Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Development of amperometric horseradish peroxidase based biosensors for clozapine and for the screening of thiol compounds

Two amperometric biosensors with immobilized horseradish peroxidase (HRP) were developed for the investigation of the clozapine drug oxidation and for thiols screening based on biosensor signal inhibition. The HRP was retained either in magnetized nanoporous silica microparticles (MMPs) or in a carbon paste (CP). The latter served for the carbon paste electrode while the MMPs were attracted in close proximity of a magnetized carbon electrode. The potential use of these configurations for drug oxidation and inhibition studies was illustrated by the enzymatic oxidation of clozapine (CLZ) in the presence of hydrogen peroxide. The biosensor signal corresponded to the electro-reduction of CLZ oxidation products namely a nitrenium ion (CLZox) generated by the enzyme HRP. Several thiols reactive towards CLZox were investigated and the biosensor signal inhibition (IC(50)) was comparatively determined. A protective effect of the nanoporous silica and carbon paste matrices towards HRP inactivation was inferred by comparing the biosensor inhibition results with those obtained with the free enzyme in solution.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

Immobilization of metallothionein as a sensitive biosensor chip for the detection of metal ions by surface plasmon resonance

A biosensor based on mammalian metallothionein (MT) for the detection of metal ions was developed and characterized. MT was immobilized onto a carboxymethylated dextran matrix as a biosensor for the detection of metal ions by surface plasmon resonance (SPR). The optimal pH for the immobilization step was determined to be 4. The temperature for the analysis was also defined, and the highest interaction was observed at 30 degrees C. The MT sensor chip binds cadmium (Cd), zinc (Zn) or nickel (Ni), but not magnesium (Mg), manganese (Mn) and calcium (Ca). Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection is at the micromolar level. The interaction between the metal ions and the sensor chip is influenced significantly by the presence of NaCl, Tween 20 and the pH of the reaction buffer. By decreasing the NaCl in the reaction buffer to 1 mM, the MT chip effectively differentiates cadmium from zinc and nickel. Kinetic parameters of the metal-MT interactions were also determined by using this chip. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel, which is the same as that determined for MT in solution. Thus, the MT chip can be an effective biosensor for the detection and measurement of several metal ions.     

A super highly sensitive glucose biosensor based on Au nanoparticles-AgCl@polyaniline hybrid material

Gold nanoparticles (AuNPs) with an average diameter of 5nm were assembled on the surface of silver chloride@polyaniline (PANI) core-shell nanocomposites (AgCl@PANI). Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) suggested that AuNPs were incorporated on AgCl@PANI through coordination bonds instead of electrostatic interaction. The resulting AuNPs-AgCl@PANI hybrid material exhibited good electroactivity at a neutral pH environment. An amperometric glucose biosensor was developed by adsorption of glucose oxidase (GOx) on an AuNPs-AgCl@PANI modified glassy carbon (GC) electrode. AuNPs-AgCl@PANI could provide a biocompatible surface for high enzyme loading. Due to size effect, the AuNPs in the hybrid material could act as a good catalyst for both oxidation and reduction of H(2)O(2). As the measurement of glucose was based on the electrochemical detection of H(2)O(2) generated by enzyme-catalyzed-oxidation of glucose, the biosensor exhibited a super highly sensitive response to the analyte with a detection limit of 4 pM. Moreover, the biosensor showed good reproducibility and operation stability. The effects of some factors, such as temperature and pH value, were also studied.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin–streptavidin interaction monitoring

A new electrochemical method to monitor biotin–streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin–streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at +0.08 V was recorded in NH₃ 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H₂SO₄ 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at −0.18 V for 2 min and anodic stripping voltammetry were carried out in NH₃ 1.0 M containing 2.0×10⁻⁵ M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25×10⁻¹⁵ to 2.24×10⁻¹² M and a limit of detection of 2.0×10⁻¹⁵ M were obtained.     

A highly sensitive nanostructure-based electrochemical sensor for electrocatalytic determination of norepinephrine in the presence of acetaminophen and tryptophan

A new electrochemical sensor for the determination of norepinephrine (NE), acetaminophen (AC) and tryptophan (Trp) is described. The sensor is based on carbon paste electrode (CPE) modified with 3,4-dihydroxybenzaldehyde-2,4-dinitrophenylhydrazone (DDP) and takes the advantages of carbon nanotubes (CNTs), which makes the modified electrode highly sensitive for the electrochemical detection of these compounds. Cyclic voltammetry (CV) at various scan rates was used to investigate the redox properties of the modified electrode. The apparent charge transfer rate constant, k(s), and transfer coefficient, α, for electron transfer between DDP and CNT paste electrode were calculated. The mediated oxidation of NE at the modified electrode was investigated by CV and the values of k, α and diffusion coefficient (D) were calculated. Under the optimum pH of 7.0, the oxidation of NE occurs at a potential about 215 mV less positive than that of the unmodified CPE. Differential pulse voltammetry (DPV) of NE at the modified electrode exhibited two linear dynamic ranges with a detection limit (3σ) of 77±2 nM. DPV was used for simultaneous determination of NE, AC and Trp at the modified electrode, and quantitation of NE in some real samples by the standard addition method.     

Mediated, amperometric biosensor for glucose-6-phosphate monitoring based on entrapped glucose-6-phosphate dehydrogenase, Mg2+ ions, tetracyanoquinodimethane, and nicotinamide adenine dinucleotide phosphate in carbon paste

In this study, an amperometric carbon paste biosensor is developed for glucose-6-phosphate (G6P) monitoring which is based on entrapped Mg2+ ions, G6P dehydrogenase, NADP+ polyethylenimine (PEI) and the electroactive mediator, tetracyanoquinodimethane (TCNQ). The calibration line had a slope of 1.55 x 10(-5) A. M-1 with a correlation coefficient of 0.9965. The limit of detection (defined as three times the standard deviation of the response of the electrode to blank phosphate buffer injections (noise)) of the G6P biosensor was 5.0 x 10(-5) M. The application of this biosensor for monitoring G6P in human blood using the standard addition method is also demonstrated. A two-parameter empirical equation which adequately describes the deactivation of the biosensor steady-state response with time is also proposed.     

Ultrasensitive indicator-free and enhanced self-signal nanohybrid DNA sensing platform based on electrochemically grown poly-xanthurenic acid/Fe2O3 membranes

This paper describes a novel electrochemical DNA biosensor for simple, rapid, and specific detection of PML/RARA fusion gene in acute promyelocytic leukemia by using 18-mer single-stranded deoxyribonucleic acid as the capture probe. Nanosized Fe(2)O(3) was first immobilized on the surface of a carbon paste electrode (CPE). Then poly-xanthurenic acid (PXa), a new electroactive material, was electrogenerated by using the pulse potentiostatic method on the Fe(2)O(3) substrate to form a unique and uniform nanorhombus structure. Due to the unique binding ability of xanthurenic acid (Xa) with Fe(2)O(3), Xa monomers tended to be adsorbed around nanosized Fe(2)O(3), and the electropolymerization efficiency was greatly improved. Owing to the presence of abundant carboxyl groups, the capture probe was covalently attached on the carboxyl-terminated PXa/Fe(2)O(3) nanorhombus membranes through the free amines of DNA sequences based on the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydrosulfosuccinimide cross-linking reaction. The covalently immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA on the PXa/Fe(2)O(3)/CPE surface. Electrochemical impedance spectroscopy was adopted for indicator-free monitoring of the hybridization reaction on the probe-captured electrode. As a result, the efficient probe immobilization platform, coupled with the ultrasensitive indicator-free impedance measurement, gave rise to a detection limit of 2.8 fmol/L and a dynamic range spanning 8 orders of magnitude. The excellent analytical properties of the proposed biosensor developed here holds great promise for ultrasensitive detection of other biorecognition events and diagnosis of diseases in practice.     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

Voltammetric determination of mefenamic acid at lanthanum hydroxide nanowires modified carbon paste electrodes

Lanthanum hydroxide nanowires modified carbon paste electrode (LNW/CPE) exhibiting an electrocatalytic response toward the oxidation of mefenamic acid (MFA) is described. The catalytic action of the LNW/CPE on the oxidation of MFA via one-electron and one-proton transfer is attributed to the formation of the porous construction and the increase of efficient surface of the electrode due to the adulteration of LNW with carbon powders. Using the LNW/CPE, a linear sweep voltammetric method for the determination of MFA and other drugs with diphenylamine parent is proposed. A linear range of 2.0 x 10(-11) to 4.0 x 10(-9)mol L(-1) is obtained along with a detection limit of 6.0 x 10(-12)mol L(-1).     

Design and development of a highly stable hydrogen peroxide biosensor on screen printed carbon electrode based on horseradish peroxidase bound with gold nanoparticles in the matrix of chitosan

The design and development of a screen printed carbon electrode (SPCE) on a polyvinyl chloride substrate as a disposable sensor is described. Six configurations were designed on silk screen frames. The SPCEs were printed with four inks: silver ink as the conducting track, carbon ink as the working and counter electrodes, silver/silver chloride ink as the reference electrode and insulating ink as the insulator layer. Selection of the best configuration was done by comparing slopes from the calibration plots generated by the cyclic voltammograms at 10, 20 and 30 mM K(3)Fe(CN)(6) for each configuration. The electrodes with similar configurations gave similar slopes. The 5th configuration was the best electrode that gave the highest slope. Modifying the best SPCE configuration for use as a biosensor, horseradish peroxidase (HRP) was selected as a biomaterial bound with gold nanoparticles (AuNP) in the matrix of chitosan (HRP/AuNP/CHIT). Biosensors of HRP/SPCE, HRP/CHIT/SPCE and HRP/AuNP/CHIT/SPCE were used in the amperometric detection of H(2)O(2) in a solution of 0.1M citrate buffer, pH 6.5, by applying a potential of -0.4V at the working electrode. All the biosensors showed an immediate response to H(2)O(2). The effect of HRP/AuNP incorporated with CHIT (HRP/AuNP/CHIT/SPCE) yielded the highest performance. The amperometric response of HRP/AuNP/CHIT/SPCE retained over 95% of the initial current of the 1st day up to 30 days of storage at 4 degrees C. The biosensor showed a linear range of 0.01-11.3mM H(2)O(2), with a detection limit of 0.65 microM H(2)O(2) (S/N=3). The low detection limit, long storage life and wide linear range of this biosensor make it advantageous in many applications, including bioreactors and biosensors.     

Xanthine biosensor based on XO/AuNP/PtNP/MWCNT hybrid nanocomposite modified GCPE

A new xanthine (X) biosensors based on a hybrid nanocomposite containing multi-walled carbon nanotubes (MWCNT), Pt nanoparticles (PtNP) and gold nanoparticle (AuNP) was presented. X biosensor was fabricated by dropping AuNP/PtNP/MWCNT onto xanthine oxidase (XO) modified glassy carbon paste electrode (GCPE). Resulted XO/AuNP/PtNP/MWCNT/GCPE biosensor showed two linearity between 2.0 and 50 µM and 0.25 and 6.0 mM for X. RSD value was calculated as 2.46 (n = 5). Finally, the biosensor was applied to the X detection in synthetic serum samples and good recovery value was obtained.     

Synergetic effect for NADH oxidation of ferrocene and zeolite in modified carbon paste electrodes. New approach for dehydrogenase based biosensors

The development of electrochemical biosensors using dehydrogenases associated with the corresponding cofactor is strongly related to the better understanding of NADH oxidation at the electrode surface. The aim is to lower the necessary overvoltage and consequently to escape interferences and electrode fouling. In this paper, we show that carbon paste electrode (CPE) modified with NaY zeolite fulfils this requirements thanks to its hydrophilic surface. Oxidation of NADH at ferrocene (FcH) modified carbon paste electrode exhibits a rather slow electrocatalytic effect. We demonstrated the existence of synergetic effect on the electrocatalytic oxidation of NADH when the CPE is doped with zeolite (NaY) and FcH mediator or with the zeolite exchanged beforehand with the mediator (Y-Ferricinium, YFcH). This cumulative effect permits to reach high sensitivity for NADH detection and offers new way for the development of enzymatic biosensors using dehydrogenases depending on NADH as cofactor.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2'-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.     

Studies on the electrochemical performance of glucose biosensor based on ferrocene encapsulated ORMOSIL and glucose oxidase modified graphite paste electrode

The electrochemical performance of a new glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) and ferrocene encapsulated palladium (Pd)-linked organically modified sol-gel glass (ORMOSIL) material incorporated within graphite paste electrode. The ORMOSIL material incorporated within graphite paste electrode behaves as an excellent electrocatalyst for the oxidation of enzymatically reduced GOD. The electrochemical behavior of new glucose biosensor has been examined by cyclic volammetry and amperometric measurements. The bioelectrocatalysis of ORMOSIL embedded within graphite paste as a function of storage time and varying concentration of ORMOSIL is reported. The initial amperometric response on glucose sensing is recorded to be 145 microA at 15% (w/w) concentration of the ORMOSIL which is decreased to 20 microA at 5% of the same keeping GOD concentration constant. The variation of electrochemical behavior of the ORMOSIL embedded within graphite paste as a function of time has also been studied based on cyclic voltammetry. The voltammograms showing the reversible electrochemistry of ORMOSIL encapsulated ferrocene is changed into a plateau shape as a function of time, however, the electrocatalytic behavior is still retained. The practical usability of new glucose sensor has been compared with earlier developed glucose sensor. The sensitivity, response time and linearity of the new glucose biosensor are found to be excellent over earlier reported glucose biosensor. The amperometric response, calibration curve and practical applications of new glucose sensor are reported.