Highly Sensitive Amperometric Glucose Biosensor Based on Glassy Carbon Electrode with Copper/Palladium Coating

A highly sensitive amperometric glucose biosensor based on immobilizing glucose oxidase in electropolymerized poly(o‐phenylenediamine) film on glassy carbon electrode coated sequentially with copper and palladium layers has been developed. The steady‐state amperometric response to glucose was determined by means of the oxidation of hydrogen peroxide generated by the enzymatic reaction at a potential of either +0.70 or +0.40 V (vs. Ag|AgCl reference). The deposited copper/palladium layer showed great enhancement in the performance of the enzyme electrode, possibly due to its better electrocatalytic activity for hydrogen peroxide oxidation and large surface area. Effects of the relative loading of palladium, enzyme and polymer on the electrode performance were examined in detail. Sensitivity and detection limit for glucose determinations at +0.70 V were about 7.3 μA/mM and 0.1 μM, respectively. A wide linear range up to 6.0 mM glucose could be achieved. Electrode performance was superior to similar works reported in the literature. The response time was less than 2 s and its lifetime was longer than three months. The permeable polyphenylenediamine film also offered good anti‐interference ability to ascorbic acid, uric acid and acetaminophen, especially when a detection potential of +0.40 V was employed.     

Electrochemical Behaviour of a Lipid Modified Enzyme Electrode

Abstract

The modification of the surface of a platinum electrode by coating with a layer of a lipid mixture (asolectin), allows the relative measurement of hydrogen peroxide in the presence of interfering analytes. The lipid-enzyme complex and the platinum amperometric sensor offer greater selectivity and extended stability of the resulting probe. Measurements of glucose with the glucose oxidase enzyme and detection of the liberated hydrogen peroxide have been performed as a model system. Linear response of the signal versus glucose concentration was observed in the range of glucose concentration 1.10^?3 ? 1.10^?5 M with a response time of 20 s. The interferences of ascorbic acid, uric acid, iron (II), paracetamol, tyrosine and glutathion can be drastically minimized by appropriate adjustment of the amount of lipid contained in the biocatalyst layer.     

Development and characterization of a microfluidic glucose sensing system based on an enzymatic microreactor and chemiluminescence detection

Chemiluminescence detection was developed as an alternative to amperometric detection for glucose analysis in a portable, microfluidics-based continuous glucose monitoring system. Amperometric detection allows easy determination of hydrogen peroxide, a product of the glucose oxidase-catalyzed reaction of glucose with oxygen, by oxidation at a microelectrode. However, (micro)electrodes in direct contact with physiological sample are subject to electrode fouling, which leads to signal drift, decreased reproducibility and shortened detector lifetimes. Moreover, there are a few species present in the body (e.g. ascorbic acid, uric acid) which can undergo oxidation at the same applied potential as hydrogen peroxide. These species can thus interfere with the glucose measurement, reducing detection specificity. The rationale for exploring chemiluminescence as opposed to amperometric detection is thus to attempt to improve the lifetime and reproducibility of glucose analysis for monitoring purposes, while reducing interference caused by other chemicals in the body. The study reported here represents a first step in this direction, namely the realization of a microfluidic device with integrated silicon photodiode for chemiluminescence detection of glucose. This microflow device uses a chaotic mixing approach to perform enzymatic conversion of glucose, followed by reaction of the hydrogen peroxide produced with luminol to produce light at 425 nm. The chemiluminescence reaction is catalyzed by horseradish peroxidase in the presence of iodophenol. The performance of the fabricated chip was characterized to establish optimal reaction conditions with respect to sample and reagent flow rates, pH, and concentrations. A linear calibration curve was obtained for current response as a function of glucose concentration in the clinically relevant range between 2 and 10 mM, with a sensitivity of 39 pA/mM (R = 0.9963, one device, n = 3) and a limit of detection of 230 μM (S/N = 3).     

The interference of HEPES buffer during amperometric detection of ATP in clinical applications

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, ³¹P NMR, and ¹H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca²⁺ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.     

Prussian-Blue-based amperometric biosensors in flow-injection analysis

Optimisation of the electrodeposition of Prussian Blue onto mirrored glassy carbon electrodes yielded a modified electrode practically insensitive to oxygen reduction. At the same time the electrode activity towards hydrogen peroxide reduction was extremely high. This allowed the detection of hydrogen peroxide by electroreduction over a wide potential range. Flow-injection investigations of this electrode inserted into a flowthrough electrochemical cell of the confined wall-jet type showed that the response for hydrogen peroxide is limited by diffusion. Glucose and alcohol biosensors were made by immobilisation of glucose oxidase and alcohol oxidase respectively, within a Nafion layer, onto the top of the Prussian-Blue-modified electrodes. By increasing the density of Nafion and decreasing the measuring potential the glucose biosensor was made completely insensitive to both ascorbate and acetominophes.     

Amperometric enzyme electrodes for the determination of l-glutamate

Electrodes for amperometric measurement of l-glutamate were prepared by immobilization of l-glutamate oxidase on an Immobilon-AV Affinity membrane and attachment to an oxygen/hydrogen peroxide sensor. The response of the hydrogen peroxide sensor was linear over the concentration range 5.0 x 10(-8)-5.0 x 10(-4)Ml-glutamate, with a limit of detection of 35nM. Attachment of a size-exclusion membrane (cut-off for molecular weight > 100) or of a hydrophobic oxygen membrane eliminated electro-oxidizable interferences, but the response was attenuated by a factor of 2-3. The response may be amplified 10-fold by co-immobilizing l-glutamate dehydrogenase with the l-glutamate oxidase. The electrode initially lost 25% of its activity but was then stable for more than 320 days and at least 200 assays. The electrode was successfully used to assay glutamate in a protein tablet and in several food products. A flow-injection system was assembled for the continuous assay of l-glutamate.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Amperometric sensing of hydrogen peroxide vapor for security screening

Rapid detection of the hydrogen peroxide precursor of peroxide explosives is required in numerous security screening applications. We describe a highly sensitive and selective amperometric detection of hydrogen peroxide vapor at an agarose-coated Prussian-blue (PB) modified thick-film carbon transducer. The sensor responds rapidly and reversibly to dynamic changes in the level of the peroxide vapor, with no apparent carry over and with a detection limit of 6 ppbv. The remarkable selectivity of the PB-based screen-printed electrode towards hydrogen peroxide leads to effective discrimination against common beverage samples. For example, blind tests have demonstrated the ability to selectively and non-invasively identify concealed hydrogen peroxide in drinking cups and bottles. The attractive performance of the new microfabricated PB-based amperometric peroxide vapor sensor indicates great potential for addressing a wide range of security screening and surveillance applications. Figure Experimental setup (left) with three electrode electrochemical Hydrogen Peroxide sensor hanging above container of “unknown” liquid. Schematic (right) demonstrating fundamental principles of operation of the sensor.     

基于等离子体聚合膜固定酶的H2O2生物传感器

以玻碳电极为基础电极,用微波等离子体技术聚合沉积聚乙二胺等离子体膜,使之形成带氨基功能团的表面,再通过戊二醛交联共价固定辣根过氧化物酶,制得H2O2生物传感器.探讨了等离子体聚合膜的形成条件(如放电功率、单体流速、单体气压和聚合时间),讨论了工作电位、介体浓度和pH值对传感器响应的影响.此外,用红外光谱对等离子体聚合膜进行了表征.该传感器在5×10-7～1.1×10-3mol/LH2O2浓度范围内有线性响应,最低检测限为0.3μmol/L.将此传感器用于实际试样回收率的测定,结果良好.     

Electrocatalysis via Direct Electrochemistry of Myoglobin Immobilized on Colloidal Gold Nanoparticles

The direct electron transfer between immobilized myoglobin (Mb) and colloidal gold modified carbon paste electrode was studied. The Mb immobilized on the colloidal gold nanoparticles displayed a pair of redox peaks in 0.1 M pH 7.0 PBS with a formal potential of –(0.108 ± 0.002) V (vs. NHE). The response showed a surface‐controlled electrode process with an electron transfer rate constant of (26.7 ± 3.7) s ^?1 at scan rates from 10 to 100 mV s^?1 and a diffusion‐controlled process involving the diffusion of proton at scan rates more than 100 mV s^?1. The immobilized Mb maintained its activity and could electrocatalyze the reduction of both hydrogen peroxide and nitrite. Thus, the novel renewable reagentless sensors for hydrogen peroxide and nitrite were developed, respectively. The activity of Mb with respect to the pseudo peroxidase with a K_M^app value of 0.65 mM could respond linearly to hydrogen peroxide concentration from 4.6 to 28 μM. The sensor exhibited a fast amperometric response to NO₂^? reduction and reached 93% of steady‐state current within 5 s. The linear range for NO₂^? determination was from 8.0 to 112 μM with a detection limit of 0.7 μM at 3σ.     

Development of Silver Nanowires Based Highly Sensitive Amperometric Glucose Biosensor

The fabrication of a highly sensitive amperometric glucose biosensor based on silver nanowires (AgNWs) is presented. The electrochemical behavior of glassy carbon electrode modified by Ag NWs exhibits remarkable catalytic performance towards hydrogen peroxide (H₂O₂) and glucose detection. The biosensor could detect glucose in the linear range from 0.005 mM to 10 mM, with a detection limit of 50 µM (S/N=3). The glucose biosensor shows high and reproducible sensitivity of 175.49 µA cm^?2 mM and good stability. In addition, the biosensor exhibits a good anti‐interference ability and favorable stability over relatively long‐term storage (more than 21 days).     

Amperometric Glucose Biosensor Based on the Deposition of Copper and Glucose Oxidase onto Glassy Carbon Transducer

ABSTRACT

The performance of a first generation glucose amperometric biosensor based on the entrapment of glucose oxidase (GOx) within a net of copper electrodeposited onto activated glassy carbon electrode, is described. The copper electrodeposited offers an efficient electrocatalytic activity towards the reduction of enzymatically-liberated hydrogen peroxide, allowing for a fast and sensitive glucose quantification. The influence of the electrodeposition conditions (pH, potential, time, copper salt and enzyme concentrations) on the response of the bioelectrode was evaluated from the amperometric signals of hydrogen peroxide and glucose. The combination of copper electrodeposition with a nation membrane allows an excellent selectivity towards easily oxidizable compounds such as uric and ascorbic acids at an operating potential of -0.050 V. The response is linear up to 2.0 × 10^?2 M glucose, the detection limit being 1.2 × 10^?3 M.     

Modeling the measurements of cellular fluxes in microbioreactor devices using thin enzyme electrodes

An analytic approach to the modeling of stop-flow amperometric measurements of cellular metabolism with thin glucose oxidase and lactate oxidase electrodes would provide a mechanistic understanding of the various factors that affect the measured signals. We divide the problem into two parts: (1) analytic formulas that provide the boundary conditions for the substrate and the hydrogen peroxide at the outer surface of the enzyme electrode layers and the electrode current expressed through these boundary conditions, and (2) a simple diffusion problem in the liquid compartment with the provided boundary conditions, which can be solved analytically or numerically, depending on the geometry of the compartment. The current in an amperometric stop-flow measurement of cellular glucose or lactate consumption/excretion is obtained analytically for two geometries, corresponding to devices developed at the Vanderbilt Institute for Integrative Biosystems Research and Education: a multianalyte nanophysiometer with effective one-dimensional diffusion and a multianalyte microphysiometer, for which plentiful data for metabolic changes in cells are available. The data are calibrated and fitted with the obtained time dependences to extract several cellular fluxes. We conclude that the analytical approach is applicable to a wide variety of measurement geometries and flow protocols.     

Trace Level Determination of Hydrogen Peroxide at a Carbon Ceramic Electrode Modified with Copper Oxide Nanostructures

An electrochemical sensor was developed for determination of hydrogen peroxide based on nanocopper oxides modified carbon sol‐gel or carbon ceramic electrode (CCE). The modified electrode was prepared by electrodeposition of metallic copper on the CCE surface and derivatized in situ to copper oxides nanostructures and characterized by scanning electron microscopy (SEM) and X‐ray diffraction (XRD) techniques. The modified electrode responded linearly to the hydrogen peroxide (H₂O₂) concentration over the range 0.78–193.98 µmol L^?1 with a detection limit of 71 nmol L^?1 (S/N=3) and the sensitivity of 0.697 A mol^?1 L cm^?2. This electrode was used as selective amperometric sensor for determination of H₂O₂ contents in hair coloring creams.     

基于表面重建螺旋型铂铱电极的葡萄糖生物传感器

通过对螺旋型铂铱电极表面进行化学腐蚀和电化学沉积铂纳米粒子实现电极表面的重建和优化,研究了螺旋型铂铱电极在不同腐蚀时间和电沉积时间下的形貌及对过氧化氢(H2O2)的催化活性.对表面重建的工作电极涂覆氧化酶和半透膜,制备出了铂纳米粒子/葡萄糖氧化酶/环氧聚氨酯酶电极,并将其用作葡萄糖传感器的工作电极.传感器计时电流检测结果表明,表面重建后的酶电极传感器对葡萄糖的检测范围扩大为2~45 mmol/L,优于裸铂铱酶电极传感器,电流响应值和灵敏度得到明显提升,同时传感器还具有良好的稳定性和选择性.     

Nonenzymatic amperometric sensor of hydrogen peroxide and glucose based on Pt nanoparticles/ordered mesoporous carbon nanocomposite

A simple and facile synthetic method to incorporate Pt nanoparticles inside the mesopores of ordered mesoporous carbons (OMCs) is reported. The Pt/OMCs nanocomposite was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and nitrogen adsorption-desorption. The results show that the incorporation of Pt nanoparticles inside the pores of OMCs does not change the highly ordered two-dimensional hexagonal mesostructure of OMCs matrix. Nonenzymatic amperometric sensor of hydrogen peroxide and glucose based on the Pt/OMCs nanocomposite-modified glassy carbon (GC) electrode is developed. Compared with the original OMCs-modified electrode, the Pt/OMCs-modified electrode displays improved current response towards hydrogen peroxide and gives linear range from 2 to 4212 μM. At an applied potential of −0.08 V, the Pt/OMCs nanocomposite gives linearity in the range of 0.5-4.5 mM glucose in neutral buffered saline solution. This glucose sensor also exhibits good ability of anti-interference to electroactive molecules. The combination the unique properties of Pt nanoparticles and the ordered mesostructure of OMCs matrix guarantees the enhanced response for hydrogen peroxide and glucose.     

Electrocatalytic Sensor for Hydrogen Peroxide Based on Immobilized Benzoquinone

An amperometric chemosensor for the detection of hydrogen peroxide is reported. The sensor is based on 1,4-benzoquinone immobilized on the gold electrode using self-assembled monolayer of short chain symmetrical dithiol as an anchor layer. Sensor analysis was performed by cyclic voltammetry at the potential range from −0.6 V till +0.9 V as well as in the anodic or cathodic potential ranges only. The results indicate oxidative electrochemical decomposition of hydrogen peroxide at the potential of ∼+0.4 V leading to the formation of oxygen while at cathodic potentials a reduction of the formed oxygen as well as of the hydrogen peroxide occur. A decrease in the oxidation potential of hydrogen peroxide on the gold electrode coated by self-assembled monolayer with 1,4-benzoquinone in comparison with that measured on the electrodes coated by the same self-assembled monolayer without 1,4-benzoquinone, indicates electrocatalytic effect of this moiety on oxidative decomposition of hydrogen peroxide. Analytical evaluation of the sensor performance was done in the voltammetric as well as in the chronoamperometric mode. The sensor exhibited linear response over the concentration range till 2.5 mM with a limit of detection ∼4 μM.     

Sensitive Flow-Injection Electrochemical Determination of Hydrogen Peroxide at a Palladium Nanoparticle-Modified Pencil Graphite Electrode

A novel flow-injection amperometric method was proposed for the sensitive and enzymeless determination of hydrogen peroxide based on its electrocatalytic reduction at a palladium nanoparticle-modified pretreated pencil graphite electrode in a laboratory-constructed electrochemical flow cell. Cyclic voltammograms of the unmodified and modified electrodes were recorded in pH 7.0 phosphate buffer containing 0.10 M KCl at a scan rate of 50?mV s^?1 for the investigation of electrocatalytic reduction of hydrogen peroxide at the palladium nanoparticle-modified pretreated pencil graphite electrode. Cyclic voltammograms of the pretreated pencil graphite electrode revealed an irreversible oxidation peak and a weak reduction peak of hydrogen peroxide at +1100?mV and –450?mV vs. an Ag/AgCl/KCl saturated reference electrode. However, the reduction of hydrogen peroxide was observed at –100?mV with an increase in current in the cyclic voltammograms of the palladium nanoparticle-modified pretreated pencil graphite electrode compared to the unmodified electrode. These results indicate that the palladium nanoparticle-modified pretreated pencil graphite electrode exhibits efficient electrocatalytic activity for the reduction of hydrogen peroxide. A linear concentration range was obtained between .01 and 10.0?mM hydrogen peroxide with a detection limit of 3.0 µM from flow injection amperometric current–time curves recorded in pH 7.0 phosphate buffer at –100?mV and a 2.0?mL min^?1 flow rate. The novelty of this work relies on its use of a laboratory-constructed flow cell constructed for the pencil graphite electrode using these inexpensive, disposable, and electrochemically reactive modified electrodes for the amperometric determination of hydrogen peroxide in a flow injection analysis system.     

Amperometric aspartate electrode

An amperometric enzyme electrode for L-aspartate determination was developed. The probe consisted of a platinum electrode which senses hydrogen peroxide produced from the reactions catalyzed by two enzymes co-immobilized on a preactivated polymeric membrane, α-Ketoglutarate in the presence of L-aspartate was transaminated to L-glutamate by aspartate aminotransferase and the glutamate produced was oxidized by glutamate oxidase, with concomitant production of hydrogen peroxide. Additional protective membranes eliminated interferences from glutamate and most electroactive compounds. The response curve of the probe was linear over the concentration range 1.0 × 10^?6 M to 2.0 × 10^?4 M aspartate and was useful for at least two months. Aspartic acid in some pharmaceutical products was determined and the results correlated well with a liquid chromatographic reference method and the manufacturer's specification.     

Amperometric Biosensor for Hydrogen Peroxide Based on Electrodeposited Sub-micrometer Gold Modified Glassy Carbon Electrode

IntroductionAmperometricbiosensorofhydrogenperoxideisofpracticalimportancebecauseofitswideapplicationsinchemical,biological,clinical,environmentalandmanyotherfields.Forimprovementofsensor抯quality,vari-ouskindsofchemicalmodificationmethodshavebeendevelopedforreducingredoxoverpotentialsofH2O2atelectrodesurfaces,increasingthedetectionsensitivity,linearrange,stabilityandlivetime.Ithasbeenshownthattheuseofsub-micrometersizedmetalparticlessuchasPt-blackcansignificantlyimprovethequalityofthebiosens…