Immobilization of glucose oxidase on Fe3O4SiO2 magnetic
生物分子固定化方法
生物传感器中生物组分的固定化方法生物传感器由两部分组成: 生物敏感元件与信号转换器。
生物传感器的选择性主要取决于敏感材料的选取,而灵敏度的高低则与转换器的类型、生物组分的固定化技术等有很大的关系。
因此固定化技术的发展就是提高传感器性能的关键因素之一。
生物传感器要呈现良好的工作性能, 其固定化技术应满足以下条件:(1) 固定化后的生物组分仍能维持良好的生物活性;(2) 生物膜与转换器须紧密接触,且能适应多种测试环境;(3) 固定化层要有良好的稳定性与耐用性;(4) 减少生物膜中生物组分的相互作用以保持其原有的高度选择性。
为了研制廉价、灵敏度高而且选择性好的生物传感器,固定化技术已成为研究者们努力探求的目标。
经过近20年的不懈探索,已建立了对各种不同生物功能材料的固定化方法。
1、1、3、1 物理吸附法此法就是通过生物分子的极性键、氢键、疏水键的作用将生物组分吸附于不溶性的惰性载体上。
文献已经报道了一些材料可用作吸附其它材料的载体,比如,石墨粉[25]、石墨-聚四氟乙烯[26]、活性碳[27]、离子交换树脂[28]等。
物理吸附法的特点就是方法简便、操作条件温与,缺点就是生物分子与载体表面的结合力弱,在表面进行任意取向的不规则分布,因此使制得的生物传感器容易发生生物分子的脱落与泄漏,从而造成传感器的灵敏度低,重现性差。
1、1、3、2 包埋法将生物组分与合成高分子经溶剂混合而使生物组分包埋于其中,制成敏感膜的方法称作包埋法。
采取的包埋方式通常包括凝胶包埋法与胶囊包埋法二种形式[29,30]。
包埋法的优点就是操作条件比较温与,膜的孔径与形状可随意控制,对生物组分活性的影响较小,缺点就是需控制很多实验因素,而且生物组分在聚合物膜内的活性会受到影响。
1、1、3、3 共价键合法将生物组分通过共价键与电极表面结合而固定的方法称作共价键合法。
该法就是利用基体表面进行活化处理,然后与生物组分偶联,从而使生物组分结合到基体表面。
葡萄糖水热碳化制备表面分子印迹基质材料多孔碳微球_赵慧君
葡萄糖水热碳化制备表面分子印迹基质材料多孔碳微球赵慧君1,2，杨永珍1,3，刘旭光1,2，许并社1,3（1. 太原理工大学新材料界面科学与工程教育部重点实验室，太原030024；2. 太原理工大学化学化工学院，太原030024；3.太原理工大学新材料工程技术研究中心，太原030024）摘 要：以葡萄糖晶体为原料，采用低温水热法和退火处理制得多孔碳微球。
利用扫描电子显微镜、红外光谱仪、X-射线衍射仪、热重分析仪和孔径分析仪对所制备的碳微球进行表征。
结果表明：通过调节低温水热反应中反应物浓度、反应时间、反应温度等参数，在葡萄糖溶液浓度0.3 mol/L、温度180 ℃、时间14 h时，得到粒径尺寸均一、表面光滑、形貌规整并且具有一定溶剂分散性的碳微球；退火后碳微球的尺寸均匀介于100~700 nm之间，孔径集中分布在1.2~1.8 nm之间，比表面积为502.6 m2/g，表面具有羟基、羰基、羧基等含氧官能团。
此多孔碳微球有望在表面分子印迹聚合物的制备过程中省去表面氧化的步骤，提高进一步接枝的反应活性，解决在表面分子印迹过程中修饰过程复杂的问题。
关键词：材料物理与化学；水热；葡萄糖；多孔碳微球；分子印迹中图分类号：TB 332文献标志码：A 文章编号：2095－2783(2012)12－0898－6Preparation of surface molecularly imprinted matrix materials porous carbon microspheres from glucose by hydrothermal carbonization methodZhao Huijun1,2，Y ang Y ongzhen1,3，Liu Xuguang 1,2，Xu Bingshe1,3(1. Key Laboratory of Interface Science and Engineering in Advanced Materials of Ministry of Education, T aiyuan University of T echnology, T aiyuan 030024, China; 2. College of Chemistry and Chemical Engineering, T aiyuan University of T echnology, T aiyuan 030024, China;3. Research Center on Advanced Materials Science and T echnology, T aiyuan University of T echnolog, T aiyuan 030024, China) Abstract: Porous carbon microspheres (PCMSs) have been prepared by hydrothermal method at low temperatures after the annealingwith glucose crystal as raw materials in the reaction vessel. The samples were characterized by electron microscopy, infrared spectroscopy, X-ray diffraction, thermogravimetry and pore size analysis. The results showed that PCMSs, with uniform size, smooth surface, regular morphology and solvent dispersion, were prepared at optimum conditions of 0.3 mol/L glucose, 14 h and 180℃. The annealed PCMSs with a specific surface area of 502.6 m2/g and sizes ranging from 100–700 nm, had pore sizes concentrated at 1.2–1.8 nm and several oxygen-containing functional groups including hydroxyl, carbonyl and carboxyl groups on their surfaces. As-obtained PCMSs will eliminate the surface activation and improve the grafting reactivity in order to solve the complexity of modification in the process of preparing molecularly imprinted surface materials.Key words: material physics and chemistry；hydrothermal；glucose；porous carbon microspheres；molecular imprinting表面分子印迹技术是把具有识别位点的印迹层结合在基质表面的印迹方法，这种方法合成的分子印迹材料的形状由基质材料决定[1]。
季铵化卤胺聚合物改性fe3o4@sio2纳米粒子的制备及抗菌性能研究
@ Si(.)2 modified with polyethyleneimine・ The quaternarized halamine polymer-functioned Fe3 O4 @ SiO2 nanoparticles were obtained after chlorination. The structure? morphology and characterization of samples were measured by FT-IR，TEM, zeta-potential measurement and VSM. It was superparamagnetic and could be separated quickly by the aid of an external magnetic field. The results of antibacterial tests showed that the antibacterial mechanism of chlorinated samples was synergistic with quaternary ammonium salt and halamine and the antibacterial property was better than the one without chlorination. 100% of S・ aureus and E. coll were inactivated within 30 min of contact. Key words: polyethyleneimine； quaternarized halamine polymer； magnetic property； antibiosis.
甲基咪唑离子液体[MIM]Cl修饰Fe3O4@SiO2磁性纳米颗粒
![甲基咪唑离子液体[MIM]Cl修饰Fe3O4@SiO2磁性纳米颗粒](https://img.taocdn.com/s3/m/0f9df5b9d1d233d4b14e852458fb770bf78a3b6b.png)
采用硅烷化法制备了甲基咪唑离子液体修饰的磁性纳米颗粒
甲基咪唑离子液体[MIM]Cl修饰Fe3O4@SiO2磁性纳米颗粒
甲基咪唑离子液体[MIM]Cl修饰Fe3O4@SiO2磁性纳米颗粒(Fe3O4@SiO2@[MIM]Cl)
中文名称：甲基咪唑离子液体[MIM]Cl修饰Fe3O4@SiO2磁性纳米颗粒
简称：Fe3O4@SiO2@[MIM]Cl
描述：采用硅烷化法制备了甲基咪唑离子液体修饰的磁性纳米颗粒:Fe3O4@SiO2@[MIM]Cl,并以其作为磁性固相萃取状：液体/粉末
贮藏：易于吸潮，需密封存放，4℃冰箱冷藏避光保存
服务：离子液体定制服务
地址：西安
厂家：西安齐岳生物科技有限公司
北大考研-工学院研究生导师简介-戴志飞
爱考机构-北大考研-工学院研究生导师简介-戴志飞戴志飞背景资料“国家杰出青年科学基金”获得者，教育部新世纪优秀人才，“龙江学者”特聘教授，“黑龙江省杰出青年科学基金”获得者。
主要从事纳米医学的研究，在多功能造影剂、分子影像、分子探针、基因/药物载体和生物传感器等方面取得了一系列研究成果。
在国际上率先研制了集超声成像与癌症光热治疗于一体的多功能造影剂，相关成果被领域顶尖期刊选为热点论文发表，【NatureMaterials】作为研究“亮点”推荐报道，同时被国内外多家专业技术网站报道与转载。
现任国家自然科学基金委专家评审组会评专家，【InsciencesJournal】高级编辑，【GlobalJPhysChem】编委，《中华核医学与分子影像杂志》编委。
迄今共发表SCI论文72篇（包括4篇封面论文），其中影响因子IF>13的论文6篇，IF>5的论文17篇。
论文被【Science】等期刊引用并给予高度评价。
申请发明专利20余项。
在科学出版社出版专著《仿生膜材料与技术》，入选当代杰出青年科学文库，参加编写专著3部。
作为第一完成人获得省自然科学一等奖1项。
研究领域1.纳米医学：纳米药物载体、药物靶向传递与控制释放2.分子影像：分子探针、多功能造影剂3.纳米生物传感器：光学、磁性和电化学等各种类型的生物传感器受教育经历1995～1998，中科院感光化学研究所（现为中科院理化所），博士1992～1995，黑龙江大学化学系，硕士1988～1992，湘潭师范学院化学系（现为湖南科技大学），本科主要科研工作经历2012～现在，北京大学工学院生物医学工程系2005～2011，哈尔滨工业大学理学院生命科学系2003～2005，美国艾默里大学医学院生物医学工程系，博士后2000～2003，德国马普研究院胶体与界面研究所，博士后1999～2000，日本关西学院大学理学部化学系，博士后1998～1999，中科院感光化学研究所，助研荣誉与获奖国家杰出青年科学基金获得者教育部新世纪优秀人才黑龙江省杰出青年科学基金获得者龙江学者特聘教授黑龙江省自然科学一等奖(排名第一)代表性论文1.Zhengbao,Zha,XiuliYue*,QiushiRen,ZhifeiDai*,UniformPolypyrroleNanoparticleswithHighPho tothermalConversionEfficiencyforPhotothermalAblationofCancerCells,AdvancedMaterials,2012, DOI:adma.201202211,inpress.2.YanMa,XiaolongLiang,ShengTong,GangBao,QiushiRenandZhifeiDai*,GoldNanoshelledNanom icellesforPotentialMRIImaging,Light-TriggeredDrugReleaseandPhotothermalTherapy,AdvancedF unctionalMaterials,2012,DOI:10.1002/adfm.201201663,inpress.3.CaixinGuo,JinliangWang,JingCheng,ZhifieiDai*,Determinationoftracecopperionswithultrahighs 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glucosebiosensing,Talanta,2012,97,438-444.9.XiaolongLiang,XiaodaLi,XiuliYue,ZhifeiDai*,Conjugationofporphyrintonanohybridcerasomesf orphotodynamictherapyofcancer,AngewandteChemieInternationalEdition,2011,50，11622-11627.VIPpaper.HighlightedinFrontispiece10.HengteKe,JinruiWang,ZhifeiDai*,YushenJin,EnzeQu,ZhanwenXing,CaixinGuo,XiuliYue,Jibin Liu,Goldnanoshelledmicrocapsules:atheranosticagentforultrasoundcontrastimagingandphototherm altherapy,AngewandteChemieInternationalEdition,2011,50(13),3017-3021.Hotpaper.Highlightedb yNatureMaterials.11.ZhengbaoZha,CelineCohn,ZhifeiDai*,WeiguoQiu,JinhongZhang,XiaoyiWu*,NanofibrousLipid MembranesCapableofFunctionallyImmobilizingAntibodiesandCapturingSpecificCells,Advanced Materials,2011,30(23),3435-3440.12.YanMa,ZhifeiDai*,ZhengbaoZha,YanguangGao,XiuliYue,SelectiveAntileukemiaeffectofstabili zednanohybridvesiclesbasedoncholesterylsuccinylsilane,Biomaterials,2011,32,9300-9307.13.XiaolongLiang,XiuliYue,ZhifeiDai*,Jun-ichiKikuch*,Photoresponsiveliposomalnanohybridcer asomes,ChemicalCommunications,2011,47,4751-4753.14.GuangleiFu,XiuliYue,ZhifeiDai*,Glucosebiosensorbasedoncovalentimmobilizationofenzymein sol-gelcompositefilmcombinedwithPrussianblue/carbonnanotubeshybrid,BiosensorsandBioelectro nics,2011,26,3973-3976.15.HengteKe,JinruiWang,ZhifeiDai*,YushenJin,EnzeQu,ZhanwenXing,CaixinGuo,andXiuliYue,B ifunctionalgoldnanorod-loadedpolymericmicrocapsulesforbothcontrast-enhancedultrasoundimagin gandphotothermaltherapy,JournalofMaterialsChemistry,2011,21,5561-5564.16.YanMa,ZhifeiDai*,YanguangGao,ZhongCao,ZhengbaoZha,XiuliYue,JunichiKikuchi,Liposomalarchitectureboostsbiocompatibilityofnanohybridcerasomes,Nanotoxicology,2011,5,622-635. 17.ZhongCao,YanMa,XiuliYue,ShouzhuLi,ZhifeiDai*,JunichiKikuchi*,Stabilizedliposomalnanoh 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壁碳纳米管修饰的葡萄糖生物传感器
２结果与讨论
２．１
不同修饰电极的传感器对葡萄糖的晌应 葡萄糖酶传感器的检测机理是葡萄糖在葡萄糖酶催化作用下生成葡萄糖酸和Ｈ。０２，通过检测Ｈ。０：
还原电流米达到测定葡萄糖浓度的目的．我们分别制作了ＭＷＮＴｓ—ＣＨＩ，ｒ—ＧＯＤ／ＧＣＥ（１）、Ｆｅ。０４～ ＣＨＩＴ—ＧＯＤ／ＧＣＥ（２）和ＭＷＮＴｓ—Ｆｅ。０４一ＣＨＩＴ—ＧＯＤ／ＧＣＥ（３）三支不同电极．在２５℃条件下，分别 将三种电极放人葡萄糖含量为１×１０～ｍｏｌ／Ｉ。的磷酸盐缓冲液（ｐＨ＝６．８）中进行扫描，得到的循环伏安 图（见图１）．由图ｌ可见，ＭＷＮＴｓ—Ｆｅ。Ｏ。一ＣＨＩＴ—ＧＯＤ／ＧＣＥ（３）较Ｆｅ。Ｏ。一ＣＨＩＴ—ＧＯＤ／ＧＣＥ（２）和 ＭＷＮＴｓ—ＣＨｌＴ—ＧＯＤ／ＧＣＥ（１）氧化还原峰电流有所提高．这主要是因为磁性纳米Ｆｅ。Ｏ。颗粒能够在 葡萄糖氧化酶的氧化还原中心和玻碳电极之间有效地传输电子，增强了酶促反应．同时，多壁碳纳米管的 引入增强了电子传递速率和酶的同定量，从而提高了电极的催化能力． ２．２扫描速度对传感器响应电流的影响 在２５℃条件下，将ＭＷＮＴｓ－－Ｆｅ。ｑ－－ＣＨＩＴ—ＧＯＤ／ＧＣ修饰电极置于葡萄糖含量为１×１０一ｍｏｌ／Ｉ。 的ＰＢＳ（ｐＨ＝６．８）溶液中，以不同扫速（Ｏ～２５０ ｍｙ／ｓ）测定，得到循环伏安图（见图２）．实验结果显示，随 着扫速的增加，氧化还原峰电流随之增大，并且均与扫描速度的平方根呈线性关系，这表明氧化还原峰电 流受扩散控制．当扫描速度达剑２５０ ｍｖ时，得到较为对称的循环伏安图，说明玻碳电极能够与固定其表面 的物质进行快速的电子交换．
［８］王存嫦。ｍ明辉．鲁亚霜．等．基于碳纳米管和铁氰酸镍纳米颗粒协同作用的葡萄糖生物传感器［Ｊ］．化学学报。２００６．
Fe3O4@SiO2磁性催化剂载体的制备及表征
Fe3O4@SiO2磁性催化剂载体的制备及表征丁冰晶;陈文民;范立维;郑屹华;吕迪;卢泽湘【摘要】以正硅酸乙酯作为硅源,采用沉淀法将四氧化三铁颗粒包覆在二氧化硅中,制备了Fe3O4@SiO2磁性催化剂载体材料.考察了氨水浓度、正硅酸乙酯浓度和四氧化三铁加入量对样品包覆率的影响,并对样品的物相和显微结构做了表征.结果表明:正硅酸乙酯可作为制备Fe3O4@SiO2磁性催化剂载体的硅源;在氨水浓度为0.4 mol/L、正硅酸乙酯浓度为0.02 mol/L、Fe3O4加入量为0.04 g的适宜反应条件下,样品包覆率为99％;二氧化硅有效包覆了四氧化三铁颗粒,但团聚较严重.【期刊名称】《无机盐工业》【年(卷),期】2014(046)011【总页数】4页(P62-64,75)【关键词】制备;磁性;Fe3O4@SiO2;硅源【作者】丁冰晶;陈文民;范立维;郑屹华;吕迪;卢泽湘【作者单位】中国蓝星(集团)股份有限公司,北京100029;中国中煤能源集团有限公司;福建农林大学资源与环境学院;福建农林大学材料工程学院化工系;福建农林大学材料工程学院化工系;福建农林大学材料工程学院化工系【正文语种】中文【中图分类】TQ138.11超细磁性核壳型Fe3O4@SiO2具有易回收、制备成本低、表面可修饰等优点［1］，是一种极具应用前景的催化剂载体材料，正受到人们的广泛关注［2-4］。
该材料通常是在超细磁性Fe3O4颗粒表面上均匀包覆一层SiO2制备而成的。
其中，SiO2的包覆层与Fe3O4@SiO2材料的分散性、抗氧化性、耐酸碱性、生物相容性、表面可修饰性等特性紧密相关［5-6］，因此Fe3O4的包覆过程成为制备该催化剂载体的关键步骤。
笔者以正硅酸乙酯为硅源，采用沉淀法制备核壳型Fe3O4@SiO2，考察了制备条件对样品包覆率的影响，并对样品的物相、显微结构和磁性进行了表征，以期为该催化剂载体的应用提供相关理论基础和实践经验。
葡萄糖氧化酶生产工艺流程
葡萄糖氧化酶生产工艺流程英文回答：Glucose oxidase is an enzyme that is widely used in various industries, including food, pharmaceutical, and diagnostic industries. It is primarily used for the production of glucose biosensors, which are used for blood glucose monitoring in diabetic patients. The production process of glucose oxidase involves several steps.Firstly, the enzyme is obtained from a suitable source, such as Aspergillus niger or Penicillium species. These fungal strains are known to produce high levels of glucose oxidase. The source organism is cultured in a suitable growth medium, which provides the necessary nutrients for enzyme production.After the culture reaches the desired biomass, thecells are harvested and the enzyme is extracted. This can be done through various methods, such as cell disruption,centrifugation, or filtration. The extracted enzyme is then purified to remove any impurities or contaminants. This is typically done using techniques like chromatography or ultrafiltration.Once the enzyme is purified, it is immobilized or stabilized to improve its stability and longevity. Immobilization can be achieved through various methods, such as covalent attachment to a solid support or encapsulation within a matrix. This immobilized enzyme can then be used for various applications, including glucose biosensor production.In the case of glucose biosensor production, the immobilized enzyme is typically combined with other components, such as an electrode and a mediator. The electrode provides a surface for the enzyme to react with glucose, while the mediator helps in transferring electrons between the enzyme and the electrode. This combination is then encapsulated within a suitable housing to protect it from external factors.The final step in the production process is quality control and testing. The produced glucose oxidase is tested for its activity, stability, and other parameters to ensure its effectiveness and reliability. This includes testingits performance in various conditions, such as pH and temperature variations.Overall, the production process of glucose oxidase involves steps like culture, extraction, purification, immobilization, and quality control. Each step is crucial to ensure the production of a high-quality enzyme for various applications, including glucose biosensor production.中文回答：葡萄糖氧化酶是一种广泛应用于食品、制药和诊断等多个行业的酶。
Fe3O4@SiO2
Ultraefficient separation and sensing of mercury and methylmercury ions in drinking water by using aminonaphthalimide-functionalizedFe3O4@SiO2core/shell magnetic nanoparticles wMinsung Park,a Sungmin Seo,a In Su Lee*b and Jong Hwa Jung*aReceived11th February2010,Accepted9th April2010First published as an Advance Article on the web22nd April2010DOI:10.1039/c002905jA newfluorogenic based aminonaphthalimide-functionalized Fe3O4@SiO2core/shell magnetic nanoparticles1has been prepared,and its abilities to sense and separate metal ions were evaluated byfluorophotometry.The nanoparticles1exhibited a high affinity and selectivity for Hg2+and CH3Hg+ions over competing metal ions.Mercury,a highly poisonous element,is a widespread pollutant which occurs from natural and anthropogenic sources.In particular,organic forms of mercury,such as methylmercury species(CH3HgX;X=halides,etc.),are much more toxic than inorganic mercury species(HgX2).1Because of their lipid solubility,methylmercury species readily cross the blood–brain barrier,accumulate in the brain,and cause damage to the central nervous system,1,2as well as other organs.The epidemics of Minamata Bay in Japan demonstrated the fatal threat of methylmercury to human health.3Thus, techniques for the separation and removal of mercury and methylmercury from pollutants are very important to prevent poisoning in environmental and biologicalfields.Although several groups4–9have recently reportedfluorescent probes for detection of Hg2+and CH3Hg+,however there are a few papers on separation and removal techniques for Hg2+and CH3Hg+in biological and environmental systems by using fluorescent nanoparticles.10,11Magnetic silica nanoparticles are of great interest for biomedical and environmental research applications because they are biocompatible,easily renewable,and are stable against degradation.12–17For example,magnetic nanoparticles have been used for bioseparation,drug targeting,cell isolation, enzyme immobilization and protein purification.However, magnetic nanoparticles have not been frequently used to separate and remove toxic environmental pollutants.In this context,the use of magnetic nanoparticles as solid supports coupled with supramolecular concepts is important for the development of hybrid nanomaterials with improved functionalities and offers a promising approach for simple and efficient separation and detection of toxic metal ions for biological,toxicological and environmental usage.18–22Herein,we report the preparation of aminonaphthalimide-functionalized Fe3O4@SiO2core/shell magnetic nanoparticles1and their applications for the detection and separation of mercury and methylmercury ions in drinking water.The Fe3O4@SiO2 core/shell nanoparticle1is a highly selective chemosensor and adsorbent for Hg2+and CH3Hg+ions.Separation experiments further established that1can be used to separate and remove Hg2+and CH3Hg+from drinking water.The encapsulation of Fe3O4nanocrystals within silica shells was conducted in a microemulsion system in water droplets and4nm sized Fe3O4nanocrystals in the external cyclohexane phase.The formation of a silica shell around the Fe3O4 nanocrystals was initiated by the successive addition of an aqueous NH4OH solution and tetraethyl orthosilicate(TEOS) and reacted for12h.Compound3was synthesized as described previously.23 Receptor2was easily prepared by treating3with 3-(triethoxysilyl)propyl isocyanate in the presence of triethyl-amine(see ESI w,Scheme S1).Then Fe3O4@SiO2core/shell nanoparticles were reacted with2in toluene under vigorous stirring overnight to covalently link2onto the surface of the Fe3O4@SiO2nanoparticles by a sol–gel reaction(Scheme1). The synthetic1was well characterized by transmission electron microscopy(TEM),FTIR spectroscopy,time-of-flight second ion mass spectroscopy(TOF-SIMS)andfluorophotometry. The TEM image of1revealed a spherical structure with a narrow size distribution(ca.20nm)with4nm of Fe3O4nano core(Fig.1A),which maintained its magnetic property (Fig.1B).For further proof of the new bond formation,we acquired FT-IR and TOF-SIMS spectra of1.In the FT-IR spectrum of1,in addition to the bands attributed to the Fe3O4@SiO2core/shell nanoparticles themselves,strong new bands at3438,2921,2853,1696,1652,1592,1531,1460,1460 and1379cmÀ1appeared,which originated from the receptor 2,in accordance with2residing on the Fe3O4@SiO2core/shell Scheme1Preparation of aminonaphthalimide-functionalized Fe3O4@SiO2core/shell magnetic nanoparticles1by sol–gel reaction.a Department of Chemistry and Research Institute of Natural Science,Gyeongsang National University,Jinju660-701,Korea.E-mail:jonghwa@gnu.ac.krb Department of Applied Chemistry,Kyung Hee University,Gyeonggi-do449-701,Korea.E-mail:insulee97@khu.ac.krw Electronic supplementary information(ESI)available:Syntheticprocedures and characterization of1,FT-IR,TOF-SIMS,fluorescencespectra.See DOI:10.1039/c002905jCOMMUNICATION /chemcomm|ChemCommnanoparticles (see ESI w ,Fig.S1).The TOF-SIMS spectrum of 1displayed the characteristic fragments of 2(m /z =551,576,605),also giving us firm evidence that 2was anchored onto the surface of the Fe 3O 4@SiO 2core/shell nanoparticles (Fig.S2,ESI w ).Spectroscopic measurements with 1were performed in 0.2M MOPS buffer,pH 7.The absorption spectrum of free 1showed a single visible absorption band at 350nm (e =6.50Â104M À1cm À1)and a corresponding fluorescence emission maximum at 520nm.As expected,1is fluorescent in its apo state (F =0.035),which resulted from the intra-molecular charge-transfer (ICT)band.Upon the addition of increasing Hg 2+concentrations,1showed a large quenching effect with a PET (photoinduced electron transfer,F o 0.0001,Fig.2A and B)effect in the emission spectra.The fluorescence quenching that occurred upon exposure to Hg 2+was fully reversible,as the addition of EDTA (0.01N,1mL)(Fig.S3,ESI w )restored the emission band.In addition,the fluorescence change was reproducible over several cycles of detection–separation.The Job plot using the fluorescence changes indicated 1:1binding for 1with Hg 2+(Fig.S4,ESI w ).From the titrations,the association constant (K )for Hg 2+coordination to 1was calculated to be 1.05Â105M À1(log K =5.02).24,25We then found that 1shows a detection limit of 1.2ppb (Fig.S5,ESI w ),more than sufficient to sense the Hg 2+concentration in drinking water with respect to the U.S.EPA limit (B 2ppb).To determine the response time of 1,the time course for the fluorescence intensity of 1at 520nm was examined (Fig.S6,ESI w ).Immediately after the addition of Hg 2+,the fluorescence intensity of 1started to decrease,and the fluorescence intensity was almost quenched after 60s (Fig.S6,ESI w ).This result suggests that the response time of this system is 60s.To evaluate further the utility of 1as an ion-selective fluorescence probe for Hg 2+,the competition-based fluorescence emission changes of 1upon addition of various biologically and environmentally relevant metal ions such as Na +,Mg 2+,Ca 2+,Cu 2+,Ag +,Co 2+,Ni 2+,Mn 2+and Pb 2+were investigated in aqueous solution (Fig.2C).Probe 1displayed a large PET effect only with Hg 2+.As expected,the fluorescence intensities of Hg 2+-bound 1were unchanged in the presence of Na +,Mg 2+,Ca 2+,Cu 2+,Ag +,Co 2+,Ni 2+,Mn 2+and Pb 2+,indicating that 1is a promising selective adsorbent for separation of Hg 2+in the mixture of metal ions.We also probed the binding ability of 1with CH 3Hg +in water/ethanol (9:1v/v)at pH 7,due to solubility.As shown in Fig.3,upon the addition of increasing CH 3Hg +concentrations,1showed a large quenching effect with PET effect in the emission spectra.When 1equiv.of CH 3Hg +was added to probe 1in 0.2M MOPS buffer,the fluorescence intensity is saturated.This result was higher sensitivity than that of Hg 2+ion.The association constant (K )for CH 3Hg +coordination to 1was calculated to be 5.25Â105M À1(log K =5.72).In addition,the detection limit of probe 1for CH 3Hg +was evaluated by monitoring the fluorescence titration of 1(0.05m M)with CH 3Hg +at ppb levels (Fig.S7,ESI w ).The fluorescence intensity of 1was nearly proportional to the amount of CH 3Hg +added and detection was possible at 0.8ppb of CH 3Hg +in water/ethanol (9:1v/v)at 251C.We observed the binding abilities of 2for metal ions on the basis of fluorescence changes upon the addition of Na +,Ca 2+,Cu 2+,Ag +,Zn 2+,Mn 2+,Pb 2+,Hg 2+and CH 3Hg +using an acetonitrile solution to optimize solubility (Fig.S8,ESI w ).Unlike probe 1,compound 2showed quenching effects with Cu 2+,Ag +,Hg 2+as well as CH 3Hg +.This difference suggests that probe 1has a potential to be utilized as a selective fluorescence chemosensor for Hg 2+.The highly selective Hg 2+recognition of fluorescence chemosensor 1is due to preorganization of 2onto the surface of Fe 3O 4@SiO 2nanoparticles.For biological and environmental applications as a fluorescence sensor,the sensing should be effective over a wide pH range.The effect of pH on 1in the absence of Hg 2+and CH 3Hg +was examined (Fig.4).At pH o 4,the fluorescence emission is largely quenched,probably due to the protonation of the nitrogen atoms of the aminonaphthalimidegroup.Fig.1(A)TEM image of the chemosensor 1.(B)Photograph of a magnet attracting 1inwater.Fig.2(A)Fluorescence titrations of 1(1.0m M)upon addition of increasing Hg 2+concentrations (0,0.01,0.02,0.05,0.08,0.1,0.2,0.25,0.4,0.6,0.8,1.0,2.0,3.0,4.0equiv.)in water at pH 7.(B)Plot for fluorescence intensity of 1as a function of Hg 2+concentration (excitation is at 350nm,and emission is monitored at 520nm).(C)Fluorescence intensity of 1(1.0m M)with Hg 2+(4equiv.)in the presence of other metal ions such as Na +,Mg 2+,Ca 2+,Cu 2+,Ag +,Co 2+,Mn 2+,Ni 2+and Pb 2+ions (10equiv.)in water at pH 7.However,minimal or no significant fluorescence changes were observed between pH 6–11.This result clearly demonstrates that 1can be used in physiological environments where a pH >4occurs.In addition,upon addition of Hg 2+and CH 3Hg +ions,a large quenching effect occurred in over the range of pH 4–11,indicating that the nanoparticle 1can be useful as a chemosensor and adsorbent for Hg 2+and CH 3Hg +at pH >4.Next,we evaluated the effectiveness of probe 1in the separation of Hg 2+or CH 3Hg +from drinking water by using short columns containing probe 1.An aliquot (1mL)of drinking water containing Hg 2+(100ppb)or CH 3Hg +(100ppb)was injected into a column (1cm Â5cm)prepared with probe 1as adsorbent.The column was eluted with 0.2M MOPS buffer solution (pH 7)at a flow rate of 1mL min À1.To determine the amount of Hg 2+and CH 3Hg +separated by the magnetic silica nanoparticles 1,the amount of Hg 2+and CH 3Hg +left in the drinking water eluate was determined by ICP-MS.Interestingly,no residual levels of Hg 2+and CH 3Hg +were detectable in drinking water by ICP-MS,suggesting that the designed magnetic silica nanoparticle 1removed almost 100%of Hg 2+and CH 3Hg +.Thus,Fe 3O 4@SiO 2core/shell nanoparticles 1can be a potentially useful and effective agent for the selective separation and rapid removal of Hg 2+and CH 3Hg +from drinking water.In summary,we have readily prepared aminonaphthalimide-functionalized Fe 3O 4@SiO 2core/shell magnetic nanoparticles 1that act as a new type of synthetic fluorogenic chemosensor for efficient separation and sensing of Hg 2+and CH 3Hg +ions in aqueous solutions over the pH range of 4–11.Chemosensor 1exhibits a high affinity and high selectivity for Hg 2+andCH 3Hg +over the other competing metal ions tested,and successfully detected and separated Hg 2+and CH 3Hg +in drinking water.These findings show considerable promise for the development of a new category of tailor-made biocompatible systems built by immobilization of appropriate fluorogenic receptors on the surface of other novel nanomaterials for study of the environmental function of toxic metal contaminants.This work was supported by a grant from the NRF (grant no.R01-2007-000-20299-0)and World Class University (WCU)Program (R32-2008-000-20003-0)supported from Ministry of Education,Science and Technology and Environmental-Fusion Project (191-091-004)supported from Ministry of Environment.Notes and references1(a )ATSDR,Toxicological Profile for Mercury ,U.S.Department of Health and Human Services,Atlanta,GA,1999;(b )ATSDR,ToxProfiles:Mercury ,U.S.Department of Health and Human Services,Atlanta,GA,2005.2(a )P.Jitaru and F.Adams,J.Phys.IV ,2004,121,185;(b )T.W.Clarkson and L.Magos,Crit.Rev.Toxicol.,2006,36,609.3F.Bakir,S.F.Damluji,L.Amin-Zaki,M.Muratadha,A.Khalidi,N.Y.Al-Rawi,S.Tikriti,H.I.Dhahir,T.W.Clarkson,J.C.Smith and R.A.Doherty,Science ,1973,181,230.4M.Santra, D.Ryu, A.Chatterjee,S.-K.Ko,I.Shin and K.H.Ahn,mun.,2009,2115.5Y.-K.Yang,S.-K.Ko,I.Shin and J.Tae,Org.Biomol.Chem.,2009,7,4590.6H.Wang and W.-H.Chan,Tetrahedron ,2007,63,8825.7X.Chen,S.-W.Kim,M.J.Jou,Y.Kim,S.-J.Kim,S.Park and J.Yoon,Org.Lett.,2008,10,5235.8S.-K.Ko,Y.-K.Yang,J.Tae and I.Shin,J.Am.Chem.Soc.,2006,128,14150.9Y.-K.Yang,K.-J.Yook and J.Tae,J.Am.Chem.Soc.,2005,127,16760.10E.Climent,M.D.Marcos,R.M-Manez,F.Sancenon,J.Soto,K.Rurack and P.Amoros,Angew.Chem.,Int.Ed.,2009,48,8519.11J.V.R-Lis,R.Casasus,es,C.Coll,M.D.Marcos,R.M-Manez,F.Sancenon,J.Soto,P.Amoros,J.E.Haskouri,N.Garro and K.Rurack,Chem.–Eur.J.,2008,14,8267.12C.Fang and M.Zhang,J.Mater.Chem.,2009,19,6258.13J.Gao,H.Gu and B.Xu,Acc.Chem.Res.,2009,42,1097.14S.J.Son,J.Reichel,B.He,M.Schuchman and S.B.Lee,J.Am.Chem.Soc.,2005,127,7316.15A.Abou-Hassan,R.Bazzi and V.Cabuil,Angew.Chem.,Int.Ed.,2009,48,7180.16N.Insin,J.B.Tracy,H.Lee,J.P.Zimmer,R.M.Westervelt and M.G.Bawendi,ACS Nano ,2008,2,197.17Y.H.Zhu,H.Da,X.Yang and Y.Hu,Colloids Surf.,A ,2003,231,123.18H.J.Kim,S.J.Lee,S.Y.Park,J.H.Jung and J.S.Kim,Adv.Mater.,2008,20,3229.19H.Y.Lee,D.R.Bae,J.C.Park,H.Song,W.S.Han and J.H.Jung,Angew.Chem.,Int.Ed.,2009,48,1239.20W.S.Han,H.Y.Lee,S.H.Jung,S.J.Lee and J.H.Jung,Chem.Soc.Rev.,2009,38,1904.21R.Martınez-Ma n ez and F.Sancenon,Coord.Chem.Rev.,2006,250,3081.22B.Descalzo,R.Martınez-Ma n ez,F.Sancenon,K.Hoffmann and K.Rurack,Angew.Chem.,Int.Ed.,2006,45,5924.23X.Guo,X.Qian and L.Jia,J.Am.Chem.Soc.,2004,126,2272.24Association constants were obtained using the computer program ENZFITTER,available from Elsevier-BIOSOFT,68Hills Road,Cambridge CB21LA,United Kingdom.25K.A.Conners,Binding Constants,The Measurement of Molecular Complex Stability ,Wiley,New York,1987.Fig.3(A)Fluorescence titrations of 1(1.0m M)upon addition of increasing CH 3Hg +concentrations (0,0.05,0.1,0.2,0.3,0.5,0.7,1.0,1.5,2.5equiv.)in water/ethanol (9:1v/v)at pH 7.(B)Plot for fluorescence intensity of 1as a function of CH 3Hg +concentration (excitation is at 350nm,and emission is monitored at 520nm).Fig.4Plot of pH values against fluorescence intensity of 1(1.0m M)in (a)the absence and (b)the presence of Hg 2+(5.0equiv.)in water and (c)CH 3Hg +(5.0equiv.)in water/ethanol (9:1v/v).。
A practical glucose biosensor based on Fe3O4 nanoparticles and chitosan-nafion composite film
Biosensors and Bioelectronics 25 (2009) 889–895Contents lists available at ScienceDirectBiosensors andBioelectronicsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i osA practical glucose biosensor based on Fe 3O 4nanoparticles and chitosan/nafion composite filmLiuqing Yang,Xiangling Ren,Fangqiong Tang ∗,Lin ZhangKey Laboratory of Photochemical Conversion and Optoelectronic Materials,Laboratory of Controllable Preparation and Application of Nanomaterials,Technical Institute of Physics and Chemistry,Chinese Academy of Sciences,Beijing 100190,PR Chinaa r t i c l e i n f o Article history:Received 14July 2009Received in revised form 19August 2009Accepted 1September 2009Available online 6 September 2009Keywords:Fe 3O 4nanoparticles Glucose oxidaseEnzyme immobilization Nafiona b s t r a c tA practical glucose biosensor was developed by combining the intrinsic peroxidase-like activity of Fe 3O 4nanoparticles (Fe 3O 4NPs)and the anti-interference ability of the nafion film.Glucose oxidase (GOD)was simply mixed with Fe 3O 4NPs and cross-linked on the Pt electrode with chitosan (Cs)medium by glutaraldehyde,and then covered with a thin nafion film.The biosensor showed high sensitivity (11.54A cm −2mM −1),low detection limit (6×10−6M),and good storage stability.A linear calibration plot was obtained in the wide concentration range from 6×10−6to 2.2×10−3M.The modified electrode could virtually eliminate the interference during the detection of glucose.Furthermore,the biosensor was successfully applied to detect glucose in human serum sample.This fabrication of glucose biosensor was of considerable interest due to its promise for simple procedure and optimizing conditions in practical application.© 2009 Elsevier B.V. All rights reserved.1.IntroductionGlucose biosensors which utilize immobilized oxidase for the conversion of the target analytes into electrochemically detectable products are one of the most widely detection methods for the determination of glucose in blood and food (Bakker,2004;Prodromidis and Karayannis,2002).Since Clark and Lyons proposed the initial concept of glucose enzyme electrodes in 1962(Clark and Lyons,1962),different approaches have been explored in the operation of glucose enzyme electrodes.By immobilizing glucose oxidase (GOD)on the electrode surface,it is available to employ such amperometric biosensors for the determination of glucose for their simplicity,high selectivity and intrinsic sensitivity.The immobilization of enzymes is one of the crucial factors in biosensor preparation (Ren et al.,2005a,b;Fan et al.,2001).Many methods have been used to immobilize enzymes and to improve the enzymatic activity.Nowadays,nanoparticles can offer many advantages,such as large surface-to-volume ratio,high surface reaction activity,and strong adsorption ability to immo-bilize desired biomolecules (Dyal et al.,2003;Cai et al.,2006).There are growing interests in the application of nanoparticles to immobilize enzyme,as well as to exploit electrically contacted enzyme electrodes or protein-functionalized electrodes.Moreover,nanoparticles have the unique ability to promote electron transfer between electrodes and the active site of the enzyme due to their∗Corresponding author.Tel.:+861082543521;fax:+861062554670.E-mail address:tangfq@ (F.Tang).suitable size and physical properties (Orozco et al.,2009).Many kinds of metal nanoparticles such as Ag (Ren et al.,2005a,b ),Au (Xiao et al.,2003;Xu et al.,2006),Pt (Bahshi et al.,2008),and semi-conductor nanoparticles (CdSe/ZnS)(Gill et al.,2008)have been reported to immobilize enzyme to prepare modified electrodes.Among a wide range of biomedical and technological applica-tions (Kurlyandskaya and Levit,2007;Zhang et al.,2008;Mavre et al.,2007),magnetic particles have been considered as an interesting material for the immobilization of desired biomolecules resulted from many superior properties:biocompatibility,superparamag-netic property,as well as better contact between biocatalyst and its substrates (Katz et al.,2004;Rossi et al.,2004;Lu and Chen,2006;Huang et al.,2008).Electrochemical detection of hybridized DNA strands have been achieved with a magnetic nanoparticle modified electrode (Zhu et al.,2006).Nanoparticles of various fer-ric oxides (hematite,magnetite,amorphous Fe 2O 3,-Fe 2O 3and ferrihydrite)incorporated into carbon paste have exhibited electro-catalytic properties towards hydrogen peroxide reduction (Hrbac et al.,2007).Kaushik et al.have fabricated a new glucose biosen-sor and a urea sensor based on iron oxide nanoparticles-chitosan nanocomposite (Kaushik et al.,2008,2009).Wang et al.have devel-oped a novel amperometric glucose biosensor by immobilizing ferritin antibody on the surface of Fe 3O 4nanoparticles (Fe 3O 4NPs)/chitosan (Cs)composite film modified glassy carbon elec-trode (GCE)for determination of ferritin (Wang and Tan,2007).However,magnetic particles have only been studied to be used to immobilize the linked enzymes in these reports.Furthermore,the mechanism of the electron transfer between the electrode and the substrate molecule,as well as the role of Fe 3O 4NPs playing in0956-5663/$–see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2009.09.002890L.Yang et al./Biosensors and Bioelectronics25 (2009) 889–895the electron transfer of these biosensors have not been explained clearly.Recently,Gao et al.(2007)have proved that Fe3O4NPs in fact possessed an intrinsic enzyme mimetic activity similar to that nat-ural peroxidase.It showed that Fe3O4NPs not only offered a good microenvironment for retaining the bioactivity of enzymes,but also acted as natural peroxidase to oxidize organic substrates.Wei et al.have further confirmed that the Fe3O4NPs possessed intrin-sic peroxidase-like activity and developed this new biosensor in the application of detection of H2O2and glucose by colorimetric method(Wei and Wang,2008).The results that Fe3O4NPs were used to enhance electrocatalytic and bioelectrocatalytic transfor-mation have been emphasized with the aim to highlight a new concept for biosensors.In this paper,we synthesized the Fe3O4NPs for biosen-sor application and explained the mechanism of the electron transfer between the electrode and the substrate molecule.The fabrication,characterization and analytical performance of the modified biosensor based on Fe3O4NPs to develop an elec-trochemical glucose sensor were also described in the paper. Moreover,the Pt/GOD/Fe3O4/Cs/nafion modified Pt electrode was used to achieve unique features of electrocatalytic reduction and elimination of interference.The new glucose sensor was used for the determination of glucose in human serum.There-fore,the new biosensor modified with Fe3O4NPs,chitosan and nafionfilm have shown potential of robust,easy-to-make,durable analytical applications in both biological and environmental detec-tion.2.Materials and methods2.1.Chemicals and reagentsGlucose oxidase was extracted from Aspergillus niger(Sigma company,118U/mg).-d-Glucose was purchased from Sigma and nafion was from DuPont company.l(+)-Ascorbic acid(AA)and sodium hydroxide(NaOH)were products of Beijing Shiji Company. Chitosan was from Guo Yao Chemical Reagent Company.Phosphate buffers containing0.1M KCl consisted of KH2PO4and Na2HPO4 (0.2M,pH6.8).All reagents were used without further purification and prepared with redistilled water.2.2.Preparation of Fe3O4nanoparticlesThe Fe3O4NPs were prepared via a previously reported co-precipitation method(Mikhaylova et al.,2004;Vereda et al.,2007). Firstly,0.32M ferric chloride and0.31M ferrous chloride were mixed in0.45M HCl solution diluted by deoxygenated water.Sec-ondly,the mixed solution of ferrous and ferric salts was then added into250mL of0.3M oxygen-free sodium hydroxide solution under vigorous stirring at room temperature in a nitrogen atmosphere. The formed black Fe3O4colloidal particles were separated by cen-trifugation and further washed with water for three times.The Fe3O4NPs were then re-dispersed in water and stored at room tem-perature.(Referred to as the Fe3O4nanoparticle stock solution witha total concentration of25mM.)2.3.Preparation of enzyme electrodesThe platinum electrode with a diameter of1mm wasfirst boiled in nitric acid for8min and washed in redistilled water. The aqueous solution of GOD(10L,1U/L)and10L Fe3O4NPs solution were added to20L glutaraldehyde(2%)to form a mix-ture and then dried on the Pt electrode.400L Fe3O4NPs was added to2mL chitosan solution(2%)in a5mL beaker.And then the platinum electrode was dipped into the mixture for5min and dried in air.Finally,2mL nafion(2.5%)was drop-coated on the Pt/GOD/Fe3O4/Cs electrode.These electrodes were stored at4◦C for24h before measurement.The enzyme electrode without Fe3O4NPs and the enzyme elec-trode modified by Fe3O4NPs while without nafion were used as comparison and prepared by the same method as described above.2.4.Apparatus and measurementsTransmission electron microscope(TEM)of Fe3O4nanoparticles was obtained with a JEM-2100electron microscope,operating at 160kV.Cyclic voltammetry and electrochemical impedance spec-troscope(EIS)were performed using a CHI604B electrochemical workstation(Shang Hai Chen Hua company)in5mM K3Fe(CN)6 and 2.5mM K3Fe(CN)6/K4Fe(CN)6(1:1)containing0.1M KCl.A conventional three-electrode system was used throughout the electrochemical experiment at room temperature with a mod-ified electrode as working electrode,a bare Pt electrode as auxiliary electrode,and a glassy carbon electrode as reference electrode against which all potentials were measured in this paper.The sensitivity of the glucose biosensor was tested by measur-ing the current response using a three-electrode cell consisting of a modified electrode as working electrode,a bare Pt electrode as auxiliary electrode and a reference electrode of Ag/AgCl.Measure-ments were conducted in a5mL phosphate buffer(pH6.8)cell at 35◦C.Afixed potential of0.4V was applied to this electronic cell. Firstly,the three-electrode cell was put into phosphate solution at 35◦C.When background current reached a constant value,differ-ent concentrations(from3×10−6to1.1×10−2M)of-d-glucose solution were added.Then response currents were noted down, and background currents were deducted.At last,the correlation between the current response and the concentrations of glucose solution was obtained.3.Results and discussion3.1.TEM characterization of the Fe3O4NPs,GOD and Fe3O4 compositeFig.1A shows the typical transmission electron microscope (TEM)of the Fe3O4NPs,which indicates that the sample is com-posed of a large quantity of well-dispersed spherical nanoparticles. The average size of these particles estimated from the TEM image is about20nm.The selected area of the same single nanoarchitecture in the inset of Fig.1A can be indexed to the crystal lattice structure of Fe3O4NPs.Fig.1B shows the TEM image of the mixture of GOD and Fe3O4NPs solution which indicates that the composite of Fe3O4 NPs with GOD are also well-dispersed.3.2.Evaluation of the electrochemical performance of the electrodes3.2.1.Cyclic voltammetric characterizationCyclic voltammetric experiment is used to evaluate the elec-trochemical performance of the electrodes.Fig.1C shows cyclic voltammograms(CVs)of different modified electrodes in5.0mM potassium ferricyanide solution.For the bare Pt electrode,the well-defined oxidation and reduction peaks caused by the Fe3+/Fe2+ redox couples are noticed in forward and reverse scan(curve(a)). After the electrode is modified by Cs,an obvious decrease in reduc-tion and oxidation peaks is observed(curve(b)).This may be due to the properties of the chitosan,as the organic membrane,which can block the electron transfer between solution and the Pt elec-trode.When Fe3O4NPs are embedded into the Cs membrane,theL.Yang et al./Biosensors and Bioelectronics25 (2009) 889–895891Fig.1.(A)TEM image of the Fe3O4NPs,the inset shows the crystal lattice of the selected-area Fe3O4NPs.(B)TEM image of the Fe3O4NPs with GOD.(C)Cyclic voltammograms (CVs)of different modified electrodes in5.0mM ferricyanide solution:(a)bare Pt electrode;(b)the electrode with Csfilm;(c)the electrode with Csfilm added GOD and Fe3O4NP(scan rate is50mV/s).(D)The Nyquist plots of the EIS of bare Pt(a),Pt/Cs(b),Pt/GOD/Fe3O4/Cs(c),in2.5mM K3Fe(CN)6/K4Fe(CN)6(1:1)containing0.1M KCl.current response of CVs could be increased(curve(c)),indicating the enhancing effect of the Fe3O4NPs on the electric conductivity of the enzyme electrode.3.2.2.Electrochemical impedance spectroscopyElectrochemical impedance spectroscopy(EIS)can provide use-ful information on the impedance changes of the electrode surface during the fabrication process.The semicircular portion at higher frequencies corresponds to the electron-transfer-limited process and its diameter is equal to the electron-transfer resistance,which controls the electron-transfer kinetics of the redox probe at the electrode interface.Meanwhile,the linear part at lower frequen-cies corresponds to the diffusion process(Feng et al.,2005).Fig.1D displays the Nyquist plots of the EIS of bare Pt(a),Pt/Cs(b), Pt/GOD/Fe3O4/Cs(c)in 2.5mM K3Fe(CN)6/K4Fe(CN)6(1:1)con-taining0.1M KCl.Cs displays an excellentfilm-forming ability, good adhesion,biocompatibility and susceptibility to chemical modification.It can perhaps overcome the existing problem of aggregation of Fe3O4nanoparticles.However,the Nyquist diam-eter of the Pt/Cs(curve(b),the value of resistance is458 )is much larger than that of the bare Pt(curve(a),the value of resis-tance is137 ),which suggests that the Cs coated on the electrode could increase the resistance of the electrodes(Miao and Tan, 2000).Whereas comparing with the Pt/Cs,the interfacial resis-tance of the Pt/GOD/Fe3O4/Cs(curve(c),the value of resistance is217 )is observed less than that of the Pt/Cs(b).The results show that the Fe3O4NPs modified electrode can decrease the resistance of the electrodes and hold high electron-transfer effi-ciency.3.3.The current response to glucose and the apparentMichaelis–Menten constantFig.2A shows the current response curves of GOD electrodes with Fe3O4nanoparticles in different concentrations of glucose. It can be seen that the electrode with Fe3O4NPs and Cs can significantly enhance the current response.In particular,as the concentration of glucose reaches4mM,the current response of the GOD electrode containing Fe3O4NPs and Cs(Pt/GOD/Fe3O4/Cs) (curve(a))is seven times higher compared with the enzyme electrode without Fe3O4NPs(Pt/GOD//Cs)(curve(c)).The rel-ative standard deviation(RSD)of the current response of the Pt/GOD/Fe3O4/Cs electrode is3.2%.Nafion is always used as a com-positefilm to improve the anti-interference of electrodes because the negatively charged group of nafion layer can repulse the neg-atively charged ascorbic acid from the surface of the composite electrode.However,nafion would block electron transfer between electrode and glucose,and then tend to reduce current response. During our research,we surprisingly found that the current response of Pt/GOD/Fe3O4/Cs/nafion electrode declined only a little after modified with nafionfilm(curve(b)).The relative standard deviation of the current response of the Pt/GOD/Fe3O4/Cs/nafion electrode is2.7%.The resulted calibration plot for glucose over the concentration range of6×10−6to2.2×10−3M is presented in Fig.2B.The linear plot reveals that such electrode can work well in glucose solu-tion with a sensitivity of11.54A cm−2mM−1,and the correlation coefficient is0.996.An estimated detection limit of6×10−6M at a signal-to-noise ratio of3is much lower than that of0.37mM in892L.Yang et al./Biosensors and Bioelectronics25 (2009) 889–895Fig.2.(A)The current response curves of GOD electrodes with and without Fe3O4 nanoparticles or Cs in different concentration of glucose:(a)Pt/GOD/Fe3O4/Cs,(b) Pt/GOD/Fe3O4/Cs/nafion,(c)Pt/GOD/Cs.(B)The linear calibration plot correspond-ing to the current response of different concentration of glucose.glucose sensor based on gold nanoparticle–chitosan compositefilm (Feng et al.,2009)and0.015mM by gold nanoparticles modified glucose sensor(Wang et al.,2008).The apparent Michaelis–Menten constant(K M),which gives an indication of the enzyme–substrate, can be obtained from the Michaelis–Menten equation(Kong et al., 2009):V=V max[S]/(K M+[S])where V is rate of reaction,V max is the maximum rate of reaction measured under saturated substrate condition,and[S]is the bulk concentration of the substrate.The value of the apparent Michaelis–Menten constant(K M)is calculated to show suitability of the enzyme in the hybrid nanobiocomposite matrix.From the inner of Fig.2B,K M value is found to be0.611mM for the immobilized GOD by using Line weaver–Burke plot(1/I ver-sus1/[C]).The value of K M is much lower than the reported14.4mM (Zou et al.,2008),6.3mM(Qiu et al.,2008)by ferrocene-modified multiwalled carbon nanotubes glucose biosensor and3.73mM by glucose biosensor based on prussian blue/chitosan hybridfilm (Wang et al.,2009).These results show that the biosensor possesses higher biological affinity to glucose.3.4.Discussion of the mechanism of the electron-transferbehaviorThe electron-transfer behavior of the GOD-electrode occurs as the following:GOD-FAD+2e−+2H+↔GOD-FADH2(1)Glucose+GOD-FAD→gluconolactone+GOD-FADH2(2) GOD-FADH2+O2→GOD-FAD+H2O2(3) 2H2O2→2H2O+O2+2e−(4) As is well known,-d-glucose,the substrate of GOD,will result in the reductive form of GOD(GOD-FADH2)in the enzyme-catalyzed reaction.In the process,it is reported that ferrocene could act as the electron-transfer mediators to communicate elec-trically with GOD and electrode(Escorcia and Dhirani,2007).In our enzyme-catalyzed system,the electrons generated from the biochemical reactions would transfer to the Pt/GOD/Fe3O4/Cs elec-trode through the Fe(II)/Fe(III)couples(Lin and Leu,2005).The results of the CVs also prove that Fe3O4NPs could provide a conduc-tive path through the composite membrane matrix.So the Fe3O4 NPs,acting as electron-transfer mediators,can help in enhancing the current response of enzyme electrode and then increasing the sensitivity of the biosensor.From Eqs.(3)and(4),it can be seen that the response current could be produced from the decompo-sition of hydrogen dioxide on the electrode with electrocatalytic properties of Fe3O4NPs.It has been proved that Fe3O4NPs with the valence-state of Fe(II)and Fe(III)was identified as the catalyst for H2O2detection.The reduction of H2O2proceeding as two-electron, two-proton reduction step which gives the product of H2O,as well as the reductive valence-state of Fe(II)on Fe3O4is oxidized to Fe(III)(Rossi et al.,2004).These processes are shown in Fig.3A. The proposed biochemical reaction during the glucose detection is shown in Fig.3B.In the enzyme-catalyzed process,Fe3O4NPs could catalyze the reaction of H2O2decomposed into H2O(Eq.(4)), then enhance the electron transfer to the electrode.So the current response of enzyme electrode could be improved.In order to prove this process,we also afforded the contrast electrode only modi-fied with the Fe3O4NPs and Cs(Pt/Fe3O4/Cs)to detect glucose. The current response of the contrast electrode is close to the back-ground current.It shows that the Fe3O4NPs cannot catalyze H2O2 and enhance the current response without the process in which GOD catalyzes-d-glucose forming H2O2in the present of oxy-gen.Besides these specific physics-chemical characters of iron in the electron-transfer of the electrode,Fe3O4NPs not only act as electron-transfer mediators and natural peroxidase,but also play an important role in the preparation of immobilized enzymes due to their desirable characteristics:large pore size and volume,and good electron conductivity(Lee et al.,2005).Fe3O4NPs also cre-ate suitable microenvironment which benefit the exposition of the active center,and increase the activity of enzyme(Zhang et al., 2005).In conclusion,with the excellent characteristics of Fe3O4 NPs as an electron-transfer mediator and artificial peroxide,theFig.3.Proposed mechanism of the electron transfer between nanocomposite and the Pt electrodes and the immobilization of glucose oxidase on nanocomposite matrix.L.Yang et al./Biosensors and Bioelectronics25 (2009) 889–895893Fig.4.Optimization of the biosensor:(A)response curves of the modified biosensor with different amounts of enzyme loading.(B)The current response of the biosensor at2.2mM glucose with increasing pH.(C)The current response of the biosensor at2.2mM glucose with increasing potential.(D)The current response of the biosensor at 2.2mM glucose with increasing temperature((a)the modified electrode with nafionfilm;(b)the modified electrode without Fe3O4NPs and nafionfilm).(E)The stability of the biosensor was investigated by amperometric measurements.new electrode modified by Fe3O4NPs can obviously amplify the current response of enzyme electrode and as a result increase the sensitivity of the biosensor.3.5.Optimization of the biosensor3.5.1.Effect of enzyme loadingThe amount of enzyme loading can affect the response current of this kind of glucose biosensor.Fig.4A shows the response curves of the modified biosensor with different enzyme loading.For each glucose concentration,with the increase of enzyme loading,the response current increases gradually.More enzymes would pro-duce more H2O2at the same concentration of glucose under the same condition(Luo et al.,2004).It shows that Fe3O4NPs could immobilize more enzymes on the electrode.However,From the Fig.4A,wefind that when the loading of enzyme is more than10U, the response current tends to be saturated.Further increment of enzyme loading would be a waste of this expensive reagent,so in the following experiments the enzyme loading is set as10U.3.5.2.Effect of pH,working potential and temperature on the biosensorInvestigation of the effect of pH value on the performance of the biosensor is of great importance,because the activity of the immobilized GOD is pH dependent(Yang and Zhu,2005).From the Eq.(1),as the proton is critical in redox behavior of GOD-FAD, the decrease of GOD response at high pH is possibly due to the decreases of proton concentration and bioactivity of the immobi-lized GOD.Fig.4B shows that the pH dependence of the sensor response is evaluated at2.2mM glucose over the pH range from 5.0to8.3.The optimum response is achieved at pH7.2.In order to keep consistent with the neutral condition of human blood,we chose phosphate buffers(pH6.8)to ensure higher sensitivity of the biosensor.Fig.4C shows the effect of working potential on the per-formance of the biosensor.The working potential is stepped from 0.2to0.6V in2.2mM glucose.The steady-state current response increases obviously with the working potential from0.2to0.4V, and then increases smoothly from0.4to0.6V.Therefore,0.4V is selected as the working potential for amperometric detection of glucose concentration.894L.Yang et al./Biosensors and Bioelectronics25 (2009) 889–895 The results of the effect of temperature on biosensor are pre-sented in Fig.4D.It shows that the current response of the modifiedbiosensor is enhanced with increasing temperature.The currentresponse reaches a maximum at approximately65◦C(curve(a)),and then goes down as the temperature turn higher.This phe-nomenon may be attributed to that the enzyme is denatured at hightemperature.In contrast,the modified electrode without Fe3O4NPs and nafionfilms shows that the response declines when tem-perature is higher than45◦C(curve(b)).The result indicates thatenzyme bioconjugated with Fe3O4NPs and the cover of nafionfilmshas good thermodynamic stability and life span.The modified glu-cose biosensor would be used in some special condition.In orderto keep consistent with the temperature of human body,35◦C isselected for this work.3.5.3.Stability of the enzyme electrodeThe stability of the biosensor is investigated by amperometricmeasurements in the presence of2.2mM glucose.Fig.4E shows thatthe current response of biosensor is retained about83%of its orig-inal response after52times uninterrupted detection.In addition,the long-term stability is also tested by measuring the glucose con-centration after a month.It is revealed that the current responseof the sensor maintains84%of the initial current response.Thismeans that Fe3O4NPs and nafionfilms ensure well stability of thebiosensor.With the help of Fe3O4NPs and nafionfilms,GOD cankeep bioactivity on the Pt electrode.3.6.The interference studyThe effect of several possible interfering substances,such as AA,uric acid(UA),sucrose,lactose on the glucose biosensor based onthe Pt/GOD/Fe3O4/Cs/Nafion electrode is investigated at0.4V ver-sus Ag/AgCl in phosphate buffer solution of pH6.8.The additionof0.3mM sucrose,lactose and UA do not cause any observableinterference to the detection of glucose.The normal physiological level of glucose is3–8mM,which ismuch higher than that of AA(0.1mM).However,the electron-transfer rate of AA is higher than that of glucose.The coexistenceof AA with glucose in the physiological system might significantlyinfluence the practical glucose detection.The signal for afixed con-centration of glucose is compared with the current value obtainedin the presence of the variable concentrations of the interferingspecies AA.The biosensor is evaluated with0.1mM AA in thedetection of2.2mM glucose.Table1compares the interferencesof the modified electrode and the electrode without nafion.Thecurrent response of the Pt/GOD/Fe3O4/Cs/nafion electrode in theabove solution is equal to the glucose solution without AA.How-ever,an increase of the current response by10%at the0.1mMAA is observed for the electrodes without nafionfilm.It is nafionfilm covered on the surface of the modified electrode that playsan important role in holding back the disturbance from interferingspecies.It indicates that the modified biosensor can give crediblecurrent signal though the interfering species reach high concentra-tions.Table1Comparison of the interference performance of electrodes with and without nafion.Interfering substances Concentration(mM)Pt/GOD/Fe3O4/Cs Pt/GOD/Fe3O4/Cs/nafionRSD(%)RSD(%)Sucrose37.5 2.3 Lactose3 4.4 1.6 Uric acid0.334 1.5 AA0.111.30.3Table2Glucose concentrations in serum samples tested by the proposed method and Auto-matic Biochemical Analyzer.Serum sample Clinical assayconcentration(mM)Proposed method(n=3)concentration(mM)RSD(%)Sample1 4.37 4.24−2.97 Sample2 6.19 6.08−1.78 Sample39.429.64 2.233.7.Application of the biosensor to serum samplesIn order to investigate the possible application of this new biosensor in clinical analysis,the modified electrode was also tested in real human serum and the results were compared with those obtained by Automatic Biochemical Analyzer as shown in Table2. The glucose in serum wasfirstly detected by Automatic Biochemi-cal Analyzer,and then400L of serum sample was diluted to4mL with PBS solution(pH6.8).The response currents were noted down. The concentration of the glucose in blood sample was calculated from the calibration curve.From the Table2,regardless of normal or high blood glucose level,we can see a good agreement between the data determined by two methods.It proved that the modified electrodes ascertained the practical application of the proposed biosensor in clinical analysis.4.ConclusionWe have proposed a novel strategy for developing a com-posite electrode consisting GOD/Fe3O4/Cs/Nafion,which showed remarkably enhanced sensitivity and selectivity for glucose in the presence of excess AA,UA as inference.The novel glucose biosensor showed relatively rapid response,high sensitivity,broad linear range,low detection limit,good reproducibility,and long-term stability.The wide detection range and high sensitivity may be assigned to the amplification of the magnitude of current response since Fe3O4NPs could catalyze the reaction of H2O2. This would account for the potential application of several char-acteristic enhancements in the determination of glucose in blood, drugs and food.Moreover,this biosensor virtually eliminated the interference and could be useful to detect glucose in samples con-taining other analytes.Therefore,this novel biosensor could be readily extended to the detection of other clinically important anti-gens by using Fe3O4NPs to develop other simple and practical biosensors.AcknowledgementsWe acknowledgefinancial support from the National Science Foundation of China(60602006,60736001)and the Knowl-edge Innovation Program of the Chinese Academy of Sciences (TYF0808).ReferencesBahshi,L.,Frasconi,M.,Vered,R.T.,Yehezkeli,O.,Willner,I.,2008.Anal.Chem.21, 8253–8259.Bakker,E.,2004.Anal.Chem.76,3285–3298.Cai,W.Y.,Xu,Q.,Zhao,X.N.,Zhu,J.J.,Chen,H.Y.,2006.Chem.Mater.18,279–284. Clark,L.C.,Lyons,C.,1962.Ann.NY Acad.Sci.102,29–45.Dyal,A.,Loos,K.,Noto,M.,Chang,S.W.,Spagnoli,C.,Shafi,K.V.P.M.,Ulman,A., Cowman,M.,Gross,A.R.,2003.J.Am.Chem.Soc.125,1684–1685.Escorcia,A.,Dhirani,A.A.,2007.J.Electroanal.Chem.601,260–268.Fan,C.H.,Wang,H.Y.,Sun,S.,Zhu,D.X.,Wagner,G.,Li,G.X.,2001.Anal.Chem.73, 2850–2854.Feng,D.,Wang,F.,Chen,Z.L.,2009.Sens.Actuators B138,539–544.Feng,J.J.,Zhao,G.,Xu,J.J.,Chen,H.Y.,2005.Anal.Biochem.342,280–286.Gao,L.Z.,Zhuang,J.,Nie,L.,Zhang,J.B.,Zhang,Y.,Gu,N.,Wang,J.,Feng,T.H.,Yang,D.L.,Perrett,S.,Yan,X.Y.,2007.Nat.Nanotechnol.,577–583.。
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ORIGINALRESEARCHPAPERImmobilizationofglucoseoxidaseonFe3O4/SiO2magneticnanoparticles
JunHuang•RongZhao•HaiWang•
WenqiZhao•LiyunDing
Received:11November2009/Revised:15January2010/Accepted:21January2010/Publishedonline:14February2010ÓSpringerScience+BusinessMediaB.V.2010
AbstractGlucoseoxidase(GOD)wascovalentlyimmobilizedontoFe3O4/SiO2magneticnanoparticles(FSMNs)usingglutaraldehyde(GA).Optimalimmo-bilizationwasatpH6with3-aminopropyltriethox-ysilaneat2%(v/v),GAat3%(v/v)and0.143gGODpergcarrier.TheactivityofimmobilizedGODwas4,570U/gatpH7and50°C.TheimmobilizedGODretained80%ofitsinitialactivityafter6hat45°Cwhilefreeenzymeretainedonly20%activity.TheimmobilizedGODmaintained60%ofitsinitialactivityafter6cyclesofrepeateduseandretained75%ofitsinitialactivityafter1monthat4°Cwhereasfreeenzymesretained62%ofitsactivity.KeywordsActivityÁFe3O4/SiO2magneticnanoparticlesÁGlucoseoxidaseÁImmobilizationÁStabilityIntroductionGlucoseoxidase(EC1.1.3.4)isaflavoproteinwhichcatalysestheoxidationofb-D-glucosebyO2toD-glucono-1,5-lactoneandH2O2.Itiswidelyusedinwineproduction,tinnedfoods,textilebleachingandasaglucosebiosensor.Enzymeimmobilizationtechnologyallowsthereuseofenzymesandimprovestheirstability.GODhasbeenimmobilizedonnumerouscarriers,suchassol–gelmatrix(Lietal.2000),poroussilicabeads(Akenetal.2000),poly-mermembrane(Raufetal.2006),polymermicro-sphere(CaoandLiu2009),nanoparticles(Caselietal.2009),andquantumdots(Huangetal.2008).Fiberopticbiosensorshavenumerousadvantagesoverconventionalsensors,whichincludehighsensitivity,fastresponse,absenceofelectricalinter-ference,remotesensing,andlowcost.Foranenzyme-based,fiberopticbiosensor,thesensorperformancecanbeimprovedbyenhancingthecatalyticeffectofenzyme.Therefore,thestudyofimmobilizedGODisimportanttothedevelopmentofafiberopticGODsensor.SiO2nanoparticlesarehighlybio-compatible
withenzymes(Krosetal.2001)andbothSiO2
nanoparticlesandFe3O4/SiO2magneticnanoparticles
(FSMNs)canbeusedtofabricateamperometricglucosesensor(YangandZhu2006;Qiuetal.2007).However,tothebestofourknowledge,therehasbeennoreportonconditionsofGODimmobilizationonSiO2nanoparticlesandFSMNs,theimmobilized
enzymepropertiesandtheirapplicationinfiberopticbiosensor.Inthiswork,GODwascovalentlyimmobilizedontoFSMNsusingglutaraldehyde.ImmobilizationconditionsincludingpH,initialconcentrationsof
ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.1007/s10529-010-0217-9)containssupplementarymaterial,whichisavailabletoauthorizedusers.
J.Huang(&)ÁR.ZhaoÁH.WangÁW.ZhaoÁL.DingKeyLaboratoryofFiberOpticSensingTechnologyandInformationProcessingofMinistryofEducation,WuhanUniversityofTechnology,430070Wuhan,Chinae-mail:hjun@whut.edu.cn
123
BiotechnolLett(2010)32:817–821DOI10.1007/s10529-010-0217-9APTS,GAandGODwereoptimized.ThepropertiesincludingoptimalpHandtemperature,thermalandstoragestabilityofimmobilizedGODwerecomparedwiththoseoffreeenzyme.Theoperationalstabilityofimmobilizedenzymewasalsoinvestigated.Theresultsindicatethatthisimmobilizedenzymeisapromisingcandidateforthedevelopmentofenzyme-basedfiberopticglucosesensor.
MaterialsandmethodsMaterialsGlucoseoxidase(GOD,EC1.1.3.4)wasfromAspergillusnigeratanactivityof100U/mgandwasobtainedfromBiosharpInc.3-Aminopropyltriethox-ysilane(APTS)waspurchasedfromAlfa-Aesar.Allotherreagentswereofanalyticalgradeandusedwithoutfurtherpurification.
PreparationoftheFSMNsThemagneticFe3O4nanoparticleswereprepared
usingthemethodofMassart(1981).Core–shellFSMNswerepreparedusingamodifiedmethodofSto¨beretal.(1968):0.5mlaqueoussolutioncontain-ing1.8mgFe3O4nanoparticleswasultrasonically
dispersedintoamixtureof80mlethanol,18mldeionizedwater,7ml25%(v/v)NH4OHand0.22ml
tetraethylorthosilicate(TEOS).Themixturewasstirredat25°Cfor6handFSMNswerecollectedbymagneticseparation.
ActivationofFSMNsFSMNsascarriersweresilanizedwithAPTSandactivatedusingglutaraldehyde(GA)beforeGODimmobilization.80llAPTSwasaddedinto4mlaqueoussolutioncontaining14mgFSMNsandthemixturewasstirredfor24hat25°C.FSMNswerecollectedwithamagnetanddispersedin3.52mlofphosphatebuffersolution(PBS,0.01M,pH7.3).0.48mlGA(25%,v/v)wasaddedtothePBSsolutioncontainingFSMNsandthereactionwasallowedtocontinuefor1.5hat25°C.Theactivatedcarriersweredispersedin6mlPBSbuffer(0.2M,pH6).
GODimmobilizationImmobilizationwasachievedbythefollowingapproach:2mgGODwasmixedwith6mlPBSbuffer(0.2M,pH6)containing14mgactivatedFSMNscarriersat4°Cfor12hwithoccasionalshaking.ThecarrierswerewashedwiththesamebuffersolutiontoremovefreeGOD.UsingtheBradfordmethod,theamountofenzymeproteinloadedontothecarrierwasevaluatedbysubtractingtheresidualproteincontentinthesolutionfromtheinitialproteincontentandthecontentofimmobilizedproteinwasquantified.