A hydrogen peroxide biosensor based on peroxidase activity of hemoglobin in polymeric film

A Hydrogen peroxide (H2O2) biosensor, based on hemoglobin (Hb) and ortho-phenylenediamine (o-PD) gold electrode, was fabricated. Hb was immobilized onto the electrode surface by electrochemical polymerize method with o-PD. The designed biosensor showed a well defined redox peak which was attributed to the direct electrochemical response of Hb. The immobilized Hb exhibited an excellent electrocatalytical response to the reduction of hydrogen peroxide, enabling the sensitivity determination of H2O2. Factors and performances such as pH, potential, influencing the designed biosensor, were studied carefully. The amperometric detection of H2O2 was carried out at -300 mV in phosphate buffer solution (PBS) (0.1 M) with pH 6.0. This biosensor showed a fast amperometric response (less then 5 s) to H2O2. The levels of the (Relative standard deviation) RSDs (< 3.5%) for the entire analyses reflected a highly reproducible sensor performance. Using the optimized conditions, the detection limit of the biosensor was 1 x 10(-7) M and linear range was from 5 x 10(-6) to 1.25 x 10(-4) M. In addition, this sensor showed long-term stability and good sensitivity.     

Electrochemical biosensor based on multi-walled carbon nanotubes and Au nanoparticles synthesized in chitosan

A new biosensor is prepared by cross-linking glucose oxidase (GOD) with glutaradehyde at the electrode combining Au nanoparticles (AuNP) with multi-walled carbon nanotubes (MWCNTs). Au nanoparticles-doped chitosan (CS) solution (AuNP-CS) is prepared by treating the CS solution followed by chemical reduction of Au (III) with NaBH4. MWCNTs are then dispersed in AuNP-CS solution. TEM, FT-IR, and UV-Vis show that the AuNP-CS solution is highly dispersed and stable. The synergistic effect between AuNP and CNTs of the AuNP-CNTs-CS material has been investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometric methods. The modified glassy carbon electrode (GCE) allows low-potential detection of H2O2 with high sensitivity and fast response time. With the immobilization of GOD, a biosensor has been constructed. In phosphate buffer solutions (PBS, pH 7.0), nearly free interference determination of glucose has been realized at 0.4 V(vs. Ag/AgCl/3.0 M KCI) with a wide linear range from 2.0 x 10(-5) to 1.5 x 10(-2) M and a fast response time within 5s. The biosensor has been used to determine glucose in human serum samples and the results are satisfactory.     

Immobilization method for the preparation of biosensors based on pH shift-induced deposition of biomolecule-containing polymer films.

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.     

A Glucose Biosensor Based on Immobilization of Glucose Oxidase in Electropolymerized o-Aminophenol Film on Platinized Glassy Carbon Electrode

A high-performance amperometric glucose biosensor has been developed, based on immobilization of glucose oxidase in an electrochemically synthesized, nonconducting poly(o-aminophenol) film on a platinized glassy carbon electrode. The large microscopic surface area and porous morphology of the platinized glassy carbon electrode result in high enzyme loading, and the enzyme entrapped in the electrodeposited platinum microparticle matrix is stabler than that on a platinum disk electrode surface. The response current of the sensor is 20-fold higher than that of the sensor prepared with a platinum disk electrode of the same geometric area. The experiments showed that the high sensitivity of the sensor is due not only to the large microscopic area but also to the high efficiency of transformation of H(2)O(2) generated by enzymatic reaction to current signal on the platinized glassy carbon electrode. The response time of the sensor is <4 s, and its lifetime is >10 months.     

DNA as a support for glucose oxidase immobilization at Prussian blue-modified glassy carbon electrode in biosensor preparation

An amperometric glucose biosensor has been developed using DNA as a matrix of Glucose oxidase (GOx) at Prussian-blue (PB)-modified glassy carbon (GC) electrode. GC electrode was chemically modified by the PB. GOx was immobilized together with DNA at the working area of the PB-modified electrode by placing a drop of the mixture of DNA and GOx. The response of the biosensor for glucose was evaluated amperometrically. Upon immobilization of glucose oxidase with DNA, the biosensor showed rapid response toward the glucose. On the other hand, no significant response was obtained in the absence of DNA. Experimental conditions influencing the biosensor performance were optimized and assessed. This biosensor offered an excellent electrochemical response for glucose concentration in micro mol level with high sensitivity and selectivity and short response time. The levels of the relative standard deviation (RSDs), (<4%) for the entire analyses reflected a highly reproducible sensor performance. Through the use of optimized conditions, a linear relationship between current and glucose concentration was obtained up to 4 x 10(-4) M. In addition, this biosensor showed high reproducibility and stability.     

Development of a flow amperometric enzymatic method for the determination of total glucosinolates in real samples

The first amperometric flow analyzer, based on the biosensor concept, capable of determining total glucosinolates in real samples, is described. Myrosinase was immobilized on aminopropyl-modified controlled pore glass, which was then used for the construction of a packed-bed reactor. Myrosinase catalyzes the hydrolysis of glucosinolates (sinigrin) to glucose (among the other products), which is then oxidized by the action of glucose oxidase to produce hydrogen peroxide. The glucose enzyme electrode is based on a multimembrane architecture and was mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3M KCl). Different membrane types and different activation procedures were tested. The system was optimized to various working parameters, either as a glucose electrode or as a glucosinolate analyzer. The interference effect of various compounds was also investigated. Application of the method to real samples was carried out using glucose/glucose, hydrolyzed sinigrin and glucose/sinigrin solution as calibrators of the glucose electrode and the glucosinolate analyzer. Deviations due to the enantioselectivity of glucose oxidase to the beta-glucose anomer were observed, and a data elaboration protocol is proposed. The possibility of the simultaneous determination of glucose and glucosinolates is also demonstrated.     

A lactate electrochemical biosensor with a titanate nanotube as direct electron transfer promoter

Hydrogen titanate (H(2)Ti(3)O(7)) nanotubes (TNTs) have been synthesized by a one-step hydrothermal processing. Lactate oxidase (LOx) enzyme has been immobilized on the three-dimensional porous TNT network to make an electrochemical biosensor for lactate detection. Cyclic voltammetry and amperometry tests reveal that the LOx enzyme, which is supported on TNTs, maintains their substrate-specific catalytic activity. The nanotubes offer the pathway for direct electron transfer between the electrode surface and the active redox centers of LOx, which enables the biosensor to operate at a low working potential and to avoid the influence of the presence of O(2) on the amperometric current response. The biosensor exhibits a sensitivity of 0.24?μA?cm(-2)?mM(-1), a 90% response time of 5?s, and a linear response in the range from 0.5 to 14?mM and the redox center of enzyme obviates the need of redox mediators for electrochemical enzymatic sensors, which is attractive for the development of reagentless biosensors.     

Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode

Cationic redox polymers containing osmium-bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically "activate" the enzymes. On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor. A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly. Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly. The formation of an anionic-cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose. Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels. The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time. A detection limit of 1.0 fmol/L in 1.0-microL droplets and a linear current-concentration relationship up to 800 fmol/L were attained following a 30-min hybridization. The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.     

An effective gold nanotubes electrode for amperometric biosensor

A sensitive and effective amperometric glucose biosensor based on gold nanotubes electrode (GNTE) was investigated. Gold nanotubes (GNTs), which were prepared by electroless plating of the metal within the pores of nanoporous polycarbonate (PC) track-etched membranes, were filled into a hollow teflon cylinder to construct a GNTE. Glucose oxidase (GOD) was immobilized on the electrode via glutaraldehyde cross-linkage method. The electrochemical properties were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The km value of the immobilized glucose oxidase on GNTE was 0.47 mM. The biosensor showed a linear range from 0.4 to 11 mM with excellent sensitivity of 8.77 microA cm(-2) mM(-1) and fast response time within 5 s.     

Glucose biosensor from covalent immobilization of chitosan-coupled carbon nanotubes on polyaniline-modified gold electrode

An amperometric glucose biosensor was prepared using polyaniline (PANI) and chitosan-coupled carbon nanotubes (CS-CNTs) as the signal amplifiers and glucose oxidase (GOD) as the glucose detector on a gold electrode (the Au-g-PANI-c-(CS-CNTs)-GOD biosensor). The PANI layer was prepared via oxidative graft polymerization of aniline from the gold electrode surface premodified by self-assembled monolayer of 4-aminothiophenol. CS-CNTs were covalently coupled to the PANI-modified gold substrate using glutaradehyde as a bifunctional linker. GOD was then covalently bonded to the pendant hydroxyl groups of chitosan using 1,4-carbonyldiimidazole as the bifunctional linker. The surface functionalization processes were ascertained by X-ray photoelectron spectroscopy (XPS) analyses. The field emission scanning electron microscopy (FESEM) images of the Au-g-PANI-c-(CS-CNTs) electrode revealed the formation of a three-dimensional surface network structure. The electrode could thus provide a more spatially biocompatible microenvironment to enhance the amount and biocatalytic activity of the immobilized enzyme and to better mediate the electron transfer. The resulting Au-g-PANI-c-(CS-CNTs)-GOD biosensor exhibited a linear response to glucose in the concentration range of 1-20 mM, good sensitivity (21 μA/(mM·cm(2))), good reproducibility, and retention of >80% of the initial response current after 2 months of storage.     

A disposable amperometric sensor screen printed on a nitrocellulose strip: a glucose biosensor employing lead oxide as an interference-removing agent

A new type of disposable amperometric sensor is devised by screen printing thick-film electrodes directly on a porous nitrocellulose (NC) strip. The chromatographic NC strip is then utilized to introduce various sample pretreatment layers. As a preliminary application, a glucose biosensor based on hydrogen peroxide detection is constructed by immobilizing glucose oxidase (GOx) on the NC electrode strip and by formulating a strong oxidation layer (i.e., PbO2) at the sample loading area, placed below the GOx reaction band. The screen-printed PbO2 paste serves as a sample pretreatment layer that removes interference by its strong oxidizing ability. Samples applied are carried chromatographically, via the PbO2 paste, to the GOx layer, and glucose is catalyzed to liberate hydrogen peroxide, which is then detected at the electrode surface. The proposed NC/PbO2 strip sensor is shown to be virtually insusceptible to interfering species such as acetaminophen and ascorbic and uric acids and to exhibit good performance, in terms of the sensor-to-sensor reproducibility (standard deviation, +/-0.026 - +/-0.086 microA), the sensitivity (slope, -0.183 microA/mM), and the linearity (correlation coefficient, 0.994 in the range of 0-10 mM).     

Electrocatalytic reduction of H2O2 on thiolate graphene oxide covalently to bonded palladium nanoparticles

The electrocatalytic reduction of hydrogen peroxide on thioalted graphene oxide (t-GO) covalent bonded to palladium nanoparticles was used as the basis of an H2O2 biosensor. Poly (diallydimethylammonium chloride)-coated t-GO-Pd on glassy carbon electrodes was easily and quickly prepared and gave sensitive measurements of H2O2 concentration. The Pd nanoparticles covalently bonded to the thiolated graphene oxide were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy. Comparable results for H2O2 determination were obtained from cyclic voltammetric and amperometric measurements. The proposed H2O2 biosensor exhibited a wide linear range of 10 microM to 10 mM, and a low detection limit of 0.22 microM (S/N = 3), at an applied potential of -0.1 V by the amperometric method.     

Selective permeation of hydrogen peroxide through polyelectrolyte multilayer films and its use for amperometric biosensors.

A platinum electrode was coated with polyelectrolyte multilayer (PEM) films to prepare an amperometric hydrogen peroxide sensor which can be used in the presence of possible interferences such as ascorbic acid, uric acid, and acetaminophen. The PEM films were prepared on the surface of a Pt disk electrode by an alternate deposition of polycation and polyanion from the aqueous solutions through electrostatic force of attraction. The Pt electrodes coated with a poly(allylamine)/poly(vinyl sulfate) or poly(allylamine)/poly(styrenesulfonate) film were used successfully for detecting H2O2 selectively in the presence of the possible interfering agents. It was suggested that H2O2 can diffuse into the PEM film smoothly while the ascorbic acid, uric acid, and acetaminophen cannot penetrate the film by a size exclusion mechanism. On the other hand, the electrodes coated with PEM films containing poly(ethyleneimine) or poly(diallyldimethylammonium chloride) were not useful for the selective determination of H2O2. The results were rationalized based on the different permeability of the films due to the different molecular density or packing in the PEM films. The PEM film-coated electrode was useful for constructing glucose biosensors by coupling with glucose oxidase.     

Enzyme-modified organic conducting salt microelectrode.

A miniaturized enzyme-modified electrode has been constructed and evaluated. The tip of a capillary-encased, carbon-fiber electrode is recessed, and tetrathiafulvalene-tetracyanoquinodimethane crystals are electrochemically deposited in the recessed tip. Flavoenzymes are placed in the recess by cross-linking with glutaraldehyde. The specific enzymes used are glucose oxidase to form a microbiosensor for glucose, and a combination of acetylcholine esterase and choline oxidase to form a microbiosensor for acetylcholine. The sensor is operated in an amperometric mode with Eapp = 150 mV versus a sodium saturated calomel electrode, and the response appears to be limited by the kinetics of the enzyme reaction. The effective maximum current density for the glucose electrode is greater than 600 microA/cm2. At low concentrations of glucose, oxygen provides a significant interference by attenuating the signal. The device is simple to prepare and has a rapid response time. Interference from ascorbate has been significantly reduced by the design and by addition of a layer of ascorbate oxidase. Although not yet suitable for use in tissue, the biosensors are suitable for detection in situations where oxygen concentrations do not frequently change.     

Fabrication of a glucose biosensor based on citric acid assisted cobalt ferrite magnetic nanoparticles

A novel and practical glucose biosensor was fabricated with immobilization of Glucose oxidase (GOx) enzyme on the surface of citric acid (CA) assisted cobalt ferrite (CF) magnetic nanoparticles (MNPs). This innovative sensor was constructed with glassy carbon electrode which is represented as (GOx)/CA-CF/(GCE). An explicit high negative zeta potential value (-22.4 mV at pH 7.0) was observed on the surface of CA-CF MNPs. Our sensor works on the principle of detection of H2O2 which is produced by the enzymatic oxidation of glucose to gluconic acid. This sensor has tremendous potential for application in glucose biosensing due to the higher sensitivity 2.5 microA/cm2-mM and substantial increment of the anodic peak current from 0.2 microA to 10.5 microA.     

Study of different carbon materials for amperometric enzyme biosensor development

Diamond-like carbon (DLC) electrodes which constitute a new research area in electrochemistry and glassy carbon (GC) electrodes were used as transducers for fabrication of glucose oxidase (GOD) biosensors. The amperometric signal of the enzyme electrode was due to the electro-oxidation of H₂O₂ generated in the enzyme layer. This work has shown that the detection limit of glucose on GOD/GC electrode is 20 μM while it is 50 μM on GOD/DLC electrode. The sensitivity of GOD/GC electrode decreases 4% after 8 days and we have good repeatability of measurements for GOD/DLC electrode in the same day.     

Aptamer based electrochemical sensor system for protein using the generation/collection mode of scanning electrochemical microscope (SECM)

To detect the target molecules, aptamers are currently focused on and the use of aptamers for biosensing is particularly interesting, as aptamers could substitute antibodies in bioanalytical sensing. So this paper describes the novel electrochemical system for protein in sandwich manner by using the aptamers and the scanning electrochemical microscope (SECM). For protein detection, sandwich system is ideal since labeling of the target protein is not necessary. To develop the electrochemical protein sensor system, thrombin was chosen as a target protein since many aptamers for it were already reported and two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system. In order to obtain the electrochemical signal, the glucose oxidase (GOD) used for labeling the detection aptamers since it has large amount of stability in aqueous solution. One aptamer was immobilized onto the gold electrode and the other aptamer for detection was labeled with GOD for generation of the electric signal. Thrombin was detected in sandwich manner with aptamer immobilized onto the gold electrode and the GOD labeled aptamer. The enzymatic signal, generated from glucose addition after the formation of the complex of thrombin, was measured. The generation-collection mode of SECM was used for amperometric H2O2 detection.     

Inert metal-modified, composite ceramic-carbon, amperometric biosensors: renewable, controlled reactive layer

A new type of sol-gel-derived, inert metal-modified, composite, amperometric biosensor is developed. The electrodes are comprised of a dispersion of biochemically and chemically modified graphite powder in a porous, organically modified silicate (Ormosil) network. The percolating carbon dispersion provides electrical conductivity, oxidoreductase enzymes (e.g., glucose oxidase, lactate oxidase, or l-amino acid oxidase) are used for biocatalysis, metallic palladium is used for electrocatalysis of the biochemical reaction product, and the porous organically modified silica provides a rigid skeleton. The hydrophobicity of this composite material guarantees that only a limited section of the electrode is wetted by the aqueous analyte, thus providing a controlled-thickness reactive layer. The thickness of the reaction layer can be tuned by the addition of hydrophilic components. The electrode can be reproducibly renewed by removing its upper layer and exposing a new, thin, porous bioreactive section. The same technology is applicable for the production of thick-film, low-leaching, disposable sensors. In this configuration, the analysis is conducted using a single drop of the analyte applied on the hydrophobic film. The sensors are found to be stable over long periods.     

Prototype amperometric biosensor for sialic acid determination

This paper describes the first report on the development, characterization, and applications of a prototype amperometric biosensor for free sialic acid (SA). The sensor was constructed by the coimmobilization of two enzymes, i.e., N-acetylneuraminic acid aldolase and pyruvate oxidase, on a polyester microporous membrane, which was then mounted on top of a platinum disk electrode. The SA biosensor operation was based on the sequential action of the two enzymes to ultimately produce hydrogen peroxide, which was then detected by anodic amperometry at the platinum electrode. The surface of the platinum electrode was coated with an electropolymeric layer to enhance the biosensor selectivity in the presence of interfering oxidizable species. Optimization of the enzyme layer composition resulted in a fast and steady current response in phosphate buffer pH 7.2 at 37 degrees C. The limit of detection was 10 microM, and the response was linear to 3.5 mM (r = 0.9987). The prepared SA biosensors retained approximately 85% of their initial sensitivity after 8 days and showed excellent response reproducibility (CV = 2.3%). Utilization of a third enzyme, sialidase, expanded the scope of the present SA biosensor to determine bound sialic acid as well. The merits of the described biosensor allowed its successful application in determining SA in biological and pharmaceutical samples. The obtained results indicated that the presented SA biosensor should be a useful bioanalytical tool in several biological and clinical applications such as screening of SA as a nonspecific tumor marker as well as monitoring of tumor therapy.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.