An amperometric horseradish peroxidase inhibition biosensor for the determination of phenylhydrazine

An amperometric horseradish peroxidase (HRP) inhibition biosensor has been substantially constructed by the help of N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS). The preparation steps and the biosensor response to phenylhydrazine were monitored by electrochemical impedance spectroscopy (EIS), cyclic voltammetry, and chronoamperometry. The proposed biosensor could be applied to determine phenylhydrazine in a 0.10 M phosphate buffer solution containing 1.2 mM hydroquinone and 0.50 mM H(2)O(2) by phenylhydrazine, inhibiting the catalytic activity of the HRP enzyme in the reduction of H(2)O(2). The system was optimized to realize a reliable determination of phenylhydrazine in the range of 2.5 x 10(-7) to 1.1 x 10(-6) M with a detection limit of 8.2 x 10(-8) M and a correlation coefficient of 0.999. The modified electrode displayed good reproducibility, sensitivity and stability for the determination of phenylhydrazine.     

An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination

In this study, a new biosensor based on the inhibition of tyrosinase for the determination of fluoride is described. To construct the biosensor tyrosinase was immobilized by using gelatine and cross-linking agent glutaraldehyde on a Clark type dissolved oxygen (DO) probe covered with a teflon membrane which is sensitive for oxygen. The phosphate buffer (50 mM, pH 7.0) at 30 °C were established as providing the optimum working conditions. The method is based on the measurement of the decreasing of dissolved oxygen level of the interval surface that related to fluoride concentration added into reaction medium in the presence of catechol. Inhibitor effect of fluoride results in decrease in dissolved oxygen concentration. The biosensor response depends linearly on fluoride concentration between 1.0 and 20 μM with a response time of 3 min.In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity and storage stability were carried out. From the experiments, the average value (x), Standard deviation (S.D) and coefficient of variation (C.V %) were found as 10.5 μM, ± 0.57 μM, 5.43%, respectively for 10 μM fluoride standard.     

A hydrogen peroxide biosensor based on multiwalled carbon nanotubes-polyvinyl butyral film modified electrode

A novel approach to construct a amperometric biosensor for determination of H₂O₂ is described. Horseradish peroxidase (HRP) as a base enzyme was immobilized into the mixture of multiwalled carbon nanotubes (MWNTs) and polyvinyl butyral (PVB). Taking the classical hydroquinone as mediator, cyclic voltammetry and amperometric measurements were used to study and optimize the performance of the resulting H₂O₂ biosensor. The effect of the concentration of MWNTs, HRP, hydroquinone, solution pH, and the working potential of amperometry on the electrochemical biosensor was systematically studied. The results showed that the fabricated biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with wide linear range from 0.000832 to 0.6 mM, and low detection limit 0.000167 mM (S/N = 3) with fast response time less than 8 s. The apparent Michaelis–Menten constant was determined to be 0.049 mM. Additionally, the biosensor exhibited high sensitivity, rapid response and good long-term stability.     

An amperometric glucose biosensor based on glucose oxidase immobilized in electropolymerized poly(o-aminophenol) and carbon nanotubes composite film on a gold electrode.

An amperometric glucose biosensor is developed that is based on immobilization of glucose oxidase (GOD) in a composite film of poly(o-aminophenol) (POAP) and carbon nanotubes (CNT), which are electrochemically co-polymerized at a gold (Au) electrode. Because of the high surface per volume ratio and excellent electrical conductivity of CNT, the biosensor based on an Au/POAP/CNT/GOD electrode has lower detection limit (0.01 mM), larger maximum response current (0.24 mA cm(-2)) and higher sensitivity (11.4 mA M(-1) cm(-2)) than the values of the biosensor based on an Au/POAP/GOD electrode. Additionally, the biosensor shows fast response time, large response current, and good anti-interferent ability for ascorbic acid, uric acid and acetaminophen. Good reproducibility and stability of the biosensor are also observed.     

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

An amperometric hydrogen peroxide biosensor based on immobilizing horseradish peroxidase to a nano-Au monolayer supported by sol-gel derived carbon ceramic electrode

A novel strategy for fabricating horseradish peroxidase (HRP)-based H(2)O(2) sensor has been developed by combining the merits of carbon sol-gel supporting matrix and nano-scaled particulate gold (nano-Au) mediator. The thiol functional group-derived carbon ceramic electrode (CCE) was first constructed using (3-mercaptopropyl) trimethoxy silane as sol-gel monomer. Then, the stable nano-Au monolayer was obtained through covalent linkage between nano-Au and thiol group on the surface of CCE. The experimental results showed that nano-Au monolayer formed not only could steadily immobilize HRP but also efficiently retain its bioactivity. Hydrogen peroxide was detected with the aid of hydroquinone mediator to transfer electrons between the electrode and HRP. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as the operating potential, mediator concentration and pH of background electrolyte were explored for optimum analytical performance of the enzyme electrode. The biosensor had a fast response of less than 8 s with linear range of 1.22 x 10(-5) to 1.10 x 10(-3)mol l(-1) and a detection limit of 6.1 x 10(-6)mol l(-1). The sensitivity of the sensor for H(2)O(2) was 0.29 A l mol(-1) cm(-2). The activation energy for enzyme reaction was calculated to be 10.1 kJ mol(-1). The enzyme electrode retained 75% of its initial activity after 5 weeks storage in phosphate buffer at pH 7.     

A novel amperometric biosensor based on spinach (Spinacia oleracea) tissue homogenate for urinary oxalate determination

In this study, a novel spinach (Spinacia oleracea) tissue homogenate-based biosensor for determination of oxalate in urine was developed. The biosensor was constructed by immobilizing tissue homogenate of spinach (S. oleracea) onto a high-sensitive teflon membrane of a dissolved oxygen (DO) probe. For the stability of the biosensor, general immobilization techniques were used to secure the spinach tissue homogenate in gelatin-glutaraldehyde cross-linking matrix. In the optimization and characterization studies, the amount of spinach tissue homogenate and gelatin, optimum pH, optimum temperature and thermal stability, interference effects, linear range and repeatability were investigated. A typical calibration curve for the sensor revealed a linear range of 1×10⁻⁵-10×10⁻⁵ M oxalate. In repeatability studies, variation coefficient (CV) was calculated as 1.8%. Of the various substrates tested, only oxalate was found to be specific, with a relative activity of 100%. The method was applied to the determination of oxalate in urine. The results showed that the method was applicable to oxalate determination in urine specifically and selectively.     

An amperometric glucose oxidase biosensor based on liposome microreactor-chitosan nanocomposite-modified electrode for determination of trace mercury

Journal of Solid State Electrochemistry - A biosensor for trace mercury ions based on glucose oxidase (GOD) immobilized on liposome microreactor and chitosan (CS) nanocomposite through...     

溶胶-凝胶壳聚糖/二氧化硅杂化复合膜固定辣根过氧化酶的H2O2电化学生物传感器的研制

利用溶胶 凝胶法制备壳聚糖 二氧化硅有机无机复合杂化膜,用于对辣根过氧化酶进行固定,制得测定H2O2的电流型生物传感器。以1mmol/LK4Fe(CN)6作为电子媒介体。研究了各种因素如壳聚糖与二氧化硅的比率、pH、温度、工作电位等对传感器响应电流的影响。计时电流法测定H2O2的线性范围为2.0×10-6～6.8×10-4mol/L,检出限为8.0×10-7mol/L。测得酶催化动力学参数米氏常数Km=0 87mmol/L。用该法对实际样品进行了测定。     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Sol-gel-derived amperometric biosensor for hydrogen peroxide based on methylene green incorporated in Nafion film

A hydrogen peroxide biosensor was fabricated by coating a sol-gel-peroxidase layer onto a Nafion-methylene green modified electrode. Immobilization of methylene green (MG) was attributed to the electrostatic force between MG(+) and the negatively charged sulfonic acid groups in Nafion polymer, whereas immobilization of horseradish peroxidase was attributed to the encapsulation function of the silica sol-gel network. Cyclic voltammetry and chronoamperometry were employed to demonstrate the feasibility of electron transfer between sol-gel-immobilized peroxidase and a glassy carbon electrode. Performance of the sensor was evaluated with respect to response time, sensitivity as well as operational stability. The enzyme electrode has a sensitivity of 13.5 muA mM(-1) with a detection limit of 1.0x10(-7) M H(2)O(2), and the sensor achieved 95% of the steady-state current within 20 s.     

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs⁻¹ in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75–400 μM and 0.5 μM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C.     

An improved biosensor for acetaldehyde determination using a bienzymatic strategy at poly(neutral red) modified carbon film electrodes

Improved biosensors for acetaldehyde determination have been developed using a bienzymatic strategy, based on a mediator-modified carbon film electrode and co-immobilisation of NADH oxidase and aldehyde dehydrogenase. Modification of the carbon film electrode with poly(neutral red) mediator resulted in a sensitive, low-cost and reliable NADH detector. Immobilisation of the enzymes was performed using encapsulation in a sol-gel matrix or cross-linking with glutaraldehyde. The bienzymatic biosensors were characterized by studying the influence of pH, applied potential and co-factors. The sol-gel and glutaraldehyde biosensors showed a linear response up to 60 μM and 100 μM, respectively, with detection limits of 2.6 μM and 3.3 μM and sensitivities were 1.7 μA mM⁻¹ and 5.6 μA mM⁻¹. The optimised biosensors showed good stability and good selectivity and have been tested for application for the determination of acetaldehyde in natural samples such as wine.     

An amperometric biosensor for glucose based on electrodeposited redox polymer/glucose oxidase film on a gold electrode.

In this paper, we described a glucose biosensor based on the co-electrodeposition of a poly(vinylimidazole) complex of [Os(bpy)2Cl](+/2+) (PVI-Os) and glucose oxidase (GOX) on a gold electrode surface. The one-step co-electrodeposition method provided a better control on the sensor construction, especially when it was applied to microsensor construction. The modified electrode exhibited the classical features of a kinetically fast redox couple bound to an electrode surface and the redox potential of the redox polymer/enzyme film was 0.14 V (vs. SCE). For a scan rate of up to 200 mV s(-1), the peak-to-peak potential separation was less than 25 mV. In the presence of glucose, a typical catalytic oxidation current was observed, which reached a plateau at 0.25 V (vs. SCE). Under the optimal experimental conditions, the steady-state electrooxidation current measured at 0.30 V (vs. SCE) was linear to the glucose concentration in the range of 0-30 mM. Successful attempts were made in blood sample analysis.     

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.     

Polypyrrole/multiwalled carbon nanotubes‐based biosensor for cholesterol estimation

The nanocomposite electrode comprising of polypyrrole (PPY) and carboxy functionalized multiwalled carbon nanotubes (MWCNT) has been electrochemically fabricated onto indium–tin–oxide (ITO) electrode using p‐toluene sulfonic acid (PTS). Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been immobilized onto this PPY– MWCNT/ITO nanocomposite electrode using N‐ethyl‐N‐(3‐dimethylaminopropyl) carbodiimide and N‐hydroxy succinimide chemistry for estimation of esterified cholesterol. The ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode has been characterized using Fourier transform infrared spectroscopy, electrochemical techniques, and scanning electron microscope. This ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode has a response time of about 9 s, linearity of 4 × 10^?4 to 6.5 × 10^?3 M/l of cholesterol oleate concentration, K_m of 0.02 mM, and thermal stability of upto 45°C. This electrode exhibits improved biosensing characteristics compared with other total cholesterol electrodes reported in literature till date and can be used to estimate cholesterol in blood serum samples. Copyright © 2011 John Wiley & Sons, Ltd.     

An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres

A new kind of magnetic dextran microsphere (MDMS) with uniform shape and narrow diameter distribution has been prepared from magnetic iron nanoparticles and dextran. Horseradish peroxidase (HRP) was successfully immobilized on the surface of an MDMS-modified glassy-carbon electrode (GCE), and the immobilized HRP displayed excellent electrocatalytic activity in the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ). The effects of experimental variables such as the concentration of HQ, solution pH, and the working potential were investigated for optimum analytical performance. This biosensor had a fast response to H₂O₂ of less than 10 s and an excellent linear relationship was obtained in the concentration range 0.20 μmol L⁻¹–0.68 mmol L⁻¹, with a detection limit of 0.078 μmol L⁻¹ (S/N = 3) under the optimum conditions. The response showed Michaelis–Menten behavior at larger H₂O₂ concentrations, and the apparent Michaelis–Menten constant was estimated to be 1.38 mmol L⁻¹. Moreover, the selectivity, stability, and reproducibility of the biosensor were evaluated, with satisfactory results. Figure Amperometric response of the biosensor to successive additions of H₂O₂ and the plot of amperometric response vs. H₂O₂ concentration     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.