Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H₂O₂ and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM^?1 cm^?2) and stability (80% remains after 30 days).     

pH‐Switchable Bioelectrocatalysis Based on Weak Polyelectrolyte Multilayers

Weak polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were assembled into {PAH/PAA}_n layer‐by‐layer films on electrodes. The cyclic voltammetry (CV) response of ferrocenecarboxylic acid (Fc(COOH)) at {PAH/PAA}₅ film electrodes assembled under the specific condition showed pH‐sensitive “on‐off” switching property. This property was further used to control the electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH) as the electron transfer mediator, so that the pH‐switchable bioelectrocatalysis could be realized. The mechanism of pH‐sensitive behavior of the system was explored and believed to originate from the pH‐dependent structure change of the films.     

Properties of Dendrimer‐Encapsulated Pt Nanoparticles Doped Polypyrrole Composite Films and Their Electrocatalytic Activity for Glucose Oxidation

A type of novel electroanalytical sensing nanobiocomposite material was prepared by electropolymerization of pyrrole containing poly(amidoamine) dendrimers‐encapsulated platinum nanoparticles (Pt‐PAMAM), and glucose oxidase (GOx). The Pt nanoparticles encapsulated in PAMAM are nearly monodisperse with an average diameter of 3 nm, and they provide electrical conductivity. Polypyrrole acts as a polymer backbone to give stable and homogeneous cast thin films, and it also defines the electrical conductivity. Both Polypyrrole and PAMAM can provide a favorable microenvironment to keep the bioactivity of enzymes such as glucose oxidase. The homogeneity of GOx/Pt‐PAMAM‐PPy nanobiocomposite films was characterized by atomic force microscopy (AFM). Amperometric biosensors fabricated with these materials were characterized electrochemically using cyclic voltammetry (CV), electrochemical impedance spectra (EIS) and amperometric measurements in the presence of hydrogen peroxide or glucose. All those show the resultant biosensor sensitivity was strongly enhanced within the nanobiocomposite film. The optimized glucose biosensor displayed a sensitivity of 164 μA mM^?1 cm^?1, a linear range of 0.2 to 600 μM, a detection limit of 10 nM, and a response time of <3 s.     

A New Technique for Chemical Deposition of Pt Nanoparticles and its Applications on Biosensor Design

A new glucose biosensor design based on glucose oxidase (GOD) immobilized by polypyrrole has been described in this paper. The polymerization of pyrrole was initiated by a hexachloroplatinate which itself was reduced into Pt nanoparticles and thus served as a catalyst for the H₂O₂ oxidation. Properties of the produced GOD modified electrode were examined and the activity of the entrapped enzyme was determined by basic application on the amperometric detection of glucose. Much better results were found comparatively with the enzyme electrode for which the enzyme was entrapped by the electrochemically polymerized polypyrrole. This kind of technique for Pt nanoparticles deposition can be generalized to many cases where polypyrrole is used.     

铂纳米颗粒修饰直立碳纳米管电极的葡萄糖生物传感器

基于Pt纳米颗粒修饰直立的碳纳米管电极制备了葡萄糖生物传感器.铂纳米颗粒是利用电位脉冲沉积法修饰到直立碳纳米管上的,可以增强电极对酶反应过程当中产生的过氧化氢的催化行为.用扫描电镜和透射电镜观察了直立碳纳米管在修饰Pt纳米颗粒前后的形态.该酶电极对葡萄糖的氧化表现出很好的响应,线性范围为1×10-5～7×10-3mol/L,响应时间小于5s,并且有很好的重现性.     

Development of amperometric glucose biosensor through immobilizing enzyme in a Pt nanoparticles/mesoporous carbon matrix

Pt nanoparticles were deposited on mesoporous carbon material CMK-3. Glucose oxidase (GOx) was immobilized in the resulting Pt nanoparticles/mesoporous carbon (Pt/CMK-3) matrix, and then the mixture was cast on a glassy carbon electrode (GCE) using gelatin as a binder. The glucose biosensor exhibited excellent current response to glucose after cross-linking with glutaraldehyde. At 0.6V (vs. SCE) the response current was linear to glucose concentration in the range of 0.04-12.2mM. The response time (time for achieving 95% of the maximum current) was 15s and the detection limit (S/N=3) was 1 microM. The Michaelis-Menten constant (K(m)(app)) and the maximum current density (i(max)) were 10.8 mM and 908 microAcm(-2), respectively. The activation energy of the enzymatic reaction was estimated to be 22.54 kJ mol(-1). The biosensor showed good stability. It achieved the maximum response current at about 52 degrees C and retained 95.1% of its initial response current after being stored for 30 days. In addition, some fabrication and operation parameters for the biosensor were optimized in this work. The biosensor was used to monitor the glucose levels of serum samples after being covered with an extra Nafion film to improve its anti-interferent ability and satisfied results were obtained.     

伴刀豆球蛋白-糖蛋白特异性识别作用构筑的多层膜及其在生物传感器制备中的应用

研究了伴刀豆球蛋白-糖蛋白特异性分子识别作用构建的多层膜及其在葡萄糖生物传感器制备中的应用.结果表明,含有糖残基的葡萄糖氧化酶可以通过识别作用组装进入多层膜,不含糖基的过氧化氢酶经过化学修饰引入麦芽糖残基,也可以通过分子识别作用组装到多层膜中.对于多层膜厚度研究的结果表明,膜的厚度随层数的增加呈线性关系.研究了多层膜的表面形貌,并讨论了膜结构与功能的关系.伴刀豆球蛋白-酶多层膜修饰的金电极的电化学实验表明,多层膜中的酶具有催化活性.过氧化氢酶/葡萄糖氧化酶串联组装的生物传感器能够有效消除样品中过氧化氢对葡萄糖测定带来的干扰.     

LBL分子沉积法制备葡萄糖氧化酶电极

采用以静电力为主的逐层分子交替沉积技术制备葡萄糖氧化酶（GOD）电极.通过带有正电荷的聚二甲基二烯丙基铵盐酸盐（PDDA）和带有负电荷的GOD交替沉积在修饰有3-巯基-1-丙基磺酸钠（MPS）的金电极表面.以甲酸二茂铁为电子媒介体,用循环伏安法检测GOD电极对葡萄糖的响应.结果表明,当GOD电极组装层数小于4时,电流响应随着层数的增加而增大,超过4层时电流响应减小.其中4层GOD修饰电极的线性范围为0.55～6.63 mmol•L－1,当pH为7.0时,响应最大.同时电极的检测重现性能良好,相对标准偏差为2.4％.     

基于温度的酶注射式葡萄糖生物传感器检测方法研究

针对酶注射式葡萄糖生物传感器在实际使用中因为标定液与被测液的温度不同而引起的测量结果不准确问题,提出一种基于温度的葡萄糖浓度检测方法.首先根据酶促反应动力学建立目前酶注射式葡萄糖生物传感器浓度检测模型,之后利用阿伦尼乌斯公式建立温度与浓度检测动力学模型中未知参数之间的关系,并将此关系代入浓度检测动力学模型中,以建立基于温度的浓度检测新模型.此模型以温度与酶促反应的电流初始斜率为输入值,以被测葡萄糖浓度为输出值,利用此模型提出了以反应混合液的温度和反应初始电流斜率推导被测液浓度的检测方法.利用改进的检测方法进行检测,不仅能够降低温差的影响,提高检测的准确性,还可以省略常规检测中的人工标定,避免人工标定所需的取样探头拆卸步骤,更加有利于在线使用.分别在25.0,30.0和42.0℃下检测1.5 mg/mL和2.5 mg/mL葡萄糖溶液,利用原检测方法与基于温度的检测方法进行检测,结果表明,基于温度的检测方法回收率均在95.0％以上,明显优于原检测方法.     

Amperometric Glucose Biosensor Based on Platinum Nanoparticles Combined Aligned Carbon Nanotubes Electrode

A sensitive amperometric glucose biosensor based on platinum nanoparticles (PtNPs) combined aligned carbon nanotubes (ACNTs) electrode was investigated. PtNPs which can enhance the electrocatalytic activity of the electrode for electrooxidating hydrogen peroxide by enzymatic reaction were electrocrystallized on 4‐aminobenzene monolayer‐grafted ACNTs electrode by potential‐step method. These PtNPs combined ACNTs' (PtNPs/ACNTs) surfaces were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The highly dispersed PtNPs on ACNTs can be obtained. The enzyme electrode exhibits excellent response performance to glucose with linear range from 1×10^?5–7×10^?3 mol L^?1 and fast response time within 5 s. Furthermore, this glucose biosensor also has good reproducibility. It is demonstrated that the PtNPs/ACNTs electrode with high electrocatalytic activity is a suitable basic electrode for preparing enzyme electrodes.     

Amperometric Glucose Biosensor Based on Pt‐Pd Nanoparticles Supported by Reduced Graphene Oxide and Integrated with Glucose Oxidase

A novel glucose biosensor was developed based on the immobilization of glucose oxidase (GOx) on reduced graphene oxide incorporated with electrochemically deposited platinum and palladium nanoparticles (PtPdNPs). Reduced graphene oxide (RGO) was more hybridized by chemical and heat treatment. Bimetallic nanoparticles were deposited electrochemically on the RGO surface for potential application of the Pd? Pt alloy in biosensor preparation. The as‐prepared hybrid electrode exhibited high electrocatalytic activities toward H₂O₂, with a wide linear response range from 0.5 to 8 mM (R²=0.997) and high sensitivity of 814×10^?6 A/mMcm². Furthermore, glucose oxidase with active material was integrated by a simple casting method on the RGO/PdPtNPs surface. The as‐prepared biosensor showed good amperometric response to glucose in the linear range from 2 mM to 12 mM, with a sensitivity of 24×10^?6 A/mMcm², a low detection limit of 0.001 mM, and a short response time (5 s). Moreover, the effect of interference materials, reproducibility and the stability of the sensor were also investigated.     

Nonenzymatic Electrochemical Glucose Sensor Based on Pt Nanoparticles/Mesoporous Carbon Matrix

A nonenzymatic amperometric sensor for sensitive and selective detection of glucose has been constructed by using highly dispersed Pt nanoparticles supported onto mesoporous carbons (MCs). The Pt nanoparticles/mesoporous carbons (Pt/MCs) composites modified electrode displayed high electrocatalytic activity towards the oxidation of glucose. At an applied potential of 0.1 V, the Pt/MCs electrode has a linear dependence (R=0.996) in the glucose concentration up to 7.5 mM with a sensitivity of 8.52 mA M^?1 cm^?2. The Pt/MCs electrode has also shown highly resistant toward poisoning by chloride ions and without interference from the oxidation of common interfering species.     

Fabrication of a Highly Sensitive Glucose Biosensor Based on Immobilization of Osmium Complex and Glucose Oxidase onto Carbon Nanotubes Modified Electrode

In this research a novel osmium complex was used as electrocatalyst for electroreduction of oxygen and H₂O₂ in physiological pH solutions. Electroless deposition at a short period of time (60 s), was used for strong and irreversible adsorption of 1,4,8,12‐tetraazacyclotetradecane osmium(III) chloride (Os(III)LCl₂) ClO₄ onto single‐walled carbon nanotubes (SWCNTs) modified GC electrode. The modified electrode shows a pair of well defined and reversible redox couple, Os(IV)/Os(III) at wide pH range (1–8). The glucose biosensor was fabricated by covering a thin film of glucose oxidase onto CNTs/Os‐complex modified electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The fabricated biosensor shows high sensitivity, 826.3 nA μM^?1cm^?2, low detection limit, 56 nM, fast response time <3 s and wide calibration range 1.0 μM–1.0 mM. The biosensor has been successfully applied to determination of glucose in human plasma. Because of relative low applied potential, the interference from electroactive existing species was minimized, which improved the selectivity of the biosensor. The apparent Michaelis‐Menten constant of GOx on the nanocomposite, 0.91 mM, exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this glucose biosensor.     

Electrochemical Immunoanalysis for Carcinoembryonic Antigen Based on Multilayer Architectures of Gold Nanoparticles and Polycation Biomimetic Interface on Glassy Carbon Electrode

This paper describes a layer‐by‐layer (LBL) self‐assembly process of chitosan (CTS) and gold nanoparticles (Au) on the pretreated negatively charged glassy carbon (GC) electrode to fabricate electrochemistry immunosensor with a nontoxic biomimetic interface, which provided an environment similar to a native system and allowed more freedom in orientation for immobilization of carcinoembryonic antibody (anti‐CEA) to monitor carcinoembryonic antigen (CEA). UV‐vis spectroscope, atomic force microscopy (AFM), and cyclic voltammetric (CV) measurements were used to follow the multilayer film formation. The performance of the biominetic interface and factors influencing the assay system were investigated in detail. The differential pulse voltammetry (DPV) current response is used for the CEA concentration assay. The dynamic range was from 0.50 to 80.00 ng mL^?1 with a detection limit of 0.27 ng mL^?1 at 3σ. In addition, the experiment results indicate that immobilization described in this proposed method exhibits a good sensitivity, selectivity, and stability.     

基于柔性衬底的ZnO葡萄糖酶电极制备及特性

通过水热法在长有ZnO籽晶层的柔性聚酰亚胺(PI)衬底上生长了整齐的ZnO纳米棒,ZnO纳米棒的晶体结构和表面形貌通过X射线衍射(XRD)、扫描电子显微镜(SEM)等进行表征.通过静电吸附方式,将葡萄糖氧化酶(GOx)固定在其表面.分别对GOx及修饰前后的ZnO纳米棒进行了紫外-可见光谱表征,发现修饰后存在ZnO的吸收峰和GOx的特征吸收峰,表明GOx固定在ZnO表面.通过对修饰样品进行傅里叶变换红外(FTIR)光谱测试发现了与GOx相关的吸收峰,这进一步表明GOx仍保持生物活性.最后在循环伏安曲线的测试中,这种在柔性衬底上制备的生物酶电极表现出非常灵敏的电流响应,为制备柔性葡萄糖生物传感器奠定了实验基础.     

Direct Electrochemistry of Glucose Oxidase Immobilized at a Novel Composite Material β‐Cyclodextrin/poly(4‐aminothiophenol)/Au Nanoparticle Modified Electrode

A novel complex material was fabricated by three steps. In the first step, gold nanoparticle (Au_nano) was prepared with the method of chemistry and dialysis. In the second step, 4‐aminothiophenol (AT) was encapsulated in the cavity of β‐cyclodextrin and formed inclusion complex, cyclodextrin/4‐aminothiophenol (CD/AT). And then inclusion complex was adsorbed to the surface of Au_nano based on the bond of Au‐S interaction. In the last step, a complex material, cyclodextrin/poly(4‐aminothiophenol)‐Au nanoparticles (CD/PAT‐Au_nano) was obtained by the polymerizing in the acid solution initiated by chlorauric acid. The CD/PAT‐Au_nano has spherical nanostructure with the average diameter of 55 nm. Glucose oxidase (GOx) was anchored with this complex material and direct electrochemistry of GOx was achieved. A couple of stable and well‐defined redox peaks were observed with the formal potential (E^0′) of ‐0.488 V (vs. SCE) in a pH 6.98 buffer solution. The GOx modified electrode also exhibited an excellent electrocatalytic activity to the reduction of glucose, a linearity range for determination of glucose is from 0.25 mM to 16.0 mM with a detection limit of 0.09 mM (S/N = 3). This protocol had potential application to fabricate the third‐generation biosensor.     

基于Dy_2(MoO_4)_3-AuNPs复合纳米材料的葡萄糖生物传感器

采用水热法合成了纳米材料钼酸镝[Dy_2(MoO_4)_3],并制备了Dy_2(MoO_4)_3-AuNPs复合材料,利用该复合材料固定葡萄糖氧化酶(GOD)构建了葡萄糖生物传感器.通过透射电子显微镜(TEM)、紫外-可见光谱(UV-Vis)和能谱分析(EDS)等手段对所制备的材料进行了表征,并利用电化学阻抗谱(EIS)和循环伏安(CV)曲线研究了该传感器的电化学性能.结果表明,Dy_2(MoO_4)_3-AuNPs复合材料具有较好的生物相容性,能增强固定化的GOD的生物活性,并促进GOD在电极表面的电子传递速率;该传感器在葡萄糖浓度为0.01~1.0 mmol/L范围内葡萄糖浓度与响应电流呈较好的线性关系,最低检出限为3.33μmol/L(S/N=3),该生物传感器还具有较好的稳定性和重现性.     

Construction of Polycation‐Based Non‐Viral DNA Nanoparticles and Polyanion Multilayers via Layer‐by‐Layer Self‐Assembly

Summary: The multilayers of polycation‐based non‐viral DNA nanoparticles and biodegradable poly(L ‐glutamic acid) (PGA) were constructed by a layer‐by‐layer (LbL) technique. Poly(ethyleneimine) (PEI) was used to condense DNA to develop non‐viral DNA nanoparticles. AFM, UV‐visible spectrometry, and TEM measurements revealed that the PEI‐DNA nanoparticles were successfully incorporated into the multilayers. The well‐structured, easily processed multilayers with the non‐viral DNA nanoparticles may provide a novel approach to precisely control the delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.

A TEM image of the cross section of a (PGA/PEI‐DNA nanoparticle)₂₀ multilayer.     

Electrochemical Biosensors Based on Layer‐by‐Layer Assemblies

Layer‐by‐layer (LBL) assemblies, which have undergone great progress in the past decades, have been used widely in the construction of electrochemical biosensors. The LBL assemblies provide a strategy to rationally design the properties of immobilized films and enhance the performance of biosensors. The following review focuses on the application of LBL assembly technique on electrochemical enzyme biosensors, immunosensors and DNA sensors.     

Amperometric Hydrogen Peroxide Biosensor Based on the Immobilization of Horseradish Peroxidase (HRP) on the Layer‐by‐Layer Assembly Films of Gold Colloidal Nanoparticles and Toluidine Blue

The precursor film was first formed on the Au electrode surface based on the self‐assembly of L ‐cysteine and the adsorption of gold colloidal nanoparticles (nano‐Au). Layer‐by‐layer (LBL) assembly films of toluidine blue (TB) and nano‐Au were fabricated by alternately immersing the electrode with precursor film into the solution of toluidine blue and gold colloid. Cyclic voltammetry (CV) and quartz crystal microbalance (QCM) were adopted to monitor the regular growth of {TB/Au} bilayer films. The successful assembly of {TB/Au}_n films brings a new strategy for electrochemical devices to construct layer‐by‐layer assembly films of nanomaterials and low molecular weight materials. In this article, {TB/Au}_n films were used as model films to fabricate a mediated H₂O₂ biosensor based on horseradish peroxidase, which responded rapidly to H₂O₂ in the linear range from 1.5×10^?7 mol/L to 8.6×10^?3 mol/L with a detection limit of 7.0×10^?8 mol/L. Morphologies of the final assembly films were characterized with scanning probe microscopy (SPM).