Amperometric Choline Biosensor Research Articles

Choline Oxidase Based Composite ZrO2@AuNPs with Cu2O@MnO2 Platform for Signal Enhancing the Choline Biosensors

AbstractA novel metal composite material based on zirconium dioxide decorated gold nanoparticles (ZrO2@AuNPs), copper (I) oxide at manganese (IV) oxide (Cu2O@MnO2) and immobilized choline oxidase (ChOx) onto a glassy carbon electrode (GCE) (ChOx/Cu2O@MnO2‐ZrO2@AuNPs/GCE) has been developed for enhancing the electro‐catalytic property, sensitivity and stability of the amperometric choline biosensor. The ChOx/Cu2O@MnO2‐ZrO2@AuNPs/GCE displayed an excellent electrocatalytic response to the oxidation of the byproduct H2O2 from the choline catalyzed reaction, which exhibited a charge transfer rate constant (Ks) of 0.97 s−1, a diffusion coefficient value (D) of 4.50×10−6 cm2 s−1, an electroactive surface area (Ae) of 0.97 cm2 and a surface concentration (γ) of 0.54×10−8 mol cm−2. The modified electrode also provided a wide linear range of choline concentration from 0.5 to 1,000.0 μM with good sensitivity (97.4 μA cm−2 mM−1) and low detection limit (0.3 μM). The apparent Michaelis‐Menten constant was found to be 0.08 mM with Imax of 0.67 μA. This choline biosensor presented high repeatability (%RSD=2.9, n=5), excellent reproducibility (%RSD=2.9, n=5), long time of use (n=28 with %I>50.0 %) and good selectivity without interfering effects from possible electroactive species such as ascorbic acid, aspirin, amoxicillin, caffeine, dopamine, glucose, sucrose and uric acid. This optimal method was successfully applied for choline measurement in prepared human blood samples which demonstrated accurate and excellent reliability in the recovery range from 96.7 to 102.0 %.

Development of choline biosensor using toluidine blue O as mediator

A new choline oxidase (ChO) and toluidine blue O (TBO) based amperometric choline biosensor was reported in this article. An amperometric choline biosensor with immobilization of TBO (as a mediator), ChO onto polypyrrole–polyvinylsulphonate (PPy–PVS) film was accomplished on the surface of a platinum electrode. ChO was immobilized on PPy–PVS film by cross-linking with glutaraldehyde (GA). TBO was used as the mediator. Choline is oxidized to betaine and hydrogen peroxide in an oxygenated environment by ChO. Mediator reduced by reaction with hydrogen peroxide. The amperometric response was based upon the electrocatalytic properties of TBO. Optimum pH and temperature values were 7.0 and 30 °C, respectively. There was linearity between 1.0 × 10−8 and 2.0 × 10−8 M (R2 = 0.9805). The detection limit of the biosensor was 1.0 × 10−9 M and response time of the biosensor was 200 s. The storage stability and reproducibility of the biosensor were also investigated. Interfering effect of several interferants such as ascorbic acid, uric acid, alanine, dopamine, paracetamol, cysteine, and glucose on the choline biosensor was examined. The developed biosensor was tested in determinations of content in a synthetic blood sample.

Highly Sensitive Choline Oxidase Enzyme Inhibition Biosensor for Lead Ions Based on Multiwalled Carbon Nanotube Modified Glassy Carbon Electrodes

AbstractThe determination of lead ions by inhibition of choline oxidase enzyme has been evaluated for the first time using an amperometric choline biosensor. Choline oxidase (ChOx) was immobilized on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) through cross‐linking with glutaraldehyde. In the presence of ChOx, choline was enzymatically oxidized into betaine at –0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. The experimental conditions were optimised regarding applied potential, buffer pH, enzyme and substrate concentration and incubation time. Under the best conditions for measurement of the lowest concentrations of lead ions, the ChOx/MWCNT/GCE gave a linear response from 0.1 to 1.0 nM Pb2+ and a detection limit of 0.04 nM. The inhibition of ChOx by lead ions was also studied by electrochemical impedance spectroscopy, but had a narrower linear response range and low sensitivity. The inhibition biosensor exhibited high selectivity towards lead ions and was successfully applied to their determination in tap water samples.

A novel sensitive amperometric choline biosensor based on multiwalled carbon nanotubes and gold nanoparticles

A novel amperometric biosensor for choline determination has been developed, exploiting the electrocatalytic properties of multiwalled carbon nanotubes (MWCNT) and gold nanoparticles (GNP). Chitosan (Chit), a natural biocompatible polymer, was used to disperse CNT, then Chit-MWCNT was dropped on the surface of a glassy carbon electrode (GCE), followed by GNP; finally, choline oxidase (ChOx) was immobilized by glutaraldehyde crosslinking. The ChOx/(GNP)4/MWCNT/GCE exhibited linear response to choline from 3 to 120µM, the sensitivity was 204µAcm−2mM−1 and the detection limit was 0.6μM. The biosensor exhibited good intra and inter-electrode precision, and excellent selectivity and stability. Electrochemical impedance spectroscopy (EIS) was also used to measure choline at 0.0V and this is the first report on choline determination by EIS. Successful measurement in milk samples was performed.

A highly sensitive choline biosensor based on bamboo-like multiwall carbon nanotubes/ionic liquid/Prussian blue nanocomposite

A highly sensitive amperometric choline biosensor was fabricated using bamboo-like multi-walled carbon nanotubes (BCNTs)/ionic liquid (IL)/Prussian blue (PB) nanocomposite modified glassy carbon (GC) electrode. The PB nanoparticles were first electrodeposited on the surface of a BCNTs/IL/GC electrode. The Ni2+ ions were then electrochemically incorporated into the PB structure to improve its stability in mild alkaline media. Choline oxidase was next immobilized on the modified electrode using a cross-linking method. Amperometric measurements of choline were performed at −0.05V vs. Ag/AgCl in 0.05M phosphate buffer, pH 7.4. The choline biosensor exhibited a high sensitivity of 345.4μAmM−1cm−2 with a detection limit of 4.5×10−7M. The amperometric response was linear from 4.5×10−7 to 1.0×10−4M. The proposed biosensor displayed a short response time of 2s, low Km value of 9.7×10−5M, good storage stability and high selectivity.

An amperometric biosensor for choline determination prepared from choline oxidase immobilized in polypyrrole-polyvinylsulfonate film

In this paper, a novel amperometric choline biosensor with immobilization of choline oxidase on electrochemically polymerized polypyrrole-polyvinylsulphonate (PPy-PVS) film has been accomplished via the entrapment technique. The effects of pH and temperature were investigated and optimum parameters were found to be 9.0 and 60 °C, respectively. There are two linear parts in the region between 1.0 × 10 (-7) - 1.0 × 10 (-6)M (R(2) = 0.997) and 1.0 × 10 (-5) - 1.0 × 10 (-3) M (R(2) = 0.986). The storage stability and operation stability of the enzyme electrode were also studied.

Indirect electrocatalytic determination of choline by monitoring hydrogen peroxide at the choline oxidase-prussian blue modified iron phosphate nanostructures

Choline, as a marker of cholinergic activity in brain tissue, is very important in biological and clinical analysis, especially in the clinical detection of the neurodegenerative disorders disease. This work presents an electrochemical approach for the detection of choline based on prussian blue modified iron phosphate nanostructures (PB–FePO4). The obtained nanostructures showed a good catalysis toward the electroreduction of H2O2, and an amperometric choline biosensor was developed by immobilizing choline oxidase on the PB–FePO4 nanostructures. The biosensor exhibited a rapid response (ca. 2s), low detection limit (0.4±0.05μM), wide linear range (2μM to 3.2mM), high sensitivity (∼75.2μAmM−1cm−2), as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid and 4-acetamidophenol did not cause obvious interference due to the low detection potential (−0.05V versus saturated calomel electrode). This nanostructure could be used as a promise platform for the construction of other oxidase-based biosensors.

Amperometric Choline Biosensor Based on Dispersion of Multi-walled Carbon Nanotubes in Poly(diallyldimethylammonium chloride) via Layer-by-Layer Assembly Technique

Amperometric Choline Biosensor Based on Dispersion of Multi-walled Carbon Nanotubes in Poly(diallyldimethylammonium chloride) via Layer-by-Layer Assembly Technique

Amperometric biosensors based on gold nanoparticles-decorated multiwalled carbon nanotubes-poly(diallyldimethylammonium chloride) biocomposite for the determination of choline

A novel amperometric choline biosensor based on the nanocomposite film composed of choline oxidase (ChOx), multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and poly(diallyldimethylammonium chloride) (PDDA) was developed for the specific detection of choline. GNp-decorated MWCNTs (MWCNTs–GNp) was synthesized by a classical chemical method, and GNp was attached on MWCNTs through the specific interaction between –SH and Au. The enzyme ChOx would be attached to the MWCNTs–GNp matrix and also be adsorbed electrostatically with PDDA which was employed not only as a dispersant but also a binder material. The microscopic structure and composition of the synthesized MWCNTs–GNp were characterized by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX), and properties of the resulting choline biosensors were monitored by electrochemical measurements. Taking the sensitivity and selectivity into consideration, 0.35 V versus Ag/AgCl was selected to detect choline. The resulting biosensor exhibited a wide linear range of 0.001–0.5 mM choline, with a remarkable sensitivity of 12.97 μA/mM, a detection limit of 0.3 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it showed good reproducibility, anti-interferant ability and long-term stability. This work presented a feasible approach for further research in biosensing and other surface functionalizing.

Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing

Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8 ± 1.8° to 45.3 ± 1.4°. A quartz crystal microbalance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm −2, and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27 nA response current by 0.238 μg immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosensors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested.

Highly sensitive choline biosensor based on carbon nanotube-modified Pt electrode combined with sol-gel immobilization

A novel amperometric choline biosensor has been fabricated with choline oxidase (ChOx) immobilized by the sol-gel method on the surface of multi-walled carbon nanotubes (MWCNT) modified platinum electrode to improve the sensitivity and the anti-interferential property of the sensor. By analyzing the electrocatalytic activity of the modified electrode by MWCNT, it was found that MWCNT could not only improve the current response to H2O2 but also decrease the electrocatalytic potential. The effects of experimental variables such as the buffer solutions, pH and the amount of loading enzyme were investigated for the optimum analytical performance. This sensor shows sensitive determination of choline with a linear range from 5.0 × 10−6 to 1.0 × 10−4 mol/L when the operating pH and potential are 7.2 and 0.15 V, respectively. The detection limit of choline was 5.0 × 10−7 mol/L. Selectivity for choline was 9.48 μA·(mmol/L)−1. The biosensor exhibits excellent anti-interferential property and good stability, retaining 85% of its original current value even after a month. It has been applied to the determination of choline in human serum.

Construction of a choline biosensor through enzyme immobilization on a poly(2‐hydroxyethyl methacrylate)‐grafted Teflon film

AbstractAn amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2‐hydroxyethyl methacrylate) (PHEMA)‐grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by γ irradiation. PHEMA‐grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme‐immobilized film, the film was stretched directly on the electrode. The PHEMA‐grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 ± 0.068 U mg−1) for use in the choline biosensor. The linear working range of this biosensor was found to be 52–348 μM, with a 40 ± 5 μM minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6- O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10 −7 M), short response time (within 10 s), high sensitivity (88.6 μA mM −1 cm −2) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H 2O 2 by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis–Menten constant was found to be (0.083 ± 0.001) ×10 −3 M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.

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

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

Amperometric Choline Sensor with Enzyme Immobilized by Gamma-Irradiation in a Biocompatible Membrane

Abstract An amperometric choline biosensor was constructed using choline oxidase immobilized on poly(2-hydroxyethylmethacrylate) membranes obtained by gamma radiation-induced polymerization at low temperature. The measurements were carried out by Clark-type oxygen or hydrogen peroxide electrodes. Calibration curves were linear in the 10-200 umol · 1−1 range for the oxygen probe and 5-250 umol · 1−1 for the H2O2-based probe. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the sensor are summarized. The immobilized enzyme membranes stored in glycine buffer or in a dry state were very stable and no significant decrease in the electrode response was observed after three months. The biosensor was employed also to analyse a choline-containing pharmaceutical product and the results were compared to those obtained by enzymatic-spectrophotometric detection.