摘要
蛋白质与表面相互作用是纳米技术、生物材料和生物技术中的基本问题。抗非特异性蛋白吸附表面已经成为如医用植入物材料,药物传递以及生物传感器等生物医学应用中的一个重要问题,但是目前能够满足实际需要的超低蛋白吸附材料并不是很多。本文系统的研究了一系列两性离子聚合物表面如硫代甜菜碱丙烯酸甲酯(SBMA),羧基甜菜碱丙烯酰胺(CBAA)和羧基甜菜碱丙烯酸甲酯(CBMA),对表面进行了优化,其目的是探索一种对血清和血浆都能够实现“零”吸附的表面,并进一步探索了其在生物传感器和癌症疾病诊断等领域的应用。
第一,通过原子转移自由基聚合反应(ATRP)得到了不同厚度的聚SBMA表面,并系统研究了不同厚度的表面与蛋白吸附的关系。实验结果表明,研究的所有聚合物表面都可以认为是超低的非特异性蛋白吸附表面。同时,对于复杂介质如未稀释血清和血浆的研究发现当聚合物厚度过薄或者过厚时,都可以看到较明显的蛋白吸附,只有当聚合物的厚度大约为62 nm时,可以观察到最低的蛋白吸附。
第二,研究了一种基于聚CBAA的表面平台,这个平台可以非常有效、灵敏以及特异性的检测血浆中的特定蛋白,同时还能提供非常稳定的超低蛋白吸附背景。抗体功能化之后的聚CBAA表面表现出对未稀释血浆有超低的蛋白吸附性质,表面等离子共振传感器(SPR)测量其吸附量低于3 ng/cm2。对未稀释血浆中一种癌症生物标记物(ALCAM)的检测表明其具有很高的灵敏度和特异性。同时,还研究了聚CBMA的金表面和二氧化硅表面的功能化。
第三,通过ATRP反应得到了不同厚度聚CBAA的表面,并且研究了这些表面与未稀释的老化血清,血清和血浆等多种蛋白在不同温度下的吸附关系。实验结果表明当聚CBAA表面的厚度为20 nm左右时,不管在25°C还是37°C下,表面对未稀释的老化血清、血清和血浆的蛋白吸附值都低于SPR的检测下限即0.3 ng/cm2。另外,功能化的实验结果表明聚CBAA表面在功能化之后能继续保持其良好的抗非特异性蛋白吸附的特性,并且可以对血清中的ALCAM抗原可以有效的进行检测。
第四,验证了一种适用于纳米颗粒的可功能化且超稳定的表面。结果表明聚CBAA包覆的纳米颗粒具有与传统的包覆层如聚乙二醇相比更稳定的特性,尽管聚乙二醇和甲基丙烯酸低聚乙二醇酯包覆的纳米颗粒在10%人血清中能稳定存在,但其在未稀释的血清中不能保持稳定。另外,生物识别因素如ALCAM可以通过氨基化学法非常简单且有效的连接到聚CBAA的表面。聚CBAA包覆的纳米颗粒表面的配体密度可以通过调整抗原/抗体的浓度而很容易的被调控。
Nonspecific protein adsorption has been a crucial issue for many biomedical applications, such as medical implants, drug delivery vehicles and biosensors. At present, there are a very limited number of effective nonfouling biomaterials available to meet the challenges of practical applications. Here, zwitterionic materials such as sulfobetaine methacrylate (SBMA), carboxybetaine acrylamide (CBAA) and carboxybetaine methacrylate (CBMA) were systematically introduced, optimized and investigated for their applications as biosensors.
Firstly, protein adsorption from single protein solutions and complex media such as 100% blood serum and plasma onto polySBMA-grafted surfaces via atom transfer radical polymerization (ATRP) at varying film thicknesses was investigated. It is interesting to observe that protein adsorption exhibits a minimum at a medium film thickness. Results show that the surface with 62 nm polySBMA brushes presents the best nonfouling character in 100% blood serum and plasma although all of these surfaces are highly resistant to nonspecific protein adsorption from single fibrinogen and lysozyme solutions. Surface resistance to 100% blood serum or plasma is necessary for many applications from blood-contacting devices to drug delivery. This work provides a new in vitro evaluation standard for the application of biomaterials in vivo.
Secondly, a zwitterionic polyCBAA biomimetic material was employed to create a unique biorecognition coating with an ultralow fouling background, enabling the sensitive and specific detection of proteins in blood plasma. Conditions for surface activation, protein immobilization, and surface deactivation of the carboxylate groups in the polyCBAA coating were determined. An antibody-functionalized polyCBAA surface platform was used to detect a target protein in blood plasma using a sensitive surface plasmon resonance (SPR) sensor. A cancer biomarker (ALCAM) was directly detected from 100% human blood plasma with extraordinary specificity and sensitivity. The total nonspecific protein adsorption on the functionalized polyCBAA surface was very low (<3 ng/cm2 for undiluted blood plasma). Furthermore, functionalization on polyCBMA-grafted gold or silicon dioxide surfaces was also investigated.
Thirdly, nonspecific protein adsorption from single protein solutions and complex media such as undiluted human blood serum and plasma onto polyCBAA-grafted surfaces was investigated. The polyCBAA grafting was done via ATRP with varying film thicknesses at different temperatures. The objective is to create a surface that experiences“zero”protein adsorption from complex undiluted human blood serum and plasma. Results show that protein adsorption from undiluted blood serum, plasma, and aged serum on the polyCBAA-grafted surface is undetectable at both 25°C and 37°C by a SPR sensor. This was achieved with a film thickness of ~20 nm. Further it is demonstrated that the polyCBAA surfaces after antibody immobilized maintain undetectable protein adsorption from undiluted human blood serum.
Finally, a new surface platform presenting an abundance of functional groups for ligand immobilization in an ultra-low fouling background necessary for targeted drug delivery and diagnostics was introduced. It is demonstrated that this surface platform, made from zwitterionic polyCBAA coated nanoparticles, is not only stable in undiluted human blood serum, but also can be conjugated to bio-molecules conveniently and effectively. In addition, a practical application was demonstrated by showing the amount of antibodies to a candidate cancer biomarker immobilized onto polyCBAA-coated nanoparticles can be easily adjusted. Results also indicate that 10% serum commonly used to evaluate the stability of nanoparticles is not sufficient and undiluted blood serum is recommended to screen nanoparticles before their in vivo experiments.
引文
[1] Ratner B D, Hoffman A S, Schoen F J, et al., Biomaterials Science, an Introduction to Materials in Medicine, 2nd ed.: Elsevier: Amsterdam, 2004
[2] Langer R, Perspectives: drug delivery - drugs on target, Science, 2001, 293 (5527): 58-59.
[3] Prime K L, Whitesides G M, Self-assembled organic monolayers-model systems for studying adsorption of proteins at surfaces, Science, 1991, 252 (5009): 1164-1167.
[4] Brash J L, Exploiting the current paradigm of blood-material interactions for the rational design of blood-compatible materials, Journal of Biomaterials Science-Polymer Edition, 2000, 11 (11): 1135-1146.
[5] Wu Y G, Simonovsky F I, Ratner B D, et al., The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: A comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion, Journal of Biomedical Materials Research Part A, 2005, 74A (4): 722-738.
[6] Huang N P, Michel R, Voros J, et al., Poly(L-lysine)-g-poly(ethylene glycol) layers on metal oxide surfaces: Surface-analytical characterization and resistance to serum and fibrinogen adsorption, Langmuir, 2001, 17 (2): 489-498.
[7] Horbett T A, Principles underlying the role of adsorbed plasma-proteins in blood interactions with foreign materials Cardiovascular Pathology, 1993, 2 (3): S137-S148.
[8] Zhang Z, Zhang M, Chen S F, et al., Blood compatibility of surfaces with superlow protein adsorption, Biomaterials, 2008, 29 (32): 4285-4291.
[9]王庭槐,生理学北京:高等教育出版社, 2004
[10] Benesch J, Svedhem S, Svensson S C T, et al., Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: Experiments with fibrinogen, heparinized plasma, and serum, Journal of Biomaterials Science-Polymer Edition, 2001, 12 (6): 581-597.
[11] Van Butsele K, Jerome R, Jerome C, Functional amphiphilic and biodegradable copolymers for intravenous vectorisation, Polymer, 2007, 48 (26): 7431-7443.
[12] Kobayashi M, Chang Y S, Oka M, A two year in vivo study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus, Biomaterials, 2005, 26 (16): 3243-3248.
[13] Kunze C, Bernd H E, Androsch R, et al., In vitro and in vivo studies on blends of isotactic and atactic poly (3-hydroxybutyrate) for development of a dura substitute material, Biomaterials, 2006, 27 (2): 192-201.
[14] Ladd J, Zhang Z, Chen S, et al., Zwitterionic polymers exhibiting high resistance to nonspecific protein adsorption from human serum and plasma, Biomacromolecules, 2008, 9 (5): 1357-1361.
[15] Vaisocherova H, Yang W, Zhang Z, et al., Ultralow fouling and functionalizable surface chemistry based on a zwitterionic polymer enabling sensitive and specific protein detection in undiluted blood plasma, Analytical Chemistry, 2008, 80 (20): 7894-7901.
[16] Huang N P, Voros J, De Paul S M, et al., Biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol): A novel polymeric interface for bioaffinity sensing, Langmuir, 2002, 18 (1): 220-230.
[17] Statz A R, Meagher R J, Barron A E, et al., New peptidomimetic polymers for antifouling surfaces, Journal of the American Chemical Society, 2005, 127 (22): 7972-7973.
[18] Cheng G, Zhang Z, Chen S F, et al., Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces, Biomaterials, 2007, 28: 4192-4199.
[19] Chen S F, Jiang S Y, A new avenue to nonfouling materials, Advanced Materials, 2008, 20 (2): 335-338.
[20] Harris J M, Poly(ethylene glycol) Chemistry : Biotechnical and Biomedical Applications Plenum Press: New York and London, 1992
[21] Bailey F E, Koleske J V, Poly(ethylene Oxide): Academic Press: New York, 1976
[22] Li L Y, Chen S F, Jiang S Y, Protein interactions with oligo(ethylene glycol) (OEG) self-assembled monolayers: OEG stability, surface packing density and protein adsorption, Journal of Biomaterials Science-Polymer Edition, 2007, 18(11): 1415-1427.
[23]张永国,聚乙二醇修饰蛋白药物及其应用,海峡药学, 2008, 20 (2): 79-82.
[24]施险峰,詹家荣,廖本仁,聚乙二醇修饰技术研究进展,上海化工, 2007, 32 (12): 30-32.
[25]何春龙,刘乐乐,莎日娜,聚乙二醇修饰药物研究进展,内蒙古医学院学报, 2007, 29 (6): 562-565.
[26]田浤,姚文兵,聚乙二醇技术在药物转运系统中的研究进展,中国药科大学学报, 2008, 39 (4): 379-384.
[27] Ostuni E, Chapman R G, Holmlin R E, et al., A survey of structure-property relationships of surfaces that resist the adsorption of protein, Langmuir, 2001, 17 (18): 5605-5620.
[28] Harder P, Grunze M, Dahint R, et al., Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption, Journal of Physical Chemistry B, 1998, 102 (2): 426-436.
[29] Herold D A, Keil K, Bruns D E, Oxidation of polyethylene glycols by alcohol-dehydrogenase, Biochemical Pharmacology, 1989, 38 (1): 73-76.
[30] Shen M C, Pan Y V, Wagner M S, et al., Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether, Journal of Biomaterials Science-Polymer Edition, 2001, 12 (9): 961-978.
[31] Zwaal R F A, Comfurius P, Vandeenen L L M, Membrane asymmetry and blood-coagulation, Nature, 1977, 268 (5618): 358-360.
[32] Johnston D S, Sanghera S, Pons M, et al., Phospholipid polymers-synthesis and spectral characteristics, Biochimica Et Biophysica Acta, 1980, 602 (1): 57-69.
[33] Ishihara K, Aragaki R, Ueda T, et al., Reduced thrombogenicity of polymers having phospholipid polar groups, Journal of Biomedical Materials Research, 1990, 24 (8): 1069-1077.
[34] Feng W, Zhu S P, Ishihara K, et al., Adsorption of fibrinogen and lysozyme on silicon grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-Initiated atom transfer radical polymerization, Langmuir, 2005, 21: 5980-5987.
[35] Zhang Z, Chen S F, Chang Y, et al., Surface grafted sulfobetaine polymers viaatom transfer radical polymerization as superlow fouling coatings, Journal of Physical Chemistry B, 2006, 110 (22): 10799-10804.
[36] Cheng N, Brown A A, Azzaroni O, et al., Thickness-dependent properties of polyzwitterionic brushes, Macromolecules, 2008, 41 (17): 6317-6321.
[37] Zhang Z, Chen S F, Jiang S Y, Dual-functional biomimetic materials: Nonfouling poly(carboxybetaine) with active functional groups for protein immobilization, Biomacromolecules, 2006, 7 (12): 3311-3315.
[38] Chen S F, Yu F C, Yu Q M, et al., Strong resistance of a thin crystalline layer of balanced charged groups to protein adsorption, Langmuir, 2006, 22 (19): 8186-8191.
[39] Holmlin R E, Chen X X, Chapman R G, et al., Zwitterionic SAMs that resist nonspecific adsorption of protein from aqueous buffer, Langmuir, 2001, 17 (9): 2841-2850.
[40] Kim D, Park S, Lee J H, et al., Antibiofouling polymer-coated gold nanoparticles as a contrast agent for in vivo x-ray computed tomography imaging, Journal of the American Chemical Society, 2007, 129 (24): 7661-7665.
[41]彭慧,王娟,杨婷婷, et al.,原子转移自由基聚合法制备聚合物刷,材料导报, 2005, 19 (3): 73-76, 96.
[42]刘海林,马晓燕,袁莉, et al.,分子自组装研究进展,材料科学与工程学报2004, 22 (2): 308-311.
[43]翟怡,张金利,李韡, et al.,自组装单层膜的制备与应用,化学进展, 2004, 16 (4): 477-484.
[44]任恕,膜受体与传感器,北京:科学出版社, 1996.
[45] Zhang S G, Emerging biological materials through molecular self-assembly, Biotechnology Advances, 2002, 20 (5-6): PII S0734-9750(0702)00026-00025.
[46]曾鹏举,刘云圻,分子自组装成膜技术,物理1999, 28 (12): 713-719.
[47] Crooks R M, Ricco A J, New organic materials suitable for use in chemical sensor arrays, Accounts of Chemical Research, 1998, 31 (5): 219-227.
[48] Wang J S, Matyjaszewski K, Controlled living radical polymerization-Atom-transfer radical polymerization in the presence of transition-metal complexes, Journal of the American Chemical Society, 1995, 117 (20): 5614-5615.
[49]唐燕春,艾长军,马敬红, et al.,原子转移自由基聚合机理及其应用,合成技术及应用, 2007, 22 (4): 38-40.
[50] Matyjaszewski K, Xia J H, Atom transfer radical polymerization, Chemical Reviews, 2001, 101 (9): 2921-2990.
[51]夏攀登,耿兵,路翠苹, et al.,原子转移自由基聚合的研究进展,河南化工, 2005, 22 (4): 5-8.
[52]易运红,张力,原子转移自由基聚合进展,化工技术与开发, 2005, 34 (3): 29-33.
[53] Patten T E, Matyjaszewski K, Atom-transfer radical polymerization and the synthesis of polymeric materials, Advanced Materials, 1998, 10: 901-915.
[54] Kotani Y, Kamigaito M, Sawamoto M, Living radical polymerization of styrene by half-metallocene iron carbonyl complexes, Macromolecules, 2000, 33 (10): 3543-3549.
[55]张源源,周秀华,活性自由基聚合法合成偶氮聚合物研究进展,化工时刊2008, 22 (6): 62-67.
[56]田威,范晓东,陈卫星, RAFT聚合法制备双亲性嵌段共聚物研究进展,化学通报, 2006, 69 (w067): 1-7.
[57] Quinn J F, Chaplin R P, Davis T P, Facile synthesis of comb, star, and graft polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization, Journal of Polymer Science Part a-Polymer Chemistry, 2002, 40 (17): 2956-2966.
[58]聂利华,姚守拙,压电液相化学分析,分析化学, 1996, 24 (2): 234-241.
[59]张辉,钟博,邓玉福,生命单元表面行为的物理表征,北京:科学出版社, 2006
[60]李贵才,朱生发,杨苹, et al.,石英晶体微天平在蛋白吸附领域的研究进展,传感器世界, 2007, 12: 6-10.
[61] Aizawa H, Kurosawa S, Tozuka M, et al., Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay, Biosensors & Bioelectronics, 2003, 18 (5-6): 765-771.
[62] Modin C, Stranne A L, Foss M, et al., QCM-D studies of attachment and differential spreading of pre-osteoblastic cells on Ta and Cr surfaces, Biomaterials, 2006, 27 (8): 1346-1354.
[63]田颖,李玉文,酶联免疫吸附测定法及在兽药饲料分析中的应用,中国饲料, 2004, 10: 33-34.
[64] http://www.uscnlife.cn/web/page/news270.htm,
[65]崔大付,张璐璐,王于杰, et al.,表面等离子谐振(SPR)生化分析仪的研制与发展,现代科学仪器, 2007, 2: 3-7.
[66]李远红,表面等离子体谐振技术及其生物传感器研究,武汉职业技术学院学报, 2003, 2 (3): 59-61.
[67] Faraday M, Experimental relations of gold (and other metals) to light, Philos. Trans., 1857, 147: 145-181.
[68] Turkevitch J, P.C.Stevenson, J.Hillier, Nucleation and growth process in the synthesis of colloidal gold, Discuss. Faraday Soc., 1951, 11: 55-75.
[69] Frens G, Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nature Phys. Sci., 1973, 241: 20-22.
[70] Yonezawa T K, Practical T, Preparation of anionic mercapto ligand-stabilizer gold nanoparticles and their immobilization. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 1999, 149: 193-199.
[71] Giersig M, Mulvaney P, Preparation of ordered colloid monolayers by electrophoretic deposition, Langmuir, 1993, 9: 3408-3413.
[72] Brust M, Walker M, Bethell D, et al., Synthesis of thiol-derivatized gold nanoparticles in a 2-phase liquid-liquid system, Journal of the Chemical Society-Chemical Communications, 1994, (7): 801-802.
[73] Daniel M C, Astruc D, Gold nanoparticles: Assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chemical Reviews, 2004, 104 (1): 293-346.
[74] Page A, Faulk W, Taylor C, Communication to the editors: an immunocolloid method for the electron microscope, Immunocytochemistry, 1971, 8: 801-802.
[75] Lyon A L, Musick M D, Natan M J, Observation of hybridization of thiol-tethered DNA using two-color surface plasmon resonance spectroscopy, Journal of the American Chemistry Society, 1997, 119: 3401-3402.
[76] Lyon A L, Musick M D, Natan M J, Surface plasmon resonance of Au colloid-modified Au film:particle size dependence, Journal of Physical Chemistry B, 1999, 103 (5826-5831).
[77] Storhoff J J, Elghanian R, Mucic R C, et al., One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes, Journal of the American Chemical Society, 1998, 120: 1959-1964.
[78] El-Sayed I H, Huang X H, El-Sayed M A, Surface plasmon resonancescattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer, Nano Letters, 2005, 5 (5): 829-834.
[79] El-Sayed I H, Huang X H, El-Sayed M A, Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles, Cancer Letters, 2006, 239 (1): 129-135.
[80] Huang X H, El-Sayed I H, Qian W, et al., Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods, Journal of the American Chemical Society, 2006, 128 (6): 2115-2120.
[81] Sanvicens N, Marco M P, Multifunctional nanoparticles - properties and prospects for their use in human medicine, Trends in Biotechnology, 2008, 26 (8): 425-433.
[82] Schneider G F, Subr V, Ulbrich K, et al., Multifunctional Cytotoxic Stealth Nanoparticles. A Model Approach with Potential for Cancer Therapy, Nano Letters, 2009, 9 (2): 636-642.
[83] Gil P R, Parak W, Composite Nanoparticles Take Aim at Cancer, ACS Nano, 2008, 2 (11): 2200-2205.
[84] Zhang Z, Chao T, Chen S F, et al., Superlow fouling sulfobetaine and carboxybetaine polymers on glass slides, Langmuir, 2006, 22 (24): 10072-10077.
[85] Azzaroni O, Trappmann B, van Rijn P, et al., Mechanically induced generation of counterions inside surface-grafted charged macromolecular films: Towards enhanced mechanotransduction in artificial systems, Angewandte Chemie-International Edition, 2006, 45 (44): 7440-7443.
[86] Li L Y, Chen S F, Zheng J, et al., Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior, Journal of Physical Chemistry B, 2005, 109 (7): 2934-2941.
[87] Feng W, Brash J L, Zhu S P, Non-biofouling materials prepared by atom transfer radical polymerization grafting of 2-methacryloloxyethyl phosphorylcholine: Separate effects of graft density and chain length on protein repulsion, Biomaterials, 2006, 27 (6): 847-855.
[88] Jones D M, Brown A A, Huck W T S, Surface-initiated polymerizations in aqueous media: Effect of initiator density, Langmuir, 2002, 18 (4): 1265-1269.
[89] Bernards M T, Cheng G, Zhang Z, et al., Nonfouling polymer brushes via surface-initiated, two-component atom transfer radical polymerization, Macromolecules, 2008, 41 (12): 4216-4219.
[90] Pinkel D, Seagraves R, Sudar D, et al., High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays, Nature Genetics, 1998, 20 (2): 207-211.
[91] Homola J, Surface Plasmon Resoncance Based Sensors, Berlin: Springer-Verlag, 2006.200
[92] Guo Y L, Zhang X M, Tan W, et al., Platelet 12-lipoxygenase Arg261Gln polymorphism: functional characterization and association with risk of esophageal squamous cell carcinoma in combination with COX-2 polymorphisms, Pharmacogenetics and Genomics, 2007, 17 (3): 197-205.
[93] Cheng G, Xite H, Zhang Z, et al., A Switchable Biocompatible Polymer Surface with Self-Sterilizing and Nonfouling Capabilities, Angewandte Chemie-International Edition, 2008, 47 (46): 8831-8834.
[94] Yoon K R, Ramaraj B, Lee S M, et al., Surface initiated-atom transfer radical polymerization of a sugar methacrylate on gold nanoparticles, Surface and Interface Analysis, 2008, 40 (8): 1139-1143.
[95] Lee B S, Chi Y S, Lee K B, et al., Functionalization of poly(oligo(ethylene glycol)methacrylate) films on gold and Si/SiO2 for immobilization of proteins and cells: SPR and QCM studies, Biomacromolecules, 2007, 8 (12): 3922-3929.
[96] Weers J G, Rathman J F, Axe F U, et al., Effect of the intramolecular charge separation distance on the solution properties of betaines and sulfobetaines, Langmuir, 1991, 7 (5): 854-867.
[97] Johnsson B, Lofas S, Lindquist G, et al., Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies, Journal of Molecular Recognition, 1995, 8 (1-2): 125-131.
[98] Vaisocherova H, Mrkvova K, Piliarik M, et al., Surface plasmon resonance biosensor for direct detection of antibody against Epstein-Baff virus, Biosensors & Bioelectronics, 2007, 22 (6): 1020-1026.
[99] Lahiri J, Isaacs L, Tien J, et al., A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a commonreactive intermediate: A surface plasmon resonance study, Analytical Chemistry, 1999, 71 (4): 777-790.
[100] Chen S F, Zheng J, Li L Y, et al., Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: Insights into nonfouling properties of zwitterionic materials, Journal of the American Chemical Society, 2005, 127 (41): 14473-14478.
[101] Lee J H, Lee H B, Andrade J D, Blood compatibility of polyethylene oxide surfaces, Progress in Polymer Science, 1995, 20 (6): 1043-1079.
[102] Milner S T, Polymer brushes, Science, 1991, 251 (4996): 905-914.
[103] Huglin M B, Rego J M, Influence of a salt on some properties of hydrophilic methacrylate hydrogels, Macromolecules, 1991, 24 (9): 2556-2563.
[104] Huglin M B, Rego J M, Thermodynamic propertiies of copolymeric hydrogels based on 2-hydroxyethyl methacrylate and a zwitterionic methacrylate, Colloid and Polymer Science, 1992, 270 (3): 234-242.
[105] Zhang Z, Vaisocherova H, Cheng G, et al., Nonfouling Behavior of Polycarboxybetaine-Grafted Surfaces: Structural and Environmental Effects, Biomacromolecules, 2008, 9 (10): 2686-2692.
[106] Schwendel D, Dahint R, Herrwerth S, et al., Temperature dependence of the protein resistance of poly- and oligo(ethylene glycol)-terminated alkanethiolate monolayers, Langmuir, 2001, 17 (19): 5717-5720.
[107] Leckband D, Sheth S, Halperin A, Grafted poly(ethylene oxide) brushes as nonfouling surface coatings, Journal of Biomaterials Science-Polymer Edition, 1999, 10 (10): 1125-1147.
[108] Yang W, Chen S F, Cheng G, et al., Film thickness dependence of protein adsorption from blood serum and plasma onto poly(sulfobetaine)-grafted surfaces, Langmuir, 2008, 24 (17): 9211-9214.
[109] Feng W, Chen R X, Brash J L, et al., Surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methacrylate: Effect of solvent on graft density, Macromolecular Rapid Communications, 2005, 26 (17): 1383-1388.
[110] Ma H W, Hyun J H, Stiller P, et al., "Non-fouling" oligo(ethylene glycol)-functionalized polymer brushes synthesized by surface-initiated atom transfer radical polymerization, Advanced Materials, 2004, 16: 338-341.
[111] Matyjaszewski K, Nanda A K, Tang W, Effect of [Cu-II] on the rate ofactivation in ATRP, Macromolecules, 2005, 38 (5): 2015-2018.
[112] Matyjaszewski K, Patten T E, Xia J H, Controlled/''living'' radical polymerization. Kinetics of the homogeneous atom transfer radical polymerization of styrene, Journal of the American Chemical Society, 1997, 119 (4): 674-680.
[113] Napper D H, Polymeric stabilization of colloidal dispersions, London: Academic Press, 1983
[114] Bergen J M, Von Recum H A, Goodman T T, et al., Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery, Macromolecular Bioscience, 2006, 6 (7): 506-516.
[115]吕伸,王杉,胶体金免疫方法的进展,武汉大学学报(自然科学版), 2000, 46 (4): 393-399.