利用层层累积技术制备基于纳米碳管的葡萄糖生物传感器

以多壁纳米碳管(MWNTS)为电子媒介体和酶的吸附载体,利用层层累积的自组装技术固定葡萄糖氧化酶(GOX)的多层(MWNTa/GOx).复合薄膜修饰电极,制备了一种新型葡萄糖生物传感器.结果表明,传感器对葡萄糖的响应电流值随着MWNTa//GOx复合薄膜层数的不同而变化,当MWNTa//GOx复合薄膜的层教为6时,响应电流值迭到最大.(MWNTs/GOx).复合薄膜修饰的葡萄糖生物传感器对30mmol/L葡萄糖的响应电流为1.63μA,响应时间仅为6.7 s.该生物传感器检测的线性范围为0.5～15 mmol/L,最低检测浓度可达0.09 mmol/L.     

基于微流控芯片的尿路感染细菌鉴定及抗生素敏感性测试

发展了一种微流控芯片纸基细菌分析技术,用于多重细菌鉴定与抗生素敏感性测试.制备了阵列培养池芯片,以滤纸作为衬底固定显色培养基和抗生素.利用PVDF疏水薄膜止流阀,将尿液样品引入芯片并分隔于不同培养池.借助于培养池阵列的空间分辨力,实现多重细菌分析.根据特异性显色结果实现细菌鉴定,通过实时显色强度分析实现细菌定量,依据抑制显色反应的最低抗生素浓度确定抗生素敏感性.以3种泌尿系统感染常见病原菌(大肠杆菌、金黄色葡萄球菌和粪肠球菌)为模拟测试对象进行分析,结果表明,芯片方法可以在18 h内实现对3种细菌的同时鉴定及6种抗生素敏感性测试.对照实验显示,芯片法与传统方法细菌鉴定和抗生素敏感性测试结果一致性分别为94.1％和93.9％.本研究建立的微流控芯片细菌分析方法简便快速,非常适合于医疗资源匮乏条件下的细菌分析.     

刀豆球蛋白层层自组装双酶电极对芳香胺物质的检测

基于生物识别组装方法将辣根过氧化物酶(HRP)和葡萄糖氧化酶(Gox)固定在金电极表面制备了HRP/Gox双酶多层膜电极.HRP/Gox双酶电极通过Gox催化氧化葡萄糖"原位"产生过氧化氢的方式分析检测芳香胺物质.探讨了葡萄糖浓度和酶组装层数对双酶电极电流响应的影响.在优化条件下,制备的双酶电极在p-苯二胺的浓度为7.6 ～53.2 μmol/L时有良好的线性响应,灵敏度为146.04 mA·L/mol.常见的干扰物葡萄糖和抗坏血酸对该双酶电极无干扰.     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

纳米铂颗粒修饰薄膜金电极的新型葡萄糖传感器研究

在没有引入电子媒介体条件下,为了提高传感器的响应灵敏度,降低工作电位,利用电化学沉积法在薄膜金电极表面修饰纳米铂颗粒,并通过戊二醛固定酶的方法制备了一种新型生物传感器。研究了在薄膜金电极上修饰纳米铂颗粒前后传感器对低浓度葡萄糖的响应影响。结果表明,纳米铂颗粒修饰后所制备的葡萄糖传感器工作电位下降为0.4 V,测定葡萄糖的检出限从100μmol/L下降到10μmol/L。传感器对10~1300μmol/L低浓度葡萄糖的响应灵敏度为50.8 nA/(cm2μmol/L);响应时间30 s;r为0.9974;传感器精密度为2.1%,并具有较好的稳定性。     

高选择性的镍基无酶葡萄糖微传感器的研制及应用

采用电刻蚀法制得微镍电极,通过循环伏安法在微镍电极上修饰过氧化聚吡咯膜,利用葡萄糖在碱性条件下在该修饰电极上的电催化氧化性质,制备了新型的抗干扰的无酶葡萄糖微传感器;研究了其电化学氧化机理,在较低的碱性(pH=12.0)和较低的氧化电位( 0.47 V)条件下,微镍修饰电极上产生的Ni(Ⅲ)能直接将葡萄糖氧化为葡萄糖酸内酯,产生的安培响应与葡萄糖浓度在5.0×10-6 ～1.1×10-3 mol/L范围内呈线性关系;检出限为2.4×10-6 mol/L.该微传感器灵敏度高(30.4 nA/μM)、选择性好(20倍AA和UA不干扰)、响应快(小于3 s)、重现性好,而且制作简单、使用方便,已用于流动注射分析(FIA)测定血清中血糖含量.     

PDMS芯片自由酶反应器检测葡萄糖

近年来,芯片酶反应器备受关注~([1]).根据芯片中酶的存在形式,可分为自由酶反应器和固定化酶反应器.固定化酶反应器能使酶的稳定性提高,减少了酶的消耗,不会对产物造成污染~([1]),但酶在芯片通道内的固定步骤比较复杂;相比而言,自由酶反应器具有更好的灵活性,制作方便,操作简单.葡萄糖(Glu)存在于人体的血浆和淋巴液中,是生命活动中不可缺少的物质,它在人体内直接参与新陈代谢过程.     

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖

应用电泳中介微分析(EMMA)技术,构建聚二甲基硅氧烷(PDMS)芯片自由酶反应器, 在线检测葡萄糖(Glu),在十字形的芯片通道上,采用自制的碳纤维微电极检测葡萄糖氧化酶(GOD)催化氧化Glu生成的H2O2,并对检测电位、GOD浓度、GOD进样时间、分离电压等参数进行了优化,测定了该自由酶反应器的线性范围和检出限,考察了其重现性及稳定性.结果表明,此自由酶反应器制作方便,操作简单,重现性好,Glu浓度在0.1～20 mmol/L之间有较好的线性关系(r=0.997),检出限为19.8 μmol/L(S/N=3).     

光纤分导微梁阵列生化传感装置研制及检测应用

研制了一套基于光杠杆原理的微悬臂梁阵列传感器平台,并通过使用设计制作的微悬臂梁阵列芯片展示其在生物化学方面的检测应用.传感器平台使用光导纤维束分别与激光器耦合作为悬臂梁阵列的扫描光源,具有良好的检测稳定性,检测信号噪声水平约为2 nm;设计制作的微悬臂梁阵列芯片具有良好的平直度,温度响应均匀一致,各梁温度改变响应灵敏度偏差不超过5.0％.将整套传感系统被用于检测水溶液中的Hg2+,检测浓度范围为1 ～ 200 ng/mL;同一浓度下微悬臂梁阵列检测结果曲线一致性良好,平均偏差小于15％.在研制仪器平台上,分别实现了自制和国外商品化芯片对1.0和0.2 ng/mL样品的检测,结果表明,制作的微悬臂梁阵列芯片的检测灵敏度相对较低,需进一步改进悬臂梁阵列制作工艺.     

硅溶胶-凝胶包埋纳米金和酶的葡萄糖生物传感器

采用溶胶-凝胶技术将金纳米粒子和葡萄糖氧化酶一次性固定于硅溶胶-凝胶的网络结构中,制备了葡萄糖生物电化学传感器并优化了传感器的制备条件。酶电极对葡萄糖具有良好的电化学响应,葡萄糖浓度在0.02~2.0 mmol/L范围内和催化电流呈线性关系,检出限为0.005 mmol/L。酶电极在4 ℃下贮存100 d后对葡萄糖的响应仅下降8%。该酶电极灵敏度高、响应快、稳定性好。     

Lifetime Improvement of Glucose Biosensor by Epoxy‐Enhanced PVC Membrane

A new approach using epoxy resin to enhance the durability and adhesion of a diffusion‐limiting membrane in amperometric biosensors is described. The polymer membrane was mainly composed of commercially available fast epoxy adhesive ATACS 5104, poly(vinyl chloride) (PVC) and plasticizers such as isopropyl myristate (IMP) and Aliquat 336 (AL). It can be readily deposited on various substrates by using coating and other thin film fabrication methods. The effect of epoxy resin in the membrane composition was investigated using a coil‐type glucose biosensor containing extra enzyme. The ideal membrane was found to include approximately 1/3 epoxy resin, 1/3 plasticizer and 1/3 PVC. Such a membrane was verified to be porous and permeable to small molecules like glucose and can tightly adhere to other beneath layers such as a Nafion membrane, which serves as the interference‐eliminating layer. These epoxy‐based glucose biosensors showed excellent electrochemical response properties including a long lifetime and can be used for microanalysis of solutions and biological fluids. With an additional PU outermost layer, the present glucose biosensors can potentially be used for in vivo measurements.     

Fast prototyping of hydrophobic disposable polymer support arrays for matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of proteins by atmospheric molding

A fast protocol for prototyping hydrophobic disposable poly(alkyl methacrylate-co-methyl methacrylate) copolymer sample support arrays for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of proteins by atmospheric molding is introduced. The sample support arrays were replicated by molding prepolymer alkyl methacrylate solutions into sandwich molds containing a micromachined silicon master, an aluminum spacer, and glass cover plates, followed by UV-initiated in situ polymerization under atmospheric pressure. The fabrication procedure enables a simultaneous fabrication/modification of single-use polymer arrays by a targeted selection of functional groups of the copolymerized monomers during molding. The one-step modification during the fabrication is demonstrated for enhanced protein adsorption to the modified materials by introduction of hydrophobic butyl-, dodecyl-, and octadecyl groups to the polymer backbone without a need for additional surface coating or derivatization. The MALDI-MS performance of the new polymer chips was tested for spectral measurements of bovine pancreas insulin, horse heart myoglobin, and bovine serum albumin. The protein adsorption to the new hydrophobic copolymer chips was studied for bovine pancreas trypsinogen; the sample desalting parameters, such as time and volume, were optimized for myoglobin as model proteins. A significant signal increase was achieved after efficient desalting of an insect Delta11-desaturase membrane protein fragment from a complex elution buffer (100 mM phosphate, 10 mM tris(hydroxyethyl)aminomethane, 0.5 M NaCl, and 10 mM ethylenediamine tetraacetic acid) on the poly(butyl methacrylate-co-methyl methacrylate) copolymer chip (monomer ratio 8:2 v/v) by simply washing the target zones. The new chips offer reduced sample manipulation and device fabrication times as well as simple operation.     

糖芯片的研究进展

糖芯片是生物芯片的一种,如基因芯片对于基因研究和蛋白质芯片对于蛋白质组研究一样,糖芯片在糖组学的研究中同样也将扮演重要的角色。本文系统介绍了糖芯片的制备流程及其应用,以及在糖芯片研发开发中的技术障碍。     

Diffusion of hydrosilanes from the control layer to the vinylsilane-rich flow membrane during the fabrication of microfluidic chips

During the fabrication of poly(dimethylsiloxane) (PDMS)-based microfluidic chips, polymethylhydrosiloxane (PMHS) species in the control layer diffuse into the flow membrane, which contains polymethylvinylsiloxane (PMVS), and the components cross-link together to form the mechanically enhanced membrane. The diffusion course was investigated by using attenuated total reflectance FTIR and the improvement of mechanical properties of the flow membrane was studied by measuring the Young's modulus and the tensile strength.     

Poly(methyl methacrylate) CE microchips replicated from poly(dimethylsiloxane) templates for the determination of cations

A novel method for the rapid fabrication of poly(methyl methacrylate) (PMMA) microfluidic chips using poly(dimethylsiloxane) (PDMS) templates has been demonstrated. The PDMS molds were fabricated by soft lithography. The dense prepolymerized solution of methyl methacrylate containing thermal and UV initiators was allowed to polymerized between a PDMS template and a piece of a 1 mm thick commercial PMMA plate under a UV lamp. The images of microchannels on the PDMS template were precisely replicated into the synthesized PMMA substrates during the UV-initiated polymerization of the prepolymerized solution on the surface of the PMMA plate at room temperature. The polymerization could be completed within 10 min under ambient temperature. The chips were subsequently assembled by thermal bonding of the channel plate and the cover sheet. The new fabrication method obviates the need for specialized replication equipment and reduces the complexity of prototyping and manufacturing. Nearly 20 PMMA chips were replicated using a single PDMS mold. The attractive performance of the new microfluidic chips has been demonstrated by separating and detecting cations in connection with contactless conductivity detection. The fabricated PMMA microchip has also been successfully employed for the determination of potassium and sodium in environmental and biological samples.     

A simple method for preparation of macroporous polydimethylsiloxane membrane for microfluidic chip-based isoelectric focusing applications

A new, simple method was reported to prepare PDMS membranes with micrometer size pores for microfluidic chip applications. The pores were formed by adding polystyrene and toluene into PDMS prepolymer solution prior to spin-coating and curing. The resulting PDMS membrane has a thickness of around 10 μm and macropores with a diameter ranging from 1 to 2 μm measured using scanning electron microscope (SEM) imaging. This PDMS membrane was validated by integrating it with PDMS microfluidic chips for protein separation using isoelectric focusing mechanism coupled with whole channel imaging detection (IEF-WCID). It has been shown that five standard pI markers and a mixture of two proteins, myoglobin and β-lactoglobulin, can be separated using these chips. The results indicated that this macroporous PDMS membrane can replace the dialysis membrane in PDMS chips for the IEF-WCID technique. The preparation method of macroporous PDMS membrane may be potentially applied in other fields of microfluidic chips.     

Patterning microbeads inside poly(dimethylsiloxane) microfluidic channels and its application for immobilized microfluidic enzyme reactors

We propose a convenient and reliable approach for immobilizing microbeads on poly(dimethylsiloxane) (PDMS) microchips. It is built upon a simple fabrication procedure of PDMS chip through directly printing the master with an office laser printer which was described in our previous work (J. Chromatogr. A 2005, 1089, 270-275). On the printed toners used as the positive relief of the master, microbeads were immobilized by a thermal treatment and then transferred to the surface of the microchip by direct molding of the prepolymer on the master. With this approach, the region-selective immobilization of microbeads and the fabrication of PDMS microchips can be accomplished at the same time. Then, using these microbeads as supports, further modification with enzyme was achieved. Surface characteristics of the microbeads-modified PDMS microchannels were investigated with scanning electron microscope, atomic force microscope, and inverse fluorescence microscope. The electrokinetic properties of the native PDMS and the modified PDMS chips were also compared. Based on this approach, an immobilized glucose oxidase (GOD) reactor was constructed and the reaction using glucose as substrate was studied. All these experiments aim to show that the proposed approach may have a good potential in the study of biochemistry and other related areas.     

微流控层流技术的研究

基于微型通道自身的层流特点而发展起来的多相层流技术,从最初的液-液微萃取开始,由于其结构加工简单、操作方便和分析功能强大,已逐渐发展成为一种加工分析方法,为微流控分析的研究应用打开了一个崭新的局面。本文概述了层流的基本原理,总结了近10年来在这方面的研究,包括层流界面间的分子扩散、转移现象和化学反应,以及层流刻蚀加工技术及其在制备纳米材料和在生命医学方面的应用。具体介绍了应用层流技术进行微芯片的加工制作,微型反应器的制备,离子、分子的分离分析,聚合物薄膜的形成和应用,微通道内有机合成反应的控制,溶液的浓度梯度控制以及在免疫检测中的应用,对细胞、生物大分子的操作控制,以及对生物试剂的预处理分析等。     

微流控层流技术的研究

基于微型通道自身的层流特点而发展起来的多相层流技术，从最初的液-液微萃取开始，由于其结构加工简单、操作方便和分析功能强大，已逐渐发展成为一种加工分析方法，为微流控分析的研究应用打开了一个崭新的局面。本文概述了层流的基本原理，总结了近10年来在这方面的研究，包括层流界面间的分子扩散、转移现象和化学反应，以及层流刻蚀加工技术及其在制备纳米材料和在生命医学方面的应用。具体介绍了应用层流技术进行微芯片的加工制作，微型反应器的制备，离子、分子的分离分析，聚合物薄膜的形成和应用，微通道内有机合成反应的控制，溶液的浓度梯度控制以及在免疫检测中的应用，对细胞、生物大分子的操作控制，以及对生物试剂的预处理分析等。     

The electrochemical behaviour of ferrocene in a photocurable poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) film for a glucose biosensor

A single-step fabrication of a glucose biosensor with simultaneous immobilization of both ferrocene mediator and glucose oxidase in a photocurable methacrylic film consisting of poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) was reported. The entrapped ferrocene showed reversible redox behaviour in the photocured film and no significant leaching of both entrapped ferrocene and enzyme glucose oxidase was observed because of the low water absorption properties of the co-polymer films. From electrochemical studies, ferrocene entrapped in the co-polymer film demonstrated slow diffusion properties. A linear glucose response range of 2-11 mM was obtained at low applied potential of +0.25 V. The glucose biosensor fabricated by this photocuring method yielded sensor reproducibility and repeatability with relative standard deviation of <10% and long-term stability of up to 14 days. The main advantage of the use of photocurable procedure is that biosensor membrane fabrication can be performed in a single step without any lengthy chemical immobilization of enzyme.