Electrochemical studies on tetrathiafulvalene-tetracyanoquinodimethane modified acetylcholine/choline sensor

A sensor for acetylcholine/choline is described using a tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) electrode modified with acetylcholine esterase (AChE) and choline oxidase (ChO) enzymes. DC cyclic voltammetry and impedance measurements of the enzyme-modified TTF-TCNQ electrode that indicate the regeneration of choline oxidase at the electrode surface are reported. Effective electrochemical rate constants for the present enzyme electrode are calculated using the expressions derived by Albery et al. (1), which show the enzyme kinetics as the rate-limiting step. The values of the effective electrochemical rate constants are close to those reported by Hale and Wightman (2). The application of the sensor is described for the determination of fluorode ion and nicotine based on the reversible inhibition of AChE activity. The range of detection of fluoride ion and nicotine is found to be 5×10^-6 to 5×10^-4M.     

A Complete Electroenzymatic Choline Microprobe Based on Nanostructured Platinum Microelectrodes and an IrOx On‐probe Reference Electrode

A microelectrode array microprobe with a choline sensing site and an on‐probe reference electrode was constructed by depositing permselective polymer films and choline oxidase (ChOx) on one microelectrode, and iridium oxide (IrOx) on another, both of which were coated previously with a nanostructured Pt deposit. Scanning electron microscopy (SEM) of the nanostructured Pt layer revealed a unique pillar‐like, “nanograss” structure. Polyphenylenediamine (PPD) and Nafion were coated sequentially on the working (i. e. sensing) electrode surface to serve as the permselective polymer films. The microsensor exhibited high sensitivity to choline (123±13 μA mM^?1 cm^?2), low detection limit (3.2±0.8 μM), and fast response time (3–5 s). The choline sensor also was tested at physiological concentrations of electroactive interfering species common to brain extracellular fluid (i. e. ascorbic acid, dopamine, DOPA, and DOPAC) and showed excellent selectivity. Selectivity likely was aided by the relatively low potential of 0.35 V vs. IrOx that was made possible by the enhanced H₂O₂ electrooxidation activity of the underlying nanostructured Pt‐coated working electrode. Thus, Pt “nanograss” appears to be an excellent electrode surface modification for creation of high performance electroenzymatic biosensors.     

A Highly Sensitive Electrochemiluminescence Choline Biosensor Based on Poly(aniline‐luminol‐hemin) Nanocomposites

Poly(aniline‐luminol‐hemin) nanocomposites are prepared on an electrode surface through electropolymerization, and a highly sensitive electrochemiluminescence (ECL) biosensor for choline is developed based on the poly(aniline‐luminol‐hemin) nanocomposites and an enzyme catalyzed reaction of choline oxidase (CHOD). The obtained nanocomposites are characterized by scanning electron microscopy (SEM), atomic absorption spectrometry (AAS) and ECL. The results indicate that hemin can be incorporated into the poly(aniline‐luminol) nanocomposites using the facile electropolymerization method, and the poly(aniline‐luminol‐hemin) nanocomposites are rod shaped porous nanostructure. Moreover, the poly(aniline‐luminol‐hemin) nanocomposites exhibit higher ECL intensity than poly(aniline‐luminol) nanocomposites in alkaline media due to the catalytic effect of hemin on the ECL of the polymerized luminol and the electron transfer ability of hemin in the nanocomposites. CHOD is immobilized on the surface of the poly(aniline‐luminol‐hemin) nanocomposites modified electrode with glutaraldehyde, and the ECL biosensor based on poly(aniline‐luminol‐hemin)/CHOD exhibits a wider linear range for the choline detection. The enhanced ECL signals are linear with the logarithm of concentration of choline over the range of 1.0×10^?11～1.0×10^?7 mol L^?1 with a low detection limit of 1.2×10^?12 mol L^?1. Moreover, the proposed biosensor is successfully applied to the detection of choline in milk.     

Inhibition biosensor for determination of nicotine

An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in tobacco sample. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1,4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Choline oxidase was immobilized on the carbon paste electrode through cross-linking with bovine serum albumin (BSA) by glutaraldehyde. In the presence of choline oxidase and its endogenous cofactor flavin-ademine dinneleotide (FAD), choline was oxidized into betaine while FAD was reduced to FADH₂ which subsequently reduced 1,4-benzoquinone into hydroquinone. The later was finally oxidized at a relatively low potential of +450 mV versus saturated calomel electrode (SCE). Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to choline within 1.25×10⁻⁴ to 1.25×10⁻³ mol l⁻¹. The nicotine measurements were carried out in 0.067 mol l⁻¹phosphate buffer of pH 7.4 at an applied potential of 450 mV versus SCE. The electrode provided a linear response to nicotine over a concentration range of 2.0×10⁻⁵ to 9.2×10⁻⁴ mol l⁻¹ with a detection limit of 1.0×10⁻⁵ mol l⁻¹. The system was applied to the determination of nicotine in tobacco samples.     

Amperometric acetylcholine and choline sensors with immobilized enzymes

Acetylcholine and choline sensors are prepared by immbilizing enzymes on nylon net attached to a hydrogen peroxide snsor. Choline oxidase is used for the choline sensor; acetylcholinesterase choline oxidase are used for acetylcholine. The platinum/silver electrode pair is polarized at +0.6 V. The assembly is protected with an acetate cellulose membrane to enhance selectivity. The ranges measured are 1–10 μmol l^?1 in 0.1–1 ml of sample. The response times are 1–2 min.     

基于碳纳米管修饰电极的胆碱电化学发光生物传感器研制

在碳纳米管(CNTs)和K3Fe(CN)6修饰的铂电极上吸附固定胆碱氧化酶,以鲁米诺为发光试剂,研制了胆碱电化学发光(ECL)生物传感器.CNTs可有效提高电极表面的电荷传输能力、提高电极表面的生物相容性和对酶分子的固载能力;K3Fe(CN)6对酶活性具有激活作用,同时对H2O2增敏的鲁米诺ECL有增强作用,均有利于提...     

The unmediated choline sensor based on layered double hydroxides in hydrogen peroxide detection mode

In this work, we have developed a novel choline biosensor on the basis of immobilization of choline oxidase (ChOx) by the attractive materials layered double hydroxides (LDHs). Amperometric detection of choline was evaluated by holding the modified electrode at 0.5 V (vs. SCE). Due to the special properties of LDHs ([Zn₃-Al-Cl]), such as chemical inertness, high porosity, and swelling property, the [Zn₃-Al-Cl]/ChOx modified electrode exhibited an enhanced analytical performance. The biosensor provided a linear response to choline over a concentration range from 3.7 × 10⁻⁶ to 6.3 × 10⁻⁴ M with a low detection limit of 3 × 10⁻⁷ M based on S/N=3. The apparent Michaelis-Menten constant was calculated to be 1.38 mM. In addition, the interaction between ChOx and LDHs has also been investigated using FT-IR spectroscopy.     

Accelerating the electron transfer of choline oxidase using ionic-liquid/NH2-MWCNTs nano-composite

A nano-composite consisting of amine functionalized multi-walled carbon nanotubes and a room temperature ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate) was prepared and used for modification of glassy carbon electrode. By immobilizing choline oxidase (ChOx) on the modified electrode, the enzyme direct electron transfer has been achieved. The modified electrode exhibited a pair of well-defined cyclic voltammetric peaks at a formal potential of ?0.395?V versus Ag/AgCl in 0.2?M phosphate buffer solution at pH 7.0. This peak was characteristic of ChOx-FAD/FADH₂ redox couple. The electrochemical parameters such as charge transfer coefficient (??) and apparent heterogeneous electron transfer rate constant (k _s) were estimated to be 0.36 and 2.74?s^?1, respectively. When the enzyme electrode was examined for the detection of choline, a relatively high sensitivity (2.59???A?mM^?1) was obtained. Under the optimized experimental conditions, choline was detected in the concentration range from 6.9?×?10^?3 to 6.7?×?10^?1?mM with a detection limit of 2.7???M. The peak currents of ChOx were reasonably stable and retained 90% of its initial current after a period of 2?months.     

Biochemical sniffer with choline oxidase for measurement of choline vapour

An enzyme-based gas sensor (bio-sniffer) for choline vapour was fabricated and tested. The bio-sniffer was constructed using a Clark-type dissolved oxygen electrode and an enzyme (choline oxidase) immobilized membrane. This bioelectronic device measures choline concentration by the oxygen consumption induced by an enzyme reaction of choline oxidase. As the assessment of sensor performances, the calibration curves for choline in the liquid and gas phases were investigated, respectively. The responses of the bioelectronic device to choline solutions of various concentrations were related within a range from 5.00 to 700 μmol·L⁻¹ with a correlation coefficient of 0.999. On the other hand, the bio-sniffer for choline vapour was placed into a gas-measuring chamber and calibrated using gas detection tubes. The calibration range was 1.00–30.0 ppm (correlation coefficient: 0.996). The response time for choline vapour was approximately 15% slower than that of biosensor for choline solution. These results indicate that the bio-sniffer is useful to monitor colourless and odourless choline gas released from coating compositions including choline. Correspondence: Kohji Mitsubayashi, Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan     

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets with high oxidase yield for analytical glucose and choline detections

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets (PBBIns-Gs) was used to modify a gold electrode to form a three-dimensional PBBIns-Gs/Au electrode that was sensitive to hydrogen peroxide (H₂O₂) in the presence of acetic acid (AcOH). The positively charged nanostructured poly(N-butyl benzimidazole) (PBBIns) separated the graphene sheets (Gs) and kept them suspended in an aqueous solution. Additionally, graphene sheets (Gs) formed “diaphragms” that intercalated Gs, which separated PBBIns to prevent tight packing and enhanced the surface area. The PBBIns-Gs/Au electrode exhibited superior sensitivity toward H₂O₂ relative to the PBBIns-modified Au (PBBIns/Au) electrode. Furthermore, a high yield of glucose oxidase (GOD) on the PBBIns-Gs of 52.3 mg GOD per 1 mg PBBIns-Gs was obtained from the electrostatic attraction between the positively charged PBBIns-Gs and negatively charged GOD. The non-destructive immobilization of GOD on the surface of the PBBIns-Gs (GOD-PBBIns-Gs) retained 91.5% and 39.2% of bioactivity, respectively, relative to free GOD for the colloidal suspension of the GOD-PBBIns-Gs and its modified Au (GOD-PBBIns-Gs/Au) electrode. Based on advantages including a negative working potential, high sensitivity toward H₂O₂, and non-destructive immobilization, the proposed glucose biosensor based on an GOD-PBBIns-Gs/Au electrode exhibited a fast response time (5.6 s), broad detection range (10 μM to 10 mM), high sensitivity (143.5 μA mM⁻¹ cm⁻²) and selectivity, and excellent stability. Finally, a choline biosensor was developed by dipping a PBBIns-Gs/Au electrode into a choline oxidase (ChOx) solution for enzyme loading. The choline biosensor had a linear range of 0.1 μM to 0.83 mM, sensitivity of 494.9 μA mM⁻¹ cm⁻², and detection limit of 0.02 μM. The results of glucose and choline measurement indicate that the PBBIns-Gs/Au electrode provides a useful platform for the development of oxidase-based biosensors.     

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

The 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.     

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. Translated from Chinese Journal of Analytical Chemistry, 2006, 34(7): 910–914 (in Chinese)     

Amperometrische Biosensoren zur Bestimmung von Cholin und Acetylcholin

Summary Amperometric biosensors for the determination of choline and acetylcholine were constructed by coupling immobilized choline oxidase or choline oxidase and acetylcholinesterase membrane with an electrode. H₂O₂ which results from the oxidase reaction is anodically monitored. The sensors' response is linear in the concentration range of 1 to 300 mol/l choline and 1 to at least 660 mol/l acetylcholine. The sample frequency is 60 h^–1 with an imprecision of less than 2% for both choline and acetylcholine. The longtime-stability of the sensors was tested to be more than 22 days. The influence of the reversible inhibitor NaF on the bienzyme-electrode response was investigated.     

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

A 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 (K_s) of 0.97 s⁻¹, a diffusion coefficient value (D) of 4.50×10⁻⁶ cm² s⁻¹, an electroactive surface area (A_e) 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 I_max 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 %.     

Ruthenized screen-printed choline oxidase-based biosensors for measurement of anticholinesterase activity

Plastic disposable choline biosensors based on ruthenized-carbon screen-printed electrodes were prepared and their use for monitoring organophosphorus pesticides and carbamates is described. The presence of 0.5% ruthenium on activated carbon mixed to form a simple graphite-based ink for the working electrode surface increased the sensitivity towards hydrogen peroxide. The choline biosensor is based on such an electrode coupled with choline oxidase immobilized by adsorption and was used to detect the inhibition effect of carbamates and organophosphorus pesticides on acetylcholinesterase. With the optimized procedure described (pH, buffer composition, incubation time, substrate concentration), concentrations of pesticides (Carbofuran) as low as 1 nM could be detected.     

Calix[4]resorcinarene Ionophore in the Ion-Selective Electrodes with Plasticized Poly(vinylchloride) Membranes

Synthetic ionophore calix[4]resorcinarene was incorporated into the poly(vinyl chloride) (PVC) plasticized membrane of an ion-selective electrode (ISE). Calixarene interactions with quaternary ammonium and alkaline metals cations in the organic and water-organic media were carefully investigated by various authors. We studied the effects of choline and Na⁺ cations present in the working solution on the ionophore which was incorporated into a membrane phase. Due to the pH sensitivity of the ionophore, a study was undertaken in a wide range of pH values. The different characteristics of calix[4]resorcinarene interactions with these cations were discovered by the ISE with the PVC plasticized membrane containing this ionophore, which respond to both the choline and Na⁺ cations due to the variation of working solution pH.     

Determination of phosphatidylcitoline in serum with use of a choline-sensitive membrane electrode

A choline-sensitive membrane electrode made with potassium tetrakis(p-chlorophenyl) borate as the exchanger and o-nitrophenyl phenyl ether as membrane solvent in poly (vinyl chloride) was almost insensitive to the surfactant sodium deoxycholate. By using this electrode, choline ( 10^?5 M) formed by the enzymatic hydrolysis of phosphatidylcholine with phospholipase D in the presence of sodium deoxycholate as activator was successfully measured. The method was applied to the determination of phosphatidylcholine in serum.     

Choline biosensors based on a bi-electrocatalytic property of MnO2 nanoparticles modified electrodes to H2O2

An interesting mode of reactivity of MnO₂ nanoparticles modified electrode in the presence of H₂O₂ is reported. The MnO₂ nanoparticles modified electrodes show a bi-direction electrocatalytic ability toward the reduction/oxidation of H₂O₂. Based on this property, a choline biosensor was fabricated via a direct and facile electrochemical deposition of a biocomposite that was made of chitosan hydrogel, choline oxidase (ChOx) and MnO₂ nanoparticles onto a glassy carbon (GC) electrode. The biocomposite is homogeneous and easily prepared and provides a shelter for the enzyme to retain its bioactivity. The results of square wave voltammetry showed that the electrocatalytic reduction currents increased linearly with the increase of choline chloride concentration in the range of 1.0 × 10⁻⁵ –2.1 × 10⁻³ M and no obvious interference from ascorbic acid and uric acid was observed. Good reproducibility and stability were obtained. A possible reaction mechanism was proposed.     

Towards immunoassay in whole blood: separationless sandwich-type electrochemical immunoassay based on in-situ generation of the substrate of the labeling enzyme

An 18 minute separationless amperometric ELISA-type sandwich immunoassay, utilizing only stable reagents and having no washing steps is described. The platform for the assay was an electron conducting redox hydrogel on a vitreous carbon electrode. Avidin and choline oxidase were co-immobilized on the redox hydrogel and the biotinylated antibody to the antigen to be assayed (the biotin-labeled F(ab′)₂ fragment of goat anti-rabbit IgG) was bound to the gel. When the antigen (goat anti-rabbit IgG) was present in the analyzed solution, then its binding to the immobilized antibody made the electrode receptive to the complementary peroxidase-labeled antibody (horseradish peroxidase-labeled F(ab′)₂ fragment of goat anti-rabbit IgG). Its binding resulted in electrical contact (“wiring”) of the horseradish peroxidase label to the redox hydrogel, and converted the non-catalytic hydrogel into an electrocatalyst for the reduction of hydrogen peroxide to water at –0.07 V (SCE) and resulted in the flow of a cathodic current. The electroreduced hydrogen peroxide was not added to the solution and was therefore not significantly accessible to hydrogen peroxide decomposing agents such as catalase. Instead, it was generated within the coating of the electrode through reacting dissolved choline with oxygen. This reaction was catalyzed by the immobilized choline oxidase. The reaction centers of choline oxidase, unlike those of horseradish peroxidase, are not connected to the electrode by the redox hydrogel.     

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 H₂O₂ 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.