Direct electrochemistry of glucose oxidase in a colloid Au-dihexadecylphosphate composite film and its application to develop a glucose biosensor

Colloid Au (Au(nano)) with a diameter of about 10 nm was prepared and used in combination with dihexadecylphosphate (DHP) to immobilize glucose oxidase (GOD) onto the surface of a graphite electrode (GE). The direct electrochemistry of GOD confined in the composite film was investigated. The immobilized GOD displayed a pair of redox peaks with a formal potential of -0.475 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 150 mV s(-1). The GOD in the composite film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction peak current of dissolved oxygen decreased, which could be developed for glucose determination. A calibration linear range of glucose was 0.5-9.3 mM with a detection limit of 0.1 mM and a sensitivity of 1.14 microA mM(-1). The glucose biosensor showed good reproducibility and stability. The general interferences that coexisted in human serum sample such as ascorbic acid and uric acid did not affect glucose determination.     

Urea Biosensor Based on Electrochromic Properties of Prussian Blue

Prussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KCl_sat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R²=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M^?1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea.     

Multilayer assembly of Prussian blue nanoclusters and enzyme-immobilized poly(toluidine blue) films and its application in glucose biosensor construction

A multilayered glucose biosensor via sequential deposition of Prussian blue (PB) nanoclusters and enzyme-immobilized poly(toluidine blue) films was constructed on a bare Au electrode using electrochemical methods. The whole configuration of the present biosensor can be considered as an integration of several independent hydrogen peroxide sensing elements. In each sensing element, the poly(toluidine blue) film functioned as both the supporting matrix for the glucose oxidase immobilization and the inhibitor for the diffusion of interferences, such as ascorbic acid and uric acid. Meanwhile, the deposited Prussian blue nanocluster layers acts as a catalyst for the electrochemical reduction of hydrogen peroxide formed from enzymatic reaction. Performance of the whole multilayer configuration can be tailored by artificially arranging the sensing elements assembled on the electrode. Under optimal conditions, the biosensors exhibit a linear relationship in the range of 1 x 10(-4) to 1 x 10(-2) mol/L with the detection limit down to 10(-5) mol/L. A rapid response for glucose could be achieved in less than 3 s. For 1 mM glucose, 0.5 mM acetaminophen, 0.2 mM uric acid, and 0.1 mM ascorbic acid have no obvious interferences (<5%) for glucose detection at an optimized detection potential. The present multilayered glucose biosensor with a high selectivity and sensitivity is promising for practical applications.     

Improved Sensing Performance of Amperometric Urea Biosensor by Using Platinum Nanoparticles

A novel Platinum nanoparticle (PtNPs) modified Poly(pyrrole-co-1-(2-Aminophenyl)pyrrole)/Urease film coated Au electrode was designed for amperometric detection of urea. PtNPs quantity, film density and pH were optimized and interference effect of some substances readily found in municipal wastewater and blood was investigated. The biosensor responded to urea with a measurement concentration range of 0.1 to 30 mM, a sensitivity of 31.8 μA mM⁻¹ cm⁻², a LOD of 7.58 μM, an accuracy of 104 % and a RSD% of only 0.82. It sensed the concentration of urea in the municipal sewage water with recovery of 97.6 % (n=3) and remained 78 % of its initial response at 28^th day. Results confirmed that PtNPs with strong conductivity improved the electron transfer ability of the working electrode.     

Sensing the Lactic Acid in Probiotic Yogurts Using an L-Lactate Biosensor Coupled with a Microdialysis Fiber Inserted in a Flow Analysis System

An amperometric biosensor for the determination of L-lactic acid in probiotic yogurts has been assembled using L-lactate dehydrogenase (EC 1.1.1.27, LDH) entrapped in 1% (v/v) neutralized Nafion® solution deposited on Variamine blue redox mediator modified screen-printed electrodes. The Variamine blue was previously covalently linked to oxidized single-walled carbon nanotubes and used for modifying screen-printed electrodes. The electrochemical cell, containing the L-lactate biosensor operating at an applied working potential of +200 mV vs. Ag|AgCl, was coupled with a microdialysis fiber and connected with a flow system, thus obtaining a microdialysis based sampling experimental set-up. Various analytical parameters, such as the cofactor concentration (2 mM, NAD⁺), the flow rate (10.5 μL/min), the applied working potential (+200 mV vs. Ag|AgCl), the working buffer (50 mM phosphate buffer +0.1 M KCl), and pH (7.5), were optimized in batch amperometric experiments. The dynamic linear working range was comprised between 2·10^?4 and 1·10^?3 M. The proposed biosensor was challenged with real samples of yogurt, properly diluted in working buffer, and the performances of the L-lactate biosensor were compared with a commercially available kit for the determination of L-lactic acid in foodstuffs from R-Biopharm GmbH, Germany, showing a good agreement.     

Fabrication of Glucose Biosensor Based on Encapsulation of Glucose‐Oxidase on Sol‐Gel Composite at the Surface of Glassy Carbon Electrode Modified with Carbon Nanotubes and Celestine Blue

A simple procedure was developed to prepare a glassy carbon electrode modified with multi walled carbon nanotubes (MWCNTs) and Celestin blue. Cyclic voltammograms of the modified electrode show stable and a well defined redox couple with surface confined characteristic at wide pH range (2–12). The formal potential of redox couple (E′) shifts linearly toward the negative direction with increasing solution pH. The surface coverage of Celestine blue immobilized on CNTs glassy carbon electrode was approximately 1.95×10^?10 mol cm^?2. The charge transfer coefficient (α) and heterogeneous electron transfer rate constants (k_s) for GC/MWCNTs/Celestine blue were 0.43 and 1.26 s^?1, respectively. The modified electrode show strong catalytic effect for reduction of hydrogen peroxide and oxygen at reduced overpotential. The glucose biosensor was fabricated by covering a thin film of sol‐gel composite containing glucose oxides (GOx) on the surface of Celestine blue /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 0.3 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. The accuracy of the biosensor for glucose detection was evaluated by detection of glucose in a serum sample, using standard addition protocol. In addition biosensor can reach 90% of steady currents in about 3.0 sec and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) was eliminated. Furthermore, the apparent Michaelis–Menten constant 2.4 mM, of GOx on the nano composite exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility of redox couple, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this procedure for modification of glucose biosensor.     

Amperometric phenol biosensor based on sol-gel silicate/Nafion composite film

An amperometric biosensor based on tyrosinase immobilized in silicate/Nafion composite film has been developed for the determination of phenolic compounds. The Nafion polymer in the composite was used not only to overcome the brittleness of the pure sol-gel-derived silicate film but also to increase the long-term stability of the biosensor. Tyrosinase was immobilized by a thin film of silicate/Nafion composite on a glassy carbon electrode. Phenolic compounds were determined by the direct reduction of biocatalytically-liberated quinone species at −200 mV versus Ag/AgCl (3 M NaCl). The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH and operating potential were explored for optimum analytical performance of the enzyme electrode. The biosensor can reach 95% of steady-state current in about 15 s. The sensitivities of the biosensor for catechol and phenol were 200 and 46 mA/M, respectively. A detection limit of 0.35 mM catechol was obtained with a signal-to-noise ratio of 3. The enzyme electrode retained 74% of its initial activity after 2 weeks of storage in 50 mM phosphate buffer at pH 7.     

Biosensor for H2O2 Response Based on Horseradish Peroxidase: Effect of Different Mediators Adsorbed on Silica Gel Modified with Niobium Oxide

Reagentless biosensors sensitive to hydrogen peroxide have been developed and compared. These biosensors are comprised of a carbon paste electrode modified with horseradish peroxidase (HRP) and one phenothiazine (methylene blue), one phenoxazine (meldola's blue) or one phenazine (phenazine methosulfate) dye adsorbed on silica gel modified with niobium oxide (SN). The enzyme was immobilized onto the graphite powder by cross‐linking with glutaraldehyde and mixing with one of the electron transfer mediators (dyes) adsorbed on SN. The amperometric response was based on the electrocatalytic properties of the dye to mediate electrons, which were generated in the enzymatic reaction of hydrogen peroxide under catalysis of HRP. The dependence on the biosensor response in terms of pH, buffer, HRP amounts and applied potential was investigated. The best results were found with a biosensor containing methylene blue dye showing an excellent operational stability (around 92% of the activity was maintained after 300 determinations). The proposed biosensor also presented good sensitivity (32.87 nA cm^{?2 μ}mol^?1 L) allowing hydrogen peroxide quantification at levels down to 0.52×10^?6 mol L^?1 an optimum response at pH 6.8 and at a potential of ?50 mV (vs. SCE) and showing a wide linear response range (from 1 to 700 μmol L^?1 for hydrogen peroxide).     

Enhanced NADH Oxidation Using Polytyramine/Carbon Nanotube Modified Electrodes for Ethanol Biosensing

Polytyramine (PT) has been electro‐deposited onto multi‐walled carbon nanotube (MWCNT) modified glassy carbon (GC) electrodes via oxidation of tyramine in 0.1 M H₃PO₄ by cycling the potential over the range of −400 mV to 1300 mV (versus Ag/AgCl). The reactivity of the resulting chemically‐modified electrodes was characterized using cyclic voltammetry in the presence and absence of reduced nicotinamide adenine dinucleotide (NADH). The modified electrodes displayed electrochemical activity due to the formation of quinone species and were catalytically active towards NADH oxidation by lowering the oxidation peak potential by 170 mV compared to the value of the MWCNT modified electrode with a peak potential of 180±10 mV (versus Ag/AgCl). The MWCNT/PT surface was further characterized using SEM and XPS methods, which indicated that a thin polymeric film had been formed on the electrode surface. The present work demonstrates the advantage of using PT as a platform that combines both the immobilization of alcohol dehydrogenase (ADH) and the mediation of NADH oxidation at a low overpotential essential to the design of high performance ethanol biosensors, all within an easily electropolymerizable film. The resulting biosensor displayed an ethanol sensitivity of 4.28±0.06 μA mM⁻¹ cm⁻², a linear range between 0.1 mM and 0.5 mM and a detection limit of 10 μM.     

Electrochemical urea biosensor based on Proteus mirabilis urease immobilized over polyaniline PANi-Glassy carbon electrode

In this study, a novel, sensitive electrochemical enzyme-based biosensor for urea detection was presented. This biosensor combines a three-electrode system consisting of a classic Glassy Carbon Electrode (GCE) as the working electrode, a platinum counter electrode, and Ag/AgCl as the reference electrode. To construct this urea platform, a GCE was modified with a polyaniline (PANi) film. Then, bacterial urease from Proteus mirabilis was immobilized on the modified GCE (P_m-Urease-PANi-GCE). For the characterization of surface modification, Cyclic Voltammetry (CV) and Scanning Electron Microscope (SEM) were applied, while the Square Wave Voltammetry (SWV) technique was performed for urea detection. The main analytical characteristics of the P_m-Urease-PANi-GCE biosensor showed a good linear range from 0.1 to 10 mM of urea, a limit of detection (LOD) of 0.1 mM, a Michaelis-Menten K_m of 0.23 mM, and a sensitivity value 46 μA/mM/cm². This biosensor allows the detection of urea in solutions, and it could be improved for further medical, environmental, or engineering applications.     

New Urea Biosensor Based on Urease Enzyme Obtained from Helycobacter pylori

The urease enzyme of Helicobacter pylori was isolated from biopsy sample obtained from antrum big curvature cell extracts. A new urea biosensor was prepared by immobilizing urease enzyme isolated from Helicobacter pylori on poly(vinylchloride) (PVC) ammonium membrane electrode by using nonactine as an ammonium ionophore. The effect of pH, buffer concentration, and temperature for the biosensor prepared with urease from H. pylori were obtained as 6.0, 5 mM, and 25 °C, respectively. We also investigated urease concentration, stirring rate, and enzyme immobilization procedures in response to urea of the enzyme electrode. The linear working range of the biosensor extends from 1 × 10(-5) to 1 × 10(-2) M and they showed an apparent Nernstian response within this range. Urea enzyme electrodes prepared with urease enzymes obtained from H. pylori and Jack bean based on PVC membrane ammonium-selective electrode showed very good analytical parameters: high sensitivity, dynamic stability over 2 months with less decrease of sensitivity, response time 1-2 min. The analytical characteristics were investigated and were compared those of the urea biosensor prepared with urease enzyme isolated from Jack bean prepared at the same conditions. It was observed that rapid determinations of human serum urea amounts were also made possible with both biosensors.     

Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator

The natural dye hematein in water solution was used as a pH-sensitive redox-active mediator for amperometric pH-sensing. The electrochemical characteristics were studied using cyclic voltammetry and chronoamperometry. Several types of urea biosensors were constructed with urease on the surface of platinum and graphite composite electrodes or in the bulk of the graphite composite. They were used for the amperometric urea determination at a working potential of 0 mV (versus SCE) using 0.5 mM hematein. Detection limits and response linearity was in the micromolar range depending on the biosensor type, concentration and pH of buffers used. An interference study of various cations, anions, and substances, which may be present in real samples demonstrated good selectivity for the determination of urea. The biosensors showed good operational (>3 h) and storage (>3 months) stability. The results of urea determination in blood and urine obtained by biosensor correlated well with those obtained by a spectrophotometric reference method.     

Structural Characterization of Graphene Nanosheets for Miniaturization of Potentiometric Urea Lipid Film Based Biosensors

The present article describes a miniaturized potentiometric urea lipid film based biosensor on graphene nanosheets. Structural characterization of graphene nanosheets for miniaturization of potentiometric urea lipid film based biosensors have been studied through atomic force microscopy (AFM) and transmission electron microscopy (TEM) measurements. UV‐Vis and Fourrier transform IR (FTIR) spectroscopy have been utilized to study the pre‐ and postconjugated surfaces of graphene nanosheets. The presented potentiometric urea biosensor exhibits good reproducibility, reusability, selectivity, rapid response times (～4 s), long shelf life and high sensitivity of ca. 70 mV/decade over the urea logarithmic concentration range from 1×10^?6 M to 1×10^?3 M.     

A miniaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip

A urea biosensor prepared by covalent binding of urease directly to the surface of an ammonium-sensitive field effect transistor (FET) is described. Nonactin incorporated in carboxylated polyvinyl chloride was used to obtain the sensitive membrane of the ammonium-sensitive FET. The grafting of urease on the polyvinylchloride-COOH membrane surface was performed through carbodiimide coupling. The activity of the immobilized enzyme was spectrometrically controlled through the time-dependent disappearance of the absorbance of NADH at 340 nm. An apparent activity of 50% was found, compared with free enzyme. The sensitivity of the urea enzyme FET is 50 mV/pUrea working in a differential mode of 2 muM to 1 mM, this sensitivity being constant during 15 days. Finally, in order to test the potentialities of the urea biosensor for the environmental applications, the detection of heavy metal ions such as Cu(II) and Hg(II) in solution was performed by measuring the remaining activity of the inhibited enzyme.     

Reagentless Glucose Biosensor Based on the Direct Electrochemistry of Glucose Oxidase on Carbon Nanotube‐Modified Electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)‐modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about ?455 mV (vs. Ag/AgCl) and shows a surface‐controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD.     

Amperometric bienzymatic biosensor for L-lactate analysis in wine and beer samples

A novel amperometric bienzymatic biosensor has been developed based on the incorporation of Lactate Oxidase (LOx) and Horseradish Peroxidase (HRP) into a carbon nanotube/polysulfone membrane by the phase inversion technique onto screen-printed electrodes (SPEs). In order to improve the sensitivity and reduce the working potential, experimental conditions have been optimized and ferrocene has also been incorporated into the membrane as a redox mediator of the enzymatic reactions, which allows the reduction of H(2)O(2) at -100 mV. Measurements were carried out in phosphate buffer solution at pH 7.5 and under batch conditions. The biosensor response time to L-lactate was only 20 s and showed a good reproducibility (RSD 2.7%). Moreover, the detection limit was 0.05 mg L(-1) of l-lactate with a linear interval range from 0.1 mg L(-1) to 5 mg L(-1). Finally, the biosensor has been applied to the determination of l-lactic acid in different wine and beer samples. Then, the results obtained with the biosensor were compared with the ones obtained using, as a reference method, a commercial kit based on spectrophotometric measurements, obtaining an excellent agreement between the results, validating our approach.     

Amperometric Tyrosinase Biosensor Based on Carbon Nanotube–Titania–Nafion Composite Film

A highly sensitive and stable amperometric tyrosinase biosensor has been developed based on multiwalled carbon nanotube (MWCNT) dispersed in mesoporous composite films of sol–gel‐derived titania and perfluorosulfonated ionomer (Nafion). Tyrosinase was immobilized within a thin film of MWCNT–titania–Nafion composite film coated on a glassy carbon electrode. Phenolic compounds were determined by the direct reduction of biocatalytically‐liberated quinone species at ?100 mV versus Ag/AgCl (3 M NaCl) without a mediator. The present tyrosinase biosensor showed good analytical performances in terms of response time, sensitivity, and stability compared to those obtained with other biosensors based on different sol–gel matrices. Due to the large pore size of the MWCNT–titania–Nafion composite, the present biosensor showed remarkably fast response time with less than 3 s. The present biosensor responds linearly to phenol from 1.0×10^?7 M to 5.0×10^?5 M with an excellent sensitivity of 417 mA/M and a detection limit of 9.5×10^?8 M (S/N=3). The enzyme electrode retained 89% of its initial activity after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

Superoxide radical biosensor based on a nano-composite containing cytochrome c

A biosensor for the quantification of superoxide radical (O(2)˙(-)) was developed based on a nano-composite containing cytochrome c (Cyt c), carboxylated multi-walled carbon nanotubes and a room temperature ionic liquid (RTIL). The immobilized Cyt c was characterized by field emission scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. Using this biosensor a formal potential of -280 mV (vs. Ag/AgCl) and electron transfer rate constant of 1.24 was recorded for the immobilized Cyt c in 0.1 M phosphate buffer solution (pH 7.0). The biosensor showed a relatively high sensitivity (7.455 A M(-1) cm(-2)) and a long term stability (180 days) towards O(2)˙(-) in the concentration range from 0.05 to 8.1 μM with a detection limit of 0.03 μM. The selectivity of the biosensor to O(2)˙(-) was verified when its response was compared with those obtained by four potential interfering substances (ascorbic acid, uric acid, acetaminophen and hydrogen peroxide).     

Flow injection analysis of sulfite ion with a potentiometric titanium phosphate-epoxy based membrane sensor

A novel pH sensor suitable for use in both aqueous and non-aqueous mediums is reported. The sensor is derived from polymer modified electrode obtained from electrochemical polymerisation of aniline in dry acetonitrile containing 0.5 M tetraphenyl borate at 2.0 V versus Ag/AgCl. The light yellow colour polymer modified electrode obtained under the present experimental condition has been characterised by scanning electron microscopy (SEM). The pH sensing of polymer modified electrode in both aqueous and non-aqueous mediums is examined and reported. As the typical examples, we used weak acid (acetic acid) and weak base (ammonium hydroxide) as analytes. The acetic acid is analysed in both aqueous and dry acetonitrile whereas ammonium hydroxide is analysed only in aqueous medium. The analysis in aqueous medium is conducted in 1 mM Tris-HCl buffer pH 7.0 and also in 0.1 M KCl. The slope of pH sensing is calculated from the data recorded in typical buffers and found to be approximately 86 mV per pH. The application of polymer modified electrode for the construction of urea biosensor is described based on immobilised urease within poly vinyl alcohol (PVA) matrix and also within organically modified sol-gel glass on the surface of polymer-modified electrode. The new urea sensor has shown maximum response of 160 mV at 25 degrees C with a lowest detection limit of 20 muM. The performance of new pH sensor and urea sensor has been studied and reported in this communication.     

Direct Electrochemistry of Glucose Oxidase on Nail‐like Carbon and Its Biosensing for Glucose

Nail‐like carbon (NLC) was synthesized by a simple hydrothermal method. It was the first time that a novel electrochemical biosensing of glucose was explored based on the glucose oxidase (GOx)‐NLC‐chitosan (CHIT) glassy carbon electrode. Morphology and structure of NLC were characterized by scanning electron microscope; meanwhile the chemical composition was determined by X‐ray diffraction and energy dispersive X‐ray spectroscopy. The cyclic voltammetry of immobilized GOx showed a pair of quasireversible redox peaks with the formal potential (E°′) of ?0.458 V and the peak‐to‐peak potential separation was 47 mV at a scan rate of 100 mV s^?1. The present biosensor has a linear range of glucose from 0.02 to 1.84 mM (correlation coefficient of 0.9991) and detection limit of 0.01 mM (S/N=3). Compared with the previous reports based on the carbon material biosensor, it has a high sensitivity of 165.5 μA mM^?1 cm^?2 and low apparent Michaelis–Menten constant of 0.506 mM. Thus, the NLC may have potential applications in the field of bioelectrochemistry, bioelectronics and biofuels.