.Iron-regulatory,iron-responsive and ferritin mRNA translation.In J Biochem cell Biol,1999,31:l139—1152.
[5]刘璐,刘瑶,严玉仙.铁蛋白.国外医学医学地理分册,2005,26(4):171—172
[6] Aust S D.Ferritin as a source of iron and protection from iron-induced toxicities.Toxicol Lett,1995,82—83:941—944
[7] Goto F,Yoshihara T,Shjgemoto N,et a1.Nature Biotechnology,1999,17:282—286
[8] Xu D,Watt G D,Harb J N,et al.Electrical conductivity of ferritin proteins by conductive AFM.Nano Lett,2005,5(4):571—577
[9] Binnig G,Quate C F,Gerber C.Atomic force microscope.Phys Rev Lett,1986,56:930—933
[10]朱杰,孙润广.生命科学仪器,2005,3(1):22—26
[11] Radmacher,et al.Direct observation of enzyme activity with the atomic force microscope.Science,1994,265:1577—1579
[12] MultiMode Scanning Probe Microscope Instruction Manual,Digital Instruments Corp.
[13] Jamsen B.effects of sleep on immune cell parameter.Curt Opin Infect Dis,1992,6:526—529
[14] Hook F,Rodahl M,Brzezinski P,et all.Measurements Using the Quartz Crystal Microbalance Technique of Ferritin Monolayers on Methyl-Thiolated Gold:Dependence of Energy Dissipation and Saturation Coverage on Salt Concentration.Colloid Interface Sci,1998,208,63—67
[15] Van Dulm,Pnorde,W Lyklema J.Ion participation in protein adsorption at solid surfaces.J Colloid Interface Sci.1981,82:77—82
[16] Wang X Q,Sun H L,Xia Y Q,et al.Lysozyme mediated calcium carbonate mineralization.Journal of Colloid and Interface Science,2009,332:96—103
[17] Hemmersam A G,Rechendorff K,Besenbacher F,et all.J Phys Chem C,2008,112:4180—4186
[18] Tilton R D,Robertson C R.Manipulation of hydrophobic interactions in protein adsorption.Langmuir,1991,7:2710—2718
[19] Su T J,Thom B R,Cui Z F.The Effect of Solution pH on the Structure of Lysozyme Layers Adsorbed at the Silica?Water Interface Studied by Neutron Reflection.Langmuir,1998,14:438—445
[20] Awschalom D D,Smyth J F,Grinstein G,et al.Macroscopic quantum tunneling in magnetic proteins.Phys Rev Lett,1992,68(20):3092—3095
[21] Harris J G E,Grimaldi J E,Awschalom D D. Excess spin and the dynamics of antiferromagnetic ferritin.Phys Rev B,1999,60(5):3453—3456
[22] Tejada J,Zhang X X,del Barco E,et al.Macroscopic resonant tunneling of magnetization in ferritin.Phys Rev Lett,1997,79(9):1754—1757
[23] Yamashita I.Fabrication of a two-dimensional array of nano-particles using ferritin molecule.Thin Solid Films,2001,393(1-2):12—18
[24] Bonard J-M,Chauvin P,Klinke C.Monodisperse multiwall carbon nanotubes obtained with ferritin as catalyst.Nano Lett,2002,2(6):665—667
[25] Mackle P,Charnock J M,Garner C D.Characterization of the manganese core of reconstituted ferritin by X-ray absorption spectroscopy.J Am Chem Soc,1993,115:8471—8472
[26] Shin M K,Kim S I,Kim S J,et al.Reinforcement of polymeric nanofibers by ferritin nanoparticles.Appl Phys Lett,2006,88:193901—193903
[27] Bhattacharyya S,Sinturel C,Salvetat J P,et al.Protein-functionalized carbon nanotube-polymer composites.Appl Phys Lett,2005,86:113104
[28]李辉,韩宝善.原子力显微镜观测并切断在Si表面拉直的DNA分子.科学通报,2003, 48(7):682—685
[29] Zhou X F,Xu H,Fan X H,et al.Compression of single conjugated-polymer nanoparticles with AFM tips.Chem Lett,2005,34:1488—1489
[30]杨培慧,郑志雯,曾谷城,等.转铁蛋白及其抗体分子间作用的原子力显微镜探测.科学通报,2006,51(2):138—141
[31] Radmaeher M,Fritz M,Cleveland J P,et al.Imaging adhesion forces and elasticity of lysozyme adsorbed on mica with the atomic force micro-scope. Langmuir,1994,103809—3814
[32] Gaboriaud F,Parcha B S,Gee M L,et al.Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging. Colloid Surface B,2008,62:206—213
[33] Schaer-Zammaretti P,Ubbink J.Imaging of lactic acid bacteria with AFM—elasticity and adhesion maps and their relationship to biological and structural data.Ultramicroscopy,2003,97:199—208
[34] Chizhik S A,Huang Z,Gorbunov V V,et al.Micromechanical properties of elastic polymeric materials as probed by scanning force microscopy. Langmuir,1998,14:2606—2609
[35] Du B,Tsui O K C,Zhang Q,et al.Study of elastic modulus and yield strength of polymer thin films using atomic force microscopy.Langmuir,2001,17:3286—3291
[36] Shulha H,Zhai X,Tsukruk V V.Molecular stiffness of individual hyperbranched macromolecules at solid surfaces.Macromolecules,2003,36: 2825—2831
[37] Digital Instruments Veeco Metrology Group.NanoScope Command Reference Manual Version 5.12 Revision B,2001
[38] Hemmersam A G,Rechendorff K,Besenbacher F,et al.pH-dependent adsorption and conformational change of ferritin studied on metal oxide surfaces by a combination of QCM-D and AFM.J Phys Chem C,2008,112:4180—4186
[39] Vinckier A,Semenza G.Measuring elasticity of biological materials by atomic force microscopy.FEBS Lett,1998,430:12—16
[40] Domke J,Radmacher M.Measuring the elastic properties of thin polymer films with the atomic force microscope.Langmuir,1998,14:3320—3325
[41] Zhou X F,Sun J L,An H J,et al.Radial compression elasticity of single DNA molecules studied by vibrating scanning polarization force microscopy.Phys Rev E,2005,71:062901
[2] Song S N,Yi X J,Zheng J Q,Dimensional crossover of Shubnikov-de Haas oscillations in thin films of gray tin.Phys Rev Lett,1990,65:227—210
[3] M F Yu,T Kowalewski, R S Ruof.Investigation of under Controlled the Radial Deformability of Individual Carbon Nanotubes Force,Phys Rev Let.2000,85(7):145
[4] Thomas A M,Rogers J F,Leedman P J.Iron-regulatory,iron-responsive and ferritin mRNA translation.In J Biochem cell Biol,1999,31:l139—1152.
[5]刘璐,刘瑶,严玉仙.铁蛋白.国外医学医学地理分册,2005,26(4):171—172
[6] Aust S D.Ferritin as a source of iron and protection from iron-induced toxicities.Toxicol Lett,1995,82—83:941—944
[7] Goto F,Yoshihara T,Shjgemoto N,et a1.Nature Biotechnology,1999,17:282—286
[8] Xu D,Watt G D,Harb J N,et al.Electrical conductivity of ferritin proteins by conductive AFM.Nano Lett,2005,5(4):571—577
[9] Binnig G,Quate C F,Gerber C.Atomic force microscope.Phys Rev Lett,1986,56:930—933
[10]朱杰,孙润广.生命科学仪器,2005,3(1):22—26
[11] Radmacher,et al.Direct observation of enzyme activity with the atomic force microscope.Science,1994,265:1577—1579
[12] MultiMode Scanning Probe Microscope Instruction Manual,Digital Instruments Corp.
[13] Jamsen B.effects of sleep on immune cell parameter.Curt Opin Infect Dis,1992,6:526—529
[14] Hook F,Rodahl M,Brzezinski P,et all.Measurements Using the Quartz Crystal Microbalance Technique of Ferritin Monolayers on Methyl-Thiolated Gold:Dependence of Energy Dissipation and Saturation Coverage on Salt Concentration.Colloid Interface Sci,1998,208,63—67
[15] Van Dulm,Pnorde,W Lyklema J.Ion participation in protein adsorption at solid surfaces.J Colloid Interface Sci.1981,82:77—82
[16] Wang X Q,Sun H L,Xia Y Q,et al.Lysozyme mediated calcium carbonate mineralization.Journal of Colloid and Interface Science,2009,332:96—103
[17] Hemmersam A G,Rechendorff K,Besenbacher F,et all.J Phys Chem C,2008,112:4180—4186
[18] Tilton R D,Robertson C R.Manipulation of hydrophobic interactions in protein adsorption.Langmuir,1991,7:2710—2718
[19] Su T J,Thom B R,Cui Z F.The Effect of Solution pH on the Structure of Lysozyme Layers Adsorbed at the Silica?Water Interface Studied by Neutron Reflection.Langmuir,1998,14:438—445
[20] Awschalom D D,Smyth J F,Grinstein G,et al.Macroscopic quantum tunneling in magnetic proteins.Phys Rev Lett,1992,68(20):3092—3095
[21] Harris J G E,Grimaldi J E,Awschalom D D. Excess spin and the dynamics of antiferromagnetic ferritin.Phys Rev B,1999,60(5):3453—3456
[22] Tejada J,Zhang X X,del Barco E,et al.Macroscopic resonant tunneling of magnetization in ferritin.Phys Rev Lett,1997,79(9):1754—1757
[23] Yamashita I.Fabrication of a two-dimensional array of nano-particles using ferritin molecule.Thin Solid Films,2001,393(1-2):12—18
[24] Bonard J-M,Chauvin P,Klinke C.Monodisperse multiwall carbon nanotubes obtained with ferritin as catalyst.Nano Lett,2002,2(6):665—667
[25] Mackle P,Charnock J M,Garner C D.Characterization of the manganese core of reconstituted ferritin by X-ray absorption spectroscopy.J Am Chem Soc,1993,115:8471—8472
[26] Shin M K,Kim S I,Kim S J,et al.Reinforcement of polymeric nanofibers by ferritin nanoparticles.Appl Phys Lett,2006,88:193901—193903
[27] Bhattacharyya S,Sinturel C,Salvetat J P,et al.Protein-functionalized carbon nanotube-polymer composites.Appl Phys Lett,2005,86:113104
[28]李辉,韩宝善.原子力显微镜观测并切断在Si表面拉直的DNA分子.科学通报,2003, 48(7):682—685
[29] Zhou X F,Xu H,Fan X H,et al.Compression of single conjugated-polymer nanoparticles with AFM tips.Chem Lett,2005,34:1488—1489
[30]杨培慧,郑志雯,曾谷城,等.转铁蛋白及其抗体分子间作用的原子力显微镜探测.科学通报,2006,51(2):138—141
[31] Radmaeher M,Fritz M,Cleveland J P,et al.Imaging adhesion forces and elasticity of lysozyme adsorbed on mica with the atomic force micro-scope. Langmuir,1994,103809—3814
[32] Gaboriaud F,Parcha B S,Gee M L,et al.Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging. Colloid Surface B,2008,62:206—213
[33] Schaer-Zammaretti P,Ubbink J.Imaging of lactic acid bacteria with AFM—elasticity and adhesion maps and their relationship to biological and structural data.Ultramicroscopy,2003,97:199—208
[34] Chizhik S A,Huang Z,Gorbunov V V,et al.Micromechanical properties of elastic polymeric materials as probed by scanning force microscopy. Langmuir,1998,14:2606—2609
[35] Du B,Tsui O K C,Zhang Q,et al.Study of elastic modulus and yield strength of polymer thin films using atomic force microscopy.Langmuir,2001,17:3286—3291
[36] Shulha H,Zhai X,Tsukruk V V.Molecular stiffness of individual hyperbranched macromolecules at solid surfaces.Macromolecules,2003,36: 2825—2831
[37] Digital Instruments Veeco Metrology Group.NanoScope Command Reference Manual Version 5.12 Revision B,2001
[38] Hemmersam A G,Rechendorff K,Besenbacher F,et al.pH-dependent adsorption and conformational change of ferritin studied on metal oxide surfaces by a combination of QCM-D and AFM.J Phys Chem C,2008,112:4180—4186
[39] Vinckier A,Semenza G.Measuring elasticity of biological materials by atomic force microscopy.FEBS Lett,1998,430:12—16
[40] Domke J,Radmacher M.Measuring the elastic properties of thin polymer films with the atomic force microscope.Langmuir,1998,14:3320—3325
[41] Zhou X F,Sun J L,An H J,et al.Radial compression elasticity of single DNA molecules studied by vibrating scanning polarization force microscopy.Phys Rev E,2005,71:062901