Covalent grafting tyrosinase and its application in phenolic compounds detection

A stepwise strategy is reported for the design of a meditor-free amperometric tyrosinase biosensor. It is based on the azide-alkyne click reaction and carbodiimide coupling. Firstly, azide-terminated alkane thiols monolayers were self-assembled on the Au electrode surface. Then, nitrophenyl groups were covalent attached to the self-assembled monolayers (SAMs) via the click reaction of copper(I)-catalyzed 1,3-dipolar cycloadditions of azide-alkyne. Finally, the nitrophenyl group terminated SAMs were converted to aminophenyl-terminated interface by electrochemical reduction, and tyrosinase was covalent immobilized onto the Au electrode via carbodiimide reaction. Based on the stepwise strategy, a meditor-free amperometric tyrosinase biosensor was farbricated, and it showed good electrocatalytic reduction ability toward phenol, pyrocatechol and m-Cresol. Their linear ranges were over the range of 0.2 to 15.0 μmol·L^?1, 0.2 to 73.0 μmol·L^?1, and 0.2 to 33.0 μmol·L^?1, respectively.     

Construction of a biointerface for glucose oxidase through diazonium chemistry and electrostatic self-assembly technique

In this study, a new procedure for the fabrication of biosensors was developed. The method is based on the covalent attachment of nitrophenyl groups to the electrode surface via diazonium salt reaction followed by their conversion to amine moieties through electrochemical reduction and electrostatic layer-by-layer (LbL) assembly technique. In this procedure, highly stable iron oxide (Fe₃O₄) nanoparticles (IONPs), chitosan (CHIt), GOx, and Nile blue (NB) were assembled on the surface of aminophenyl modified glassy carbon electrode (AP/GCE) by LbL assembly technique. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the interfaces. The surface coverage of the active GOx and Michaelis–Menten constant (K _M) of the immobilized GOx were Γ?=?3.38?×?10^?11 mol cm^?2 and 2.54 mM, respectively. The developed biosensor displayed a well-defined amperometric response for glucose determination with high sensitivity (8.07 μA mM^?1) and low limit of detection (LOD) of 19.0 μM. The proposed approach allows simple biointerface regeneration by increasing pH which causes disruption of the ionic interactions and release of the electrostatic attached layers. The biosensor can then be reconstructed again using fresh enzyme. Simple preparation, good chemical and mechanical stabilities, and easy surface renewal are remarkable advantages of the proposed biosensor fabrication procedure.     

An H2O2 Biosensor Based on Immobilization of Horseradish Peroxidase Labeled Nano‐Au in Silica Sol‐Gel/Alginate Composite Film

Abstract

A novel approach to assemble an H₂O₂ amperometric biosensor was introduced. The biosensor was constructed by entrapping horseradish peroxidase (HRP) labeled nano‐scaled particulate gold (nano‐Au) (HRP‐nano‐Au electrostatic composite) in a new silica sol‐gel/alginate hybrid film using glassy carbon electrode as based electrode. This suggested strategy fully merged the merits of sol‐gel derived inorganic‐organic composite film and the nano‐Au intermediator. The silica sol‐gel/alginate hybrid material can improve the properties of conventional sol‐gel material and effectively prevent cracking of film. The entrapment of HRP in the form of HRP‐nano‐Au can not only factually prevent the leaking of enzyme out of the film but also provide a favorable microenvironment for HRP. With hydroquinone as an electron mediator, the proposed HRP electrode exhibited good catalytic activity for the reduction of H₂O₂. The parameters affecting both the qualities of sol‐gel/alginate hybrid film and the biosensor response were optimized. The biosensor exhibited high sensitivity of 0.40 Al mol^?1 cm^?2 for H₂O₂ over a wide linear range of concentration from 1.22×10^?5 to 1.46×10^?3 mol L^?1, rapid response of <5 s and a detection limit of 0.61×10^?6 mol L^?1. The enzyme electrode has remarkable stability and retained 86% of its initial activity after 45 days of storage in 0.1 mol L^?1 Tris‐HCl buffer solutions at pH 7.     

Non-enzymatic amperometric sensor for hydrogen peroxide based on a biocomposite made from chitosan, hemoglobin, and silver nanoparticles

We report on a novel non-enzymatic sensor for hydrogen peroxide (HP) that is based on a biocomposite made up from chitosan (CS), hemoglobin (Hb), and silver nanoparticles (AgNPs). The AgNPs were prepared in the presence of CS and glucose in an ultrasonic bath, and CS is found to act as a stabilizing agent. They were then combined with Hb and CS to construct a carbon paste biosensor. The resulting electrode gave a well-defined redox couple for Hb, with a formal potential of about ?0.17?V (vs. SCE) at pH?6.86 and exhibited a remarkable electrocatalytic activity for the reduction of HP. The sensor was used to detect HP by flow injection analysis, and a linear response is obtained in the 0.08 to 250?μM concentration range. The detection limit is 0.05?μM (at S/N?=?3). These characteristics, along with its long-term stability make the sensor highly promising for the amperometric determination of HP.

One-step construction of an electrode modified with electrodeposited Au/SiO2 nanoparticles, and its application to the determination of NADH and ethanol

A new electrode was developed by one-step potentiostatic electrodeposition (at ?2.0 V for 20 s) of Au/SiO₂ nanoparticles on a glassy carbon electrode. The resulting electrode (nano-Au/SiO₂/GCE) was characterized by scanning electronic microscopy, X-ray photoelectron spectroscopy and electrochemical techniques. The electrochemical behavior of dihydronicotinamide adenine dinucleotide (NADH) at the nano-Au/SiO₂/GCE were thoroughly investigated. Compared to the unmodified electrode, the overpotential decreased by about 300 mV, and the current response significantly increased. These changes indicated that the modified electrode showed excellent catalytic activity in the oxidation of NADH. A linear relationship was obtained in the NADH concentration range from 1.0?×?10^?6 to 1.0?×?10^?4 mol?L^?1. In addition, amperometric sensing of ethanol at the nano-Au/SiO₂/GCE in combination with alcohol dehydrogenase and nicotinamide adenine dinucleotide was successfully demonstrated. A wide linear response was also found for ethanol in the range from 5.0?×?10^?5 to 1.0?×?10^?3 mol?L^?1 and 1.0?×?10^?3 to 1.0?×?10^?2 mol?L^?1, respectively. The method was successfully applied to determine ethanol in beer and biological samples.     

Amperometric Glucose Biosensor Based on Platinum Nanoparticles Combined Aligned Carbon Nanotubes Electrode

A sensitive amperometric glucose biosensor based on platinum nanoparticles (PtNPs) combined aligned carbon nanotubes (ACNTs) electrode was investigated. PtNPs which can enhance the electrocatalytic activity of the electrode for electrooxidating hydrogen peroxide by enzymatic reaction were electrocrystallized on 4‐aminobenzene monolayer‐grafted ACNTs electrode by potential‐step method. These PtNPs combined ACNTs' (PtNPs/ACNTs) surfaces were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The highly dispersed PtNPs on ACNTs can be obtained. The enzyme electrode exhibits excellent response performance to glucose with linear range from 1×10^?5–7×10^?3 mol L^?1 and fast response time within 5 s. Furthermore, this glucose biosensor also has good reproducibility. It is demonstrated that the PtNPs/ACNTs electrode with high electrocatalytic activity is a suitable basic electrode for preparing enzyme electrodes.     

An enzymatic glucose biosensor based on a glassy carbon electrode modified with manganese dioxide nanowires

A glassy carbon electrode was modified with β-manganese dioxide (β-MnO₂), and glucose oxidase (GOx) was immobilized on its surface. The β-MnO₂ nanowires were prepared by a hydrothermal method and characterized by scanning electron microscopy and powder X-ray diffraction. They were then dispersed in Nafion solution and cast on the glassy carbon electrode (GCE) to form an electrode modified with β-MnO₂ nanowires that exhibits improved sensitivity toward hydrogen peroxide. If GOx is immobilized in the surface, the β-MnO₂ acts as a mediator, and Nafion as a polymer backbone. The fabrication process was characterized by electrochemical impedance spectroscopy, and the sensor and its materials were characterized by cyclic voltammetry and amperometry. The biosensor enables amperometric detection of glucose with a sensitivity of 38.2 μA?·?mM^?1?·?cm^?2, and a response time of?<?5 s. This study also demonstrates the feasibility of realizing inexpensive, reliable, and high-performance biosensors using MnO₂ nanowires.

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

Integrated Microcentrifuge Carbon Entrapped Glucose Oxidase Poly (N-Isopropylacrylamide) (pNIPAm) Microgels for Glucose Amperometric Detection

This study demonstrates a miniaturized integrated glucose biosensor based on a carbon microbeads entrapped by glucose oxidase (GOx) immobilized on poly (N-isopropylacrylamide) (pNIPAm) microgels. Determined by the Lowry protein assay, the pNIPAm microgel possesses a high enzyme loading capacity of 31?mg/g. The pNIPAm GOx loaded on the microgel was found to maintain a high activity of approximately 0.140?U determined using the 4-aminoantipyrine colorimetric method. The integrated microelectrochemical cell was constructed using a microcentrifuge vial housing packed with (1:1, w/w) carbon entrapped by pNIPAm GOx microgels, which played the dual role of the microbioreactor and the working electrode. The microcentrifuge vial cover was used as a miniaturized reference electrode and an auxiliary electrode holder. The device can work as biosensor, effectively converting glucose to H₂O₂, with subsequent amperometric detection at an applied potential of ?0.4?V. The microelectrochemical biosensor was used to detect glucose in wide linear range from 30?µM to 8.0?mM, a low detection limit of 10?µM, a good linear regression coefficient (R²) of 0.994, and a calibration sensitivity of 0.0388?µA/mM. The surface coverage of active GOx, electron transfer rate constant (ks), and Michaelis–Menten constant (K_M^app) of the immobilized GOx were 4.0?×?10^?11?mol/cm², 5.4?s^?1, and 0.086?mM, respectively. To demonstrate the applicability and robustness of the biosensor for analysis of high sample matrix environment, glucose was analyzed in root beer. The microelectrochemical device was demonstrated for analysis of small sample (<50?µL), while affording high precision and fast signal measurement (≤5?s).     

Poly(2-amino-5-(4-pyridinyl)-1, 3, 4-thiadiazole) film modified electrode for the simultaneous determinations of dopamine, uric acid and nitrite

Poly(2-amino-5-(4-pyridinyl)-1,3,4-thiadiazole) (PAPT) modified glassy carbon electrode (GCE) was fabricated and used for the simultaneous determinations of dopamine (DA), uric acid (UA) and nitrite (NO₂ ^?) in 0.1 mol?L^?1 phosphate buffer solution (PBS, pH 5.0) by using cyclic voltammetry and differential pulse voltammetry (DPV) techniques. The results showed that the PAPT modified GCE (PAPT/GCE) not only exhibited electrocatalytic activities towards the oxidation of DA, UA and NO₂ ^? but also could resolve the overlapped voltammetric signals of DA, UA and NO₂ ^? at bare GCE into three strong and well-defined oxidation peaks with enhanced current responses. The peak potential separations are 130 mV for DA–UA and 380 mV for UA–NO₂ ^? using DPV, which are large enough for the simultaneous determinations of DA, UA and NO₂ ^?. Under the optimal conditions, the anodic peak currents were correspondent linearly to the concentrations of DA, UA and NO₂ ^? in the ranges of 0.95–380 μmol?L^?1, 2.0–1,000 μmol?L^?1 and 2.0–1,200 μmol?L^?1 for DA, UA and NO₂ ^?, respectively. The correlation coefficients were 0.9989, 0.9970 and 0.9968, and the detection limits were 0.2, 0.35 and 0.6 μmol?L^?1 for DA, UA and NO₂ ^?, respectively. In 0.1 mol?L^?1 PBS pH 5.0, the PAPT film exhibited good electrochemical activity, showing a surface-controlled electrode process with the apparent heterogeneous electron transfer rate constant (k _s) of 25.9 s^?1 and the charge–transfer coefficient (α) of 0.49, and thus displayed the features of an electrocatalyst. Due to its high sensitivity, good selectivity and stability, the modified electrode had been successfully applied to the determination of analytes in serum and urine samples.     

Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H₂O₂ needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H₂O₂ concentration in solution has on HRP activity. The H₂O₂ in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L^?1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10^?3 mol L^?1 glucose, and 0.1 mol L^?1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p‐phenylenediamine, 2‐aminophenol, o‐phenylenediamine, m‐phenylenediamine, 1‐naphthylamine, o‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.     

Acetylcholinesterase Immobilization on 3‐Mercaptopropionic Acid Self Assembled Monolayer for Determination of Pesticides

Studies on the immobilization of acetylcholinesterase onto a SAM gold electrode and the use of the fabricated biosensor for the determination of carbaryl and parathion are presented. The influence of pH, ionic strength, enzyme loading and concentration of glutaraldehyde on the response of the biosensor was investigated . The amperometric biosensor developed in this study provided linearity to parathion and carbaryl in the 2.0 a 30.0×10^?6 mol L^?1 concentration range. The detection limits under the optimum working conditions were found to be 9.3 μg L^?1 for parathion and 9.0 μg L^?1 for carbaryl. The enzyme electrode was found to be stable for 7 days.     

Glucose biosensor based on graphite electrodes modified with glucose oxidase and colloidal gold nanoparticles

The electrochemistry of glucose oxidase (GOx) immobilized on a graphite rod electrode modified by gold nanoparticles (Au-NPs) was studied. Two types of amperometric glucose sensors based on GOx immobilized and Au-NPs modified working electrode (Au-NPs/GOx/graphite and GOx/Au-NPs/graphite) were designed and tested in the presence and the absence of N-methylphenazonium methyl sulphate in different buffers. Results were compared to those obtained with similar electrodes not containing Au-NPs (GOx/graphite). This study shows that the application of Au-NPs increases the rate of mediated electron transfer. Major analytical characteristics of the amperometric biosensor based on GOx and 13 nm diameter Au-NPs were determined. The analytical signal was linearly related to glucose concentration in the range from 0.1 to 10 mmol L^?1. The detection limit for glucose was found within 0.1 mmol L^?1 and 0.08 mmol L^?1 and the relative standard deviation in the range of 0.1–100 mol L^?1 was 0.04–0.39%. The τ_1/2 of V _max characterizes the storage stability of sensors: this parameter for the developed GOx/graphite electrode was 49.3 days and for GOx/Au-NPs/graphite electrode was 19.5 days. The sensor might be suitable for determination of glucose in beverages and/or in food.     

Direct Electroanalytical Determination of Fluvastatin in a Pharmaceutical Dosage Form: Batch and Flow Analysis

Abstract

The reduction of luvastatin (FLV) at a hanging mercury-drop electrode (HMDE) was studied by square-wave adsorptive-stripping voltammetry (SWAdSV). FLV can be accumulated and reduced at the electrode, with a maximum peak current intensity at a potential of approximately ?1.26 V vs. AgCl/Ag, in an aqueous electrolyte solution of pH 5.25. The method shows linearity between peak current intensity and FLV concentration between 1.0 × 10^?8 and 2.7 × 10^?6 mol L^?1. Limits of detection (LOD) and quantification (LOQ) were found to be 9.9 × 10^?9 mol L^?1 and 3.3 × 10^?8 mol L^?1, respectively.

Furthermore, FLV oxidation at a glassy carbon electrode surface was used for its hydrodynamic monitoring by amperometric detection in a flow-injection system. The amperometric signal was linear with FLV concentration over the range 1.0 × 10^?6 to 1.0 × 10^?5 mol L^?1, with an LOD of 2.4 × 10^?7 mol L^?1 and an LOQ of 8.0 × 10^?7 mol L^?1. A sample rate of 50 injections per hour was achieved.

Both methods were validated and showed to be precise and accurate, being satisfactorily applied to the determination of FLV in a commercial pharmaceutical.     

Pure copper vs. mixed copper and palladium hexacyanoferrates for glucose biosensing applications

A glucose amperometric biosensor was developed. Glucose oxidase enzyme was immobilized by means of a Nafion membrane on glassy carbon modified with an electrochemically deposited mixed Cu and Pd hexacyanoferrate (CuPdHCF). According to the data provided by X-ray atomic spectroscopy measurements, this Cu- and Pd-based hexacyanoferrate is likely to be a mixture of single CuHCF and PdHCF pure phases. The biosensor performances were evaluated by recording the steady-state currents due to submillimolar additions of glucose to a potassium buffer solution (pH 5.5) and exploiting the electrocatalytic reduction of the enzymatically produced hydrogen peroxide. The CuPdHCF-based biosensor exhibited a sensitivity of 8.1?±?0.6 A M^?1 m^?2, a limit of detection of 1.4?×?10^?5 M, and a linear response range extending between 5?×?10^?5 and 4?×?10^?4 M, with a dynamic response range up to 4?×?10^?3 M glucose. Electrode sensitivity and signal stability resulted more satisfactory as compared to those of a CuHCF-based biosensor fabricated according to the same procedure. The selectivity was investigated through an interference study. The response to easily oxidizable species was found to be low enough to allow glucose determination in biological samples.     

Microbiosensor for Salicylate Based on Modified Carbon Fibre

ABSTRACT

A microbiosensor is proposed for a quick and easy amperometric determination of salicylate. The methodology involves the use of the enzyme salicylate hydroxylase (SH) to convert salicylate to catechol, which is then oxidised at the carbon fibre electrode. The covalent immobilisation of the enzyme onto a carbon fibre electrode via carbodiimide results in an amperometric biosensor with high sensitivity, low detection limit and good stability. The response of the biosensor is linearly proportional to the salicylate concentration between 1.0 10^?7 and 2.0 10^?6 mol L^?1, at an applied potential of 300 mV vs SCE, with a response time of 3.5 s and a detection limit of 3.3 10^?8 mol L^?1. The relative standard deviation for the determination was 4.1% for n=10. The biosensor was applied to determine salicylate in urine and pharmaceutical samples and compared to the reference method with a good correlation.     

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM^?1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Synthesis of flower-like cuo hierarchical nanostructures as an electrochemical platform for glucose sensing

Flower-like CuO hierarchical nanostructures were synthesized on copper foil substrate through a simple wet chemical route in alkaline media at room temperature. SEM images collected at different reaction times revealed the transformation of initially formed Cu(OH)₂ nanowires to flower-like CuO nanostructures. The hierarchical structure of the as-prepared CuO showed high electrocatalytic activity towards the oxidation of glucose making it a promising electrode material for the development of non-enzymatic glucose sensor. The amperometric sensor exhibited a wide linear response to glucose ranging from 4.5 × 10^?5 to 1.3 × 10^?3 mol L^?1 (R ² = 0.99317) at fixed potential of 0.3 V. The detection limit was 6.9 × 10^?6 mol L^?1 (LOD = 3σ/s) with a sensitivity of 1.71 μA μmol^?1 cm^?2. Moreover, the developed sensor offers a fast amperometric response, good selectivity and stability.