Synergic effect of multi-walled carbon nanotubes and gold nanoparticles towards immunosensing of ricin with carbon nanotube-gold nanoparticles-chitosan modified screen printed electrode

An amperometric immunosensor for the specific detection of Ricinus communis is reported. Screen printed electrodes (SPEs) were modified with gold nanoparticles (GNPs) loaded multiwalled carbon nanotubes (MWCNTs)-chitosan (Ch) film. The ratio of MWCNT and GNP was optimised to get best electrochemically active electrode. Sandwich immunoassay format was used for the immunosensing of ricin. The revealing antibodies tagged with the enzyme alkaline phosphatase (ALP) converts the substrate 1-naphthyl phosphate into 1-naphthol that was determined with the amperometric technique. The amperometric current obtained was correlated with the concentration of ricin. The prepared GNP-MWCNT-Ch-SPE showed high stability due to the Ch film, short response time with good reproducibility and increased shelf life of the electrodes immobilised with antibodies. The electrochemical activity of the electrode improved because of optimization of composition of CNTs and gold nanoparticles. Under the optimal conditions, the modified electrode showed a wide linear response to the concentration of ricin in the range of 2.5-25 ng mL(-1) with a limit of detection of 2.1 ng mL(-1) and with a relative standard deviation of 5.1% and storage life of 32 days.     

Determination of underivatized amino acids by high-performance liquid chromatography and electrochemical detection at an amino acid oxidase immobilized CuPtCI6 modified electrode

An amperometric sensor for amino acids based on the immobilization of amino acid oxidase on the surface of a CuPtCl(6)/GC is described. The amperometric current is due to the oxidation of H2O2 liberated during the enzyme reaction on the surface of the enzyme electrode. The electrode response characteristics as well as kinetic parameters have been evaluated. The enzyme electrode was characterized as an electrochemical biosensor, which was used as detector in high performance liquid chromatography (HPLC) for the determination of a mixture of amino acids with satisfactory results.     

An integrated bienzyme glucose oxidase-fructose dehydrogenase-tetrathiafulvalene-3-mercaptopropionic acid-gold electrode for the simultaneous determination of glucose and fructose

A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA-AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF-FDH-GOD-MPA-AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.     

Detection of zeptomolar concentrations of alkaline phosphatase based on a tyrosinase and horse-radish peroxidase bienzyme biosensor

A bienzyme biosensor based on tyrosinase and horse-radish peroxidase is described in a flow injection analysis and cyclic voltammetry for measurement of phenol. Tyrosinase and horse-radish peroxidase were immobilized on the surface of a glassy carbon electrode by bovine serum albumin and glutaric dialdehyde. Phenol was oxidized by tyrosinase and horse-radish peroxidase via catechol to o-quinone in the presence of oxygen and hydrogen peroxide. The o-quinone was reduced to produce catechol (the substrate recycling) on the electrode surface. The enhanced sensitivity of the bienzyme electrode to phenol was observed in the flow injection system comparing with tyrosinase and horse-radish peroxidase monoenzyme electrodes. The mechanisms for enhanced amperometric response to phenol of bienzyme electrode were discussed. The biosensor was used to detect alkaline phosphatase (ALP). A detection limit of 1.4×10⁻¹⁵ M ALP (140 zmol/100 μl) was obtained after 1 h incubation with phenyl phosphate.     

Hydrogen peroxide biosensor based on electrodeposition of zinc oxide nanoflowers onto carbon nanotubes film electrode

A new amperometric biosensor for hydrogen peroxide was developed based on adsorption of horseradish peroxidase at the glassy carbon electrode modified with zinc oxide nanoflowers produced by electrodeposition onto multi-walled carbon nanotubes （MWNTs） film. The morphology of the MWNTs/nano-ZnO electrode has been investigated by scanning electron microscopy （SEM）, and the electrochemical performance of the electrode has also been studied by amperometric method. The resulting electrode offered an excellent detection for hydrogen peroxide at -0.11 V with a linear response range of 9.9×10^-7 to 2.9×10^-3 mol/L with a correlation coefficient of 0.991, and response time 〈5 s. The biosensor displays rapid response and expanded linear response range, and excellent stability.     

Detection of phenolic compounds in flow systems based on tyrosinase-modified reticulated vitreous carbon electrodes

The fabrication and performance of a reticulated vitreous carbon (RVC)-based tyrosinase flow-through electrode, in which the enzyme was covalently immobilized, is reported. The bioelectrode was tested as an amperometric detector for phenolic compounds. Variables affecting the construction of the enzyme flow-through electrode such as the RVC chemical pretreatment procedure, the enzyme immobilization method in the RVC matrix, the enzyme loading and the pH value of the buffer solution used, were optimized by flow-injection with amperometric detection. A good immobilization of the enzyme in the RVC matrix, in spite of the hydrodynamic conditions, was found. The same tyrosinase-RVC electrode could be used with no significant loss of the amperometric response for around 20 days, and reproducible responses could be achieved with different electrodes constructed in the same manner. Moreover, the operational stability of the bioelectrode was tested under continuous monitorization conditions. Calibration plots by flow injection with amperometric detection at -0.20 V were obtained for phenol, 2,4-dimethylphenol; 3-chlorophenol; 4-chlorophenol; 4-chloro-3-methylphenol and 2-aminophenol, with detection limits ranging from 2 mug l(-1) (4-chloro-3-methylphenol) to 2 mg l(-1).     

Voltammetric behavior and ion chromatographic detection of nitrite at a dispersed platinum glassy carbon electrode

Cyclic voltammetry was used to investigate the electrochemical behavior of nitrite at a platinum-modified glassy carbon electrode (Pt-CME) in phosphate buffer (pH 4.5). Experiments in flow injection analysis and ion-chromatography (IC) were performed to characterize the electrode as an amperometric sensor for the determination of nitrite ions. The effects of several common interferences on the amperometric signal were estimated. The electrode stability, precision, limit of detection, and linear range were evaluated at a constant applied potential of 1.1 V. Calibration plots were linear from 0.03 μM to 0.5 mM with a slope of about 21 μA/mM and a correlation coefficient of 0.9987. The limit of detection (LOD) was 15 nM (0.7 ppb) in a 100 μL injection. The electrode response was sufficiently stable: over 6 h of operating time a 2.8% signal loss was observed. The determination of nitrite in milk and pear juices was achieved by IC and the relevant results were compared with those obtained with a standard spectrophotometric method.     

A novel two-enzyme amperometric electrode for lactose determination.

Coimmobilization of beta-galactosidase and glucose oxidase in a redox polymer, polyvinylferrocenium perchlorate (PVF+ ClO4-), led to the development of an enzyme electrode for the determination of lactose. The amperometric response of the electrode was measured at +0.70 V vs. SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the substrate and buffer concentrations as well as the pH on the electrode response were elucidated.     

Quantitative determinations of SiC and SiO2 in new ceramic materials by Fourier transform infrared spectroscopy

In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to concentrations of the mouse IgG down to 1 ng mL(-1), with a linear range of 3-200 ng mL(-1) (S.D.<2; n=3), and very low non-specific adsorption.     

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.     

Electrochemical oxidative determination of 4-nitrophenol based on a glassy carbon electrode modified with a hydroxyapatite nanopowder

A non-enzymatic amperometric glucose is reported that is based on an glassy carbon electrode modified with a Cu-CuO nanowire (NW) composite. The morphology and the composition of the nanowire were characterized by scanning electron microscopy and X-ray diffraction, respectively. The modified electrode efficiently catalyzes the oxidation of glucose at less-positive potential (0.30 V) in 0.10 M NaOH solution in the absence of any enzymes or redox mediators. The sensor was successfully used for the amperometric sensing of glucose. Linear response was obtained over the concentration range from 0.1 to 12 mM. The common interfering agents ascorbic acid and uric acid do not interfere with the determination of glucose. The modified electrode features high sensitivity, low working potential, excellent stability, and fast amperometric sensing of glucose. Thus it is promising for the future development of non-enzymatic glucose sensors.     

Poly[Ni(II)Protoporphyrin IX] Modified Electrode for Amperometric Detection of Acetylcholine (Ach) and Choline (Ch)

Abstract

Poly[Ni(II)Protoporphyrin IX] (pNiPP) modified electrode can be used as an alternative method for direct amperometric detection of Acetylcholine (Ach) and Choline (Ch). This response is highly dependent on the electrode potential, that must be higher than +0.4 V versus Ag/AgCl with a maximum at +0.6 V.

Other amino derivatives such as ethanolamine and tetrabutyl ammonium hydroxide did not show amperometric response in the same experimental conditions. On the contrary, KCl shows an increasing amperometric signal in flow injection for a concentration range of 10^?3 to 1 M, indicating certain sensitivity of membrane to Cl^? ions. The lowest detection limit corresponds to the highest cationic volume. The response for acetylcholine is slightly higher than for choline and is almost completely non‐linear for both substrates. The proposed model for the response is Y=(A×X)/(B+X) according to AICc Akaike method.

Choline, in a complex solution, was analyzed by HPLC with the chemically modified electrode as detector.     

Microdrop analysis of a bead-based immunoassay

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.     

Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes

A simple and automated methodology for a sensitive electrochemical detection of enzyme immunoassays that employ alkaline phosphatase (AP) as label has been developed. A flow injection system with programmable pump, valve and cell functions, amperometric detection of indigo and screen-printed electrodes (SPEs) are responsible for the advantages of this methodology. Amperometric detection at a low potential of indigo, the product of the enzymatic hydrolysis of the substrate 3-indoxyl phosphate (IP), is combined with a flow injection system. This incorporates in the flow cell a disposable screen-printed board provided with a graphite working electrode. No electrode pretreatment is necessary to obtain reproducible signals. The system was applied to the determination by an enzyme-linked immunosorbent assays (ELISA) of pneumolysin (PLY), a toxin related to respiratory infections. Linear calibration curves for low and high concentration ranges were obtained. These were also performed in a proteic matrix and linearity was also obtained.     

Amperometric microcells for alkaline phosphatase assay

To develop simple electrochemical immunoassays, a screen printed amperometric microcell with graphite working and Ag/AgCl reference electrodes was tested for the determination of alkaline phosphatase enzyme (ALP) and anti-humanIgG conjugated ALP (alpha-hIgG-ALP) activity in 5-10 microl samples. To ensure reproducible, steady state conditions, the working electrode surface was coated with mass-transport controlling hydrogel layer. The kinetic response curves of the hydrogel coated electrodes were linear. In addition, the hydrogel layer reduced the nonspecific adsorption of the alpha-hIgG-ALP conjugate on the working electrode surface. The measurements were made in the range of 2 divided by 4000 mU ml(-1) enzyme activities using ascorbic acid 2-phosphate (AAP) as the enzyme substrate. AAP is commercially available, non-toxic and has excellent stability. The sensitivity of the determinations was about 71% of the sensitivity which could be achieved using p-aminophenylphosphate (PAPP), a not easily accessible and unstable enzyme substrate. The experimentally determined kinetic parameters of the ALP enzyme catalyzed reactions were the same with the bare and hydrogel layer coated electrodes.     

Determination of underivatized amino acids by high-performance liquid chromatography and electrochemical detection at an amino acid oxidase immobilized CuPtCl6 modified electrode

An amperometric sensor for amino acids based on the immobilization of amino acid oxidase on the surface of a CuPtCl₆/GC is described. The amperometric current is due to the oxidation of H₂O₂ liberated during the enzyme reaction on the surface of the enzyme electrode. The electrode response characteristics as well as kinetic parameters have been evaluated. The enzyme electrode was characterized as an electrochemical biosensor, which was used as detector in high performance liquid chromatography (HPLC) for the determination of a mixture of amino acids with satisfactory results. Received: 31 Jaunary 2000 / Revised: 31 March 2000 / Accepted: 3 April 2000     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

Amperometric selective biosensing of dimethyl- and diethyldithiocarbamates based on inhibition processes in a medium of reversed micelles

An amperometric tyrosinase electrode has been used for biosensing of dimethyl- and diethyldithiocarbamates based on the inhibition effects of these substances on the catalytic activity of the enzyme. A working medium consisting of reversed micelles, and phenol as the substrate has been used. The tyrosinase electrode was constructed by direct adsorption of the enzyme on the surface of a graphite-disk electrode. Reversible inhibition processes are shown to be involved for ziram, diram and zinc diethyldithiocarbamate. Following a simple regeneration of the enzyme electrode, an acceptable reproducibility for the measurements of the inhibition response was obtained. Experimental variables, such as temperature, phenol concentration and the presence of chloroform, affecting the inhibition processes, were optimized. The type of enzyme inactivation for each inhibitor tested was studied, and the inhibition constants were calculated. Detection limits of 0.074, 1.3 and 1.7 μmol l⁻¹ were achieved for ziram, diram and zinc diethyldithiocarbamate, respectively. Other carbamates belonging to families different from dimethyl- and diethyldithiocarbamates showed no amperometric response at the tyrosinase electrode, except for pyrimidine-derivative carbamates. The developed analytical methodology was applied to determine ziram in spiked apple samples.     

Catalytic oxidation of thiols at preheated glassy carbon electrode modified with abrasive immobilization of multiwall carbon nanotubes: applications to amperometric detection of thiocytosine, l-cysteine and glutathione

The performance of preheated glassy carbon electrode modified with carbon nanotubes is described. First glassy carbon electrode is heated for 5 min at 50 °C, then abrasive immobilization of multiwall carbon nanotubes on a preheated glassy carbon electrode was achieved by gentle rubbing of electrode surface on a filter paper supporting carbon nanotubes. Carbon nanotubes (CNTs)-modified glassy carbon electrodes exhibit strong and stable electrocatalytic response toward thiols oxidation in wide pH range. These properties permit an important decrease in over voltage for the oxidation of thiocytosine, glutathione and l-cysteine, as well as a dramatic increase in the peak currents in comparison with bare glassy carbon electrode. Furthermore, the thiols amperometric response of the coated electrodes is extremely stable, with more than 95% of the initial activity after 30 min stirring of 0.1 mM thiols. The electrocatalytic behavior is further exploited as a sensitive detection scheme for thiols detection by hydrodynamic amperometry. The substantial decrease in the overvoltage of the thiols oxidation associated with a stable amperometric response and antifouling properties of nanotubes films allow the development of highly sensitive thiols sensor without using any redox mediator. Such ability of carbon nanotubes to promote the thiols electron transfer reaction, short response time (5 s) and long-term stability, low detection limit, extended linear concentration range, high sensitivity suggest great promise for thiols amperometric sensors and detector for chromatographic analysis of thiol derivatives.     

Use of a Boron-Doped Diamond Electrode for Amperometric Assay of Bromide and Iodide Ions

The electrochemical assay of bromide and iodide ions at boron-doped diamond (BDD) electrode was investigated by cyclic voltammetry (CV) and chronoamperometry (CA). Comparison experiments were carried out using a glassy carbon (GC) electrode. The BDD electrode exhibited well-resolved and irreversible reduction voltammograms, while the GC electrode provided only an ill-defined response. Cyclic voltammetric signals at BDD electrode for 10 mM Br⁻ and I⁻ were observed at 561 and 125 mV vs. SCE; the values shifted negatively for 228.7 and 187.5 mV, respectively, compared to those at GC electrode. It was also found that the peak current of Br⁻ and I⁻ was in direct proportion to the scan rate, which is indicative of a surface confined reduction process. Sensitive amperometric responses for Br⁻ and I⁻ were obtained covering the linear ranges 0.666 μM–1 mM and 13.3 nM–1 mM, respectively, and their detection limits were 0.53 μM and 1.67 nM, respectively, under the optimum pH and applied potential. The amperometric response was very reproducible and stable with satisfactory recovery results. __________ From Zhurnal Analiticheskoi Khimii, Vol. 60, No. 11, 2005, pp. 1193–1199. Original English Text Copyright ? 2005 by Jing Wu, Xiaoli Li, Cunxi Lei, Xumei Wu, Guoli Shen, and Rugin Yu. This article was submitted by the authors in English.