Plant tissue-based membrane biosensor for l-ascorbic acid

Coupling of a slice of the mesocarp of squash (Cucurbita pepo) or cucumber (Cucumis sativus) to a Clark-type oxygen electrode allows 0.02–0.57 mmol l^?1l-ascorbic acid to be determined amperometrically. The method is based on monitoring the decrease in the curretn of oxygen at an applied potential of ?650 mV vs. Ag/AgCl; oxygen is consumed in the analyte oxidation catalyzed by ascorbate oxidase in the plant tissue. One tissue slice serves for 50–80 measurements at 30°C and pH 6. Spare slices can be stored for at least a year in aqueous 50% glycerol without substantial loss of enzyme activity. The biosensor is highly selective towards ascorbic acid with a response time of 70–90 s, the relative standard deviation being about 3%. Satisfactory results were obtained in the analysis of some fruit juices and vitamin tablets.     

Fabrication of multiwalled carbon nanotubes/polyaniline modified Au electrode for ascorbic acid determination

An ascorbate oxidase (AsOx) (E.C.1.10.3.3) purified from Lagenaria siceraria fruit was immobilized covalently onto a carboxylated multiwalled carbon nanotubes and polyaniline (c-MWCNT/PANI) layer electrochemically deposited on the surface of an Au electrode. The diffusion coefficient of ascorbic acid was determined as 3.05 × 10(-4) cm(2) s(-1). The behavior of different electrolytes on electro-deposition was also studied. An ascorbate biosensor was fabricated using a AsOx/c-MWCNT/PANI/Au electrode as a working electrode, Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode connected through a potentiostat. Linear range, response time and detection limit were 2-206 μM, 2 s and 0.9 μM respectively. The biosensor showed optimum response at pH 5.8 and in a broader temperature range (30-45 °C), when polarized at +0.6 V. The biosensor was employed for determination of ascorbic acid level in sera, fruit juices and vitamin C tablets. The sensor was evaluated with 91% recovery of added ascorbic acid in sera and 6.5% and 11.4% within and between batch coefficients of variation respectively for five serum samples. There was a good correlation (r = 0.98) between fruit juice ascorbic acid values by the standard 2,6-dichlorophenolindophenol (DCPIP) method and the present method. The enzyme electrode was used 200 times over a period of two months, when stored at 4 °C. The biosensor has advantages over earlier enzyme sensors in that it has no leakage of enzyme, due to the covalent coupling of enzyme with the support, lower response time, wider working range, higher storage stability and no interference by serum substances.     

Chemically amplified kojic acid responses of tyrosinase-based biosensor, based on inhibitory effect to substrate recycling driven by tyrosinase and l-ascorbic acid

A tyrosinase-based chemically amplified biosensor, based on the substrate recycling of polyphenols driven by tyrosinase-catalyzed oxidation and chemical reduction by l-ascorbic acid (AsA), has been utilized for the highly sensitive detection of inhibitors of tyrosinase such as kojic acid, benzoic and SCN(-) ion. The amplified current response of immobilized tyrosinase-coupling oxygen electrode due to the recycling was suppressed by the addition of inhibitors, and a highly amplified response to kojic acid over other inhibitors was observed in the presence of 5 mM AsA. The amplification factor (AF) of kojic acid is substantially proportional to the AF of substrates, and the AF for 1 x 10(-7)M kojic acid was increased by up to a factor of 143 when 1 x 10(-5)M dopamine was used as a competitive substrate in the presence of 5 mM AsA. The amplified calibration curve of kojic acid obtained with 5 mM AsA was shifted towards more than a two decades lower concentration range compared with that of the non-amplified response, and the detection limit of kojic acid was lowered to 7 x 10(-8)M.     

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 x 10(-4) to 1.5 x 10(-2) mol L(-1) with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.     

Chemically amplified current response of vitamin C based on the cyclic reaction between l-ascorbic acid and dehydroascorbic acid, using dithiothreithol and ascorbate oxidase

A cyclic reaction between the reduced and the oxidized forms of vitamin C takes place by combining the ascorbate oxidase reaction and the reduction of l-ascorbic acid to dehydroascorbic acid by dithiothreithol. l-Ascorbic acid is oxidized by the dissolved oxygen and its consumption is not compensated by the chemical regeneration of l-ascorbic acid from dehydroascorbic acid. The dissolved oxygen then continues to decrease during the cyclic reaction, consequently, the decrease of the reduction current of oxygen is amplified. The amplification factor of 5 x 10(-5)Ml-ascorbic acid at 5 x 10(-3)M dithiothreithol and pH 7.5 was found to be 10.6 when enzyme activity is 0.64 units/ml and the reaction time is 10 min.     

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.     

Determination of ascorbic acid content of some fruit juices and wine by voltammetry performed at pt and carbon paste electrodes

A method was developed for assessing ascorbic acid concentration in fruit juices and wine by differential pulse voltammetry. The oxidation peak for ascorbic acid occurs at about 530 mV (versus SCE) on a Pt strip working electrode and at about 470 mV on a carbon paste working electrode. The influence of the operational parameters like the pulse amplitude and the pulse period on the analytical signal was investigated. The obtained calibration graph shows a linear dependence between the peak height and ascorbic acid concentration within the range 0.31-20 mM with a Pt working electrode, and within the range 0.07-20 mM with a carbon paste working electrode. The equation of the calibration graph was y = 21.839x + 35.726, r2 = 0.9940, when a Pt strip electrode was used (where y represents the value of the current intensity measured for the peak height, expressed as μA and x the analyte concentration, as mM). R.S.D. = 2.09%, n = 10, C(ascorbic acid) = 2.5 mM. The equation of the calibration graph was y = 3.4429x + 5.7334, r2 = 0.9971, when a carbon paste electrode was used (where y represents the value of intensity measured for the peak height, expressed as μA and x the analyte concentration, as mM). R.S.D. = 2.35%, n = 10, C(ascorbic acid) = 2.5 mM. The developed method was applied to ascorbic acid assessment in fruit juices and wine. The ascorbic acid content determined ranged between 6.83 mg/100 mL juice for soft drinks (Fanta Madness) and 54.74 mg/100 mL for citrus (lemon) juices obtained by squeezing fruit. Different ascorbic acid concentrations (from standard solutions) were added to the analysed samples, the degree of recovery being comprised between 94.74 and 104.97%. The results of ascorbic acid assessment by differential pulse voltammetry were compared with those obtained by cyclic voltammetry. The results obtained by the two methods were in good agreement.     

Anti‐Interferent Properties of Oxidized Nickel Based on Layered Double Hydroxide in Glucose Amperometric Biosensors

An amperometric biosensor based on Pt electrodes modified with a thin film of a Ni, Al layered double hydroxide (LDH), submitted to a preliminary oxidative treatment in order to have the nickel centers at the oxidation state +4, and glucose oxidase (GOx) is presented. The oxidized LDH acts both as a system to support the enzyme and as a barrier to anions since it acquires an overall negative charge, as demonstrated by electrochemical impedance spectroscopy. Even if the biosensor response is due to the detection of H₂O₂ at anodic potentials, glucose can be accurately determined in the presence of ascorbic acid or other interferences, commonly present in real matrices in anionic form, since they can not reach the electrode surface. The effectiveness of the developed biosensor has been demonstrated by measuring glucose in samples of fruit juices containing ascorbic acid at high levels.     

Diffusion coefficient measurements in microfluidic devices

A glassy carbon electrode (GCE) modified with Pd/IrO(2) provides excellent electrocatalytic oxidation of hydrogen peroxide. Glucose oxidase (GOD) and xanthine oxidase (XOD) were co-immobilized on the modified electrode with a thin film Nafion coated on the enzyme layer to form a glucose (Glu)/hypoxanthine (Hx) sensor, without interference from electroactive species such as ascorbic acid (AA) and uric acid (UA). Its response was evaluated with respect to the enzyme amount on the electrode, pH and temperature of the electrolyte. The prepared bienzymic biosensor, used as the detector of HPLC gave a detection limit of 1.0x10(-6) mol l(-1) Glu and 2.0x10(-7) mol l(-1) Hx (Hx) with a linear concentration range of 5.0x10(-6)-2.5x10(-3) mol l(-1) and 1.0x10(-6)-5.0x10(-4) mol l(-1), respectively. Coupled with microdialysis, it was used to monitor the concentrations of Glu and Hx in rat brain.     

Spectrophotometric flow injection determination of l-ascorbic acid with a packed reactor containing ferric hydroxide

A flow injection system with spectrophotometric detection is proposed for determining l-ascorbic acid in pharmaceutical formulations. In this system a column containing Fe(OH)(3) immobilized in polyester resin (packed reactor) is inserted before the detector. Fe(III)-1,10-phenanthroline complex is reduced by l-ascorbic acid to produce Fe(II)-1,10-phenanthroline complex which is monitored at 510 nm. Under the optimum analytical conditions, the linearity of the calibration equation for l-ascorbic acid ranged from 5.0x10(-6) to 6.0x10(-5) M of added amount. The detection limit was 5.0x10(-7) M and recoveries between 98.5-102.0% were obtained. No interference was observed from the common excipients of pharmaceutical formulations and other active substances such as acetylsalicylic acid, caffeine and thiamine.     

Enantioselective screen-printed amperometric biosensor for the determination of D-amino acids

D-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of D-amino acids has been constructed by the immobilization of D-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for D-alanine in the concentration range from 5 to 200 microM. Excellent enantioselectivity towards D-amino acids was discovered. During the experiment, D-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.     

Chronoamperometric determination of paracetamol using an avocado tissue (Persea americana) biosensor

A biosensor based on vaseline/graphite modified with avocado tissue (Persea americana) as the source of polyphenol oxidase was developed and used for the chronoamperometric determination of paracetamol in pharmaceutical formulations. This enzyme catalyses the oxidation of paracetamol to N-acetyl-p-benzoquinoneimine whose electrochemical reduction back to paracetamol was obtained at a potential of -0.12 V. After addition of paracetamol reference solutions in glass cell and stirring for 60 s for the accumulation of N-acetyl-p-benzoquinoneimine at the electrode surface under open-circuit conditions, the current response was monitored by 120 s without stirring. The currents obtained at 70 s were proportional to the paracetamol concentration from 1.2x10(-4) to 5.8x10(-3) mol l(-1) (r=0.9927) with a detection limit of 8.8x10(-5) mol l(-1). The recovery of paracetamol from two samples ranged from 97.9 to 100.7% and a relative standard deviation lower than 0.5% for a solution containing 5.0x10(-3) mol l(-1) paracetamol in 0.10 mol l(-1) phosphate buffer solution (pH 7.0; n=10) was obtained. The results obtained for paracetamol in pharmaceutical formulations using the proposed biosensor and those obtained using a pharmacopoeial procedure are in agreement at the 95% confidence level.     

Amperometric Vitamin C Biosensor Based on the Immobilization of Ascorbate Oxidase into the Biocompatible Sandwich-Type Composite Film

Ascorbate oxidase (AO), a biologically active macromolecule, was successfully immobilized into a biocompatible sandwich-type composite film for developing the vitamin C (VC) biosensor, and the content of VC in commercial juices was amperometrically determined. The biocompatible and conducting poly(3,4-ethylenedioxythiophene) composite film and highly stable and selective multiwalled carbon nanotubes ?CNafion composite film were prepared as inner and outer films of biosensor. AO molecules were immobilized between these two composite films. The as-fabricated biosensor displayed an excellent bioelectrocatalytic performance towards the oxidation of VC, a fast current response, a low working potential, a high sensitivity, a wide linear range, and a low detection limit. Moreover, the working mechanism of the biosensor was proposed, and its kinetics was also discussed. In addition, the specificity, reproducibility, and feasibility of the as-fabricated biosensor were also evaluated. Good results of the VC determination in commercial juices indicated that the as-fabricated biosensor was a potential candidate for the electrochemical determination of VC in agricultural crops. Inner and outer films provided a promising platform for the immobilization of biologically active species.     

Amperometric choline biosensor fabricated through electrostatic assembly of bienzyme/polyelectrolyte hybrid layers on carbon nanotubes

We report a flow injection amperometric choline biosensor based on the electrostatic assembly of the choline oxidase (ChO) enzyme and a bienzyme of ChO and horseradish peroxidase (HRP) onto multi-wall carbon nanotubes (MWCNT) modified glassy carbon (GC) electrodes. These choline biosensors were fabricated by immobilization of enzymes on the negatively charged MWCNT surface through alternately assembling a cationic poly(diallydimethylammonium chloride) (PDDA) layer and an enzyme layer. Using this layer-by-layer assembling approach, a bioactive nanocomposite film of PDDA/ChO/PDDA/HRP/PDDA/CNT (ChO/HRP/CNT) and PDDA/ChO/PDDA/CNT (ChO/CNT) was fabricated on the GC surface. Owing to the electrocatalytic effect of carbon nanotubes, the measurement of faradic responses resulting from enzymatic reactions has been realized at low potential with acceptable sensitivity. The ChO/HRP/CNT biosensor is more sensitive than the ChO/CNT one. Experimental parameters affecting the sensitivity of biosensors, e.g., applied potential, flow rate, etc., were optimized and potential interference was examined. The response time for this choline biosensor is fast (few seconds). The linear range of detection for the choline biosensor is from 5.0 x 10(-5) to 5.0 x 10(-3) M and the detection limit is about 1.0 x 10(-5) M.     

The study of nafion/xanthine oxidase/au colloid chemically modified biosensor and its application in the determination of hypoxanthine in myocardial cells in vivo

A novel hypoxanthine (Hx) microsensor was constructed. In this work, Nafion xanthine oxidase (XOD) and Au colloid were immobilized onto the surface of a Pt microelectrode. The enzyme biosensor displayed a quick and sensitive response to Hx. Under physiological conditions, a low detection limit, with high selectivity and sensitivity for Hx determination were obtained. The oxidation current [investigated using current-time (I-t) plots] was linear with Hx concentration ranging from 2.0 x 10(-7) to 2.0 x 10(-5) mol L(-1) with a calculated detection limit of 1.0 x 10(-7) mol L(-1) (S/N of 3). The biosensor should be promising for in vivo measurement of Hx without interferences and fouling. The change of Hx concentration in cardiac myocytes stimulated by L-arginine (L-Arg) and acetylcholine (Ach) was also studied.     

A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine

A new amperometric biosensor based on urate oxidase-peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H₂O₂ from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 μM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 °C were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9×10⁻³ U cm⁻² for urate oxidase and 512.7 U cm⁻² for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out.     

Amperometric enzyme electrode for theophylline.

An amperometric biosensor for theophylline, based on the recently isolated enzyme theophylline oxidase, is described. The enzyme is entrapped, together with a ferricytochrome C cofactor, within a polymeric (Nafion) coating. The anodic detection (at +0.4 V versus Ag-AgCl) is facilitated by the addition of a redox-mediating hexacyanoferrate(III) ion. The influence of various experimental variables is described. The limit of detection is 2 x 10(-6) mol dm-3 theophylline, with linearity prevailing up to 3 x 10(-4) mol dm-3. The fast response and wash times permit rapid flow-injection measurements, with a frequency of 180 samples h-1 and a relative standard deviation of 3.0-4.0%. Prospects of using this electrode for clinical diagnostics are discussed.     

Carbon paste electrode modified with copper(II) phosphate immobilized in a polyester resin for voltammetric determination of <Emphasis Type="SmallCaps"> l </Emphasis>-ascorbic acid in pharmaceutical formulations

A carbon paste electrode modified with copper(II) phosphate immobilized in a polyester resin (CuP-Poly) is proposed for voltammetric determination of L-ascorbic acid in pharmaceutical formulations. The modified electrode allows the detection of L-ascorbic acid at lower anodic potentials than observed at unmodified electrodes. Several parameters that can influence the voltammetric response of the proposed electrode such as carbon paste composition, pH, scan rate, and possible interference were investigated. The peak current was proportional to the concentration of ascorbic acid in the range 2.0 x 10(-5) to 3.2 x 10(-3) mol L(-1) with a detection limit of 1.0 x 10(-5) mol L(-1). The stability and repeatability of the electrode for the determination of L-ascorbic acid are also discussed. Amperometric response was also recorded for electrocatalytic oxidation of the L-ascorbic acid. Concentrations of the vitamin C in pharmaceutical formulations (tablets) measured using the modified electrode and a titrimetric method are in agreement at the 95% confidence level and within an acceptable range of error.     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Enzyme-functionalized gold nanowires for the fabrication of biosensors

Gold nanowires were prepared by an electrodeposition strategy using nanopore polycarbonate (PC) membrane, with the average diameter of the nanowires about 250 nm and length about 10 microm. The nanowires prepared were dispersed into chitosan (CHIT) solution and stably immobilized onto glassy carbon electrode (GCE) surface. The electrochemical behavior of gold nanowire modified electrode and its application to the electrocatalytic reduction of hydrogen peroxide (H(2)O(2)) were investigated. The modified electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. Moreover, the good biocompatibility of nanometer-sized gold, the vast surface area of the nanowire-structure make it ideal for adsorption of enzymes for the fabrication of biosensors. Glucose oxidase was adsorbed onto the nanowire surface to fabricate glucose biosensor as an application example. The detection of glucose was performed in phosphate buffer (pH 6.98) at -0.2 V. The resulting glucose biosensor exhibited sensitive response, with a short response time (<8 s), a linear range of 10(-5)-2 x 10(-2) M and detection limit of 5 x 10(-6) M.