Choline Sensor Research Articles

Determination of trace cholinergic compounds and the evaluation of their relative receptor activity with the use of a receptor biosensor and an ion-selective choline electrode

A comparative assessment of the analytical capabilities of a tensoresistive biosensor based on muscarinic acetylcholine receptors and a potentiometric choline sensor was performed. It was found that the receptor biosensor is promising for the determination of ultratrace amounts of cholinergic agents with a detection limit of 10–7–10–10 M. The selectivity of the potentiometric choline sensor is equal to the selectivity of the receptor biosensor; however, the sensitivity of the former is lower by two orders of magnitude. Nevertheless, it can be successfully used for evaluating the relative receptor activity of cholinergic agents.

Transport of choline in rat brain slices

Enzyme-modified amperometric microsensors have been utilized in the investigation of acetylcholine and choline diffusion in solution and choline uptake and diffusion in rat brains. A small amount of the substance of interest was introduced by pressure injection and transport to the sensor was monitored. The apparent diffusion coefficients for acetylcholine and choline in agarose gel perfused with physiological solutions were determined to be 5.2±0.7×10 −6 cm 2/s and 6.1±0.8×10 −6 cm 2/s, respectively. Choline transport was monitored in two brain regions: the caudate and anterior hypothalamus. The transport time of choline in the caudate was concentration dependent, but was unaffected by the presence of a competitive, high-affinity uptake inhibitor, hemicholinium-3. The apparent diffusion coefficient ( D) and uptake rate ( k) for choline in the caudate and anterior hypothalamus were calculated using a model for point source diffusion coupled with first-order uptake kinetics. The effect of the sensors' response time on the measurements was removed by deconvolution. The D and k were 1.8±0.1×10 −6 cm 2/s and 2.0±0.1×10 −2 s −1 in the caudate and 1.9±0.1×10 −6 cm 2/s and 3.2±0.6×10 −2 s −1 in the anterior hypothalamus. The reduced diffusion coefficient determined in brain tissue compared to agar gel is consistent with the increased tortuosity of the brain microenvironment. A substance in brain tissue, presumably acetylcholinesterase, prevents the use of differential measurements of acetylcholine because choline sensors became sensitive to acetylcholine.

Enzyme modified amperometric sensors for choline and acetylcholine with tetrathiafulvalene tetracyanoquinodimethane as the electron-transfer mediator

To understand more about acetylcholine, an important neurotransmitter, and its precursor choline, acetylcholine and choline amperometric sensors have been fabricated. The performance of the sensors has been improved so that measurements of choline and acetylcholine have a high sensitivity and selectivity. The sensor is constructed from a bevelled and recessed carbon-fiber microelectrode with a radius of 16.5 μm. An organic conducting salt, tetrathiafulvalene tetracyanoquinodimethane (TTFTCNQ), is employed as an electron-transfer mediator. A recess at the tip of the microelectrode was partially filled with TTFTCNQ and covered with cross-linked choline oxidase and acetylcholine esterase. A thin Nafion polymer was also coated to increase the electrodes' mechanical stability. The resulting acetylcholine and choline sensors have a response time of 8.2 s and a linear range up to 50 and 100 μM respectively. Interference from the electroactive compounds ascorbic acid, uric acid, dopamine, 5-hydroxytryptamine (5-HT), and DOPAC was low at their maximal physiological concentrations. Data obtained in slices of rat brain indicate that these sensors are a useful and practical means of monitoring choline concentration changes in the rat brain microenvironment.

An Enzymatic Chemiluminescence Optrode for Choline Detection Under Flow Injection Conditions

Abstract A sensitive choline sensor was developed on the basis of a chemiluminometric bienzyme optrode. Choline oxidase (ChOx) and fungal peroxidase (FPOD) were covalently immobilized on a preactivated nylon membrane. The addition of polyethylenimine to the mixed enzyme solution before the immobilization combined with the crosslinking by gaseous glutardialdehyde improves both the long-term stability and the sensitivity. Choline can be detected in the range between 10−6 and 10−3 M.

A Clark-type oxidase enzyme-based amperometric microbiosensor for sensing glucose, galactose, or choline

Microbiosensors for glucose, galactose, or choline were constructed by attaching the respective oxidase enzyme to the tip of a Clark-type oxygen microelectrode. The enzyme is immobilized on the electrode tip in a polyacrylamide matrix and then coated with a polyurethane membrane. The analyte concentration in the sample controls the amount of oxygen consumed by the electrode and, hence, the biosensor's output. These microbiosensors had tip diameters of 15–40 μm, response times of 0.5–5s, and could detect as little as 2 μM of analyte. The linear range of response was dependent on the thickness of the polyurethane coating, and extended up to 10 mM for glucose and galactose biosensors. The glucose sensors were the most stable and could remain operational for up to 6 months. Galactose sensors remained operational for at least 1 month. Choline sensors remained operational for about 2 weeks and were generally less sensitive. The specific activity of the enzyme was a key determinant in the longevity and linearity of the biosensor response. In continuous operation tests, the glucose sensors were relatively drift-free and showed little deterioration of response over 72 h. These sensors exhibited little stirring dependence. As a result a glucose sensor accurately measured the glucose gradient in an unmixed, semisolid gel. These microsensors should prove to be versatile tools for measuring specific analytes in unstirred environments with a spatial resolution of 100 μm or less, and with extremely rapid response times.

Biosensors based on oxidases immobilized in various conducting polymers

The electrodeposited organic polymers polypyrrole, poly( N-methylpyrrole), poly( o-phenylenediamine) and polyaniline are compared as matrices for the immobilization of glucose oxidase in the preparation of amperometric glucose biosensors. Enzyme entrapment in the polymer layer is obtained by electrodeposition of polymers from solutions of monomers containing dissolved enzyme. For all examined sensors a useful and almost linear range of response to glucose is observed up to at least 20 mM of glucose. The best sensitivity of response is obtained for a glucose sensor made of poly( o-phenylenediamine) and polypyrrole. A linear response up to 20 mM glucose is also obtained in flow-injection measurements for a glucose/polypyrrole sensor. Poly( o-phenylenediamine) is also used for satisfactory immobilization of choline oxidase in the preparation of a choline sensor, whereas a lactate biosensor has been prepared by immobilization of lactate oxidase in polypyrrole.

Design of a choline sensor via direct coating of the transducer by photopolymerization of the sensing layer

Abstract A choline electrode has been designed based on the direct coating of the transducer tip through in situ polymerization of the sensing layer. Three types of photocrosslinkable polymer, poly(vinyl alcohol)-bearing styrylpyridinium groups (PVA-SbQ), were used. These polymers, characterized by a high stability, possess both a definite degree of polymerization and SbQ content. They were mixed with choline oxidase (ChOD) in a definite ratio PVA-SbQ/ChOD. A minute amount of this solution was directly deposited on a platinum electrode, dried, then photopolymerized. The degree of polymerization of the PVA backbone and the quantity of styrylpyridinium were found to influence the sensitivity of the resulting electrodes. The detection limit was as low as 2.5 × 10−9 M and the linear dynamic range extended up to about 10−4 M choline. Moreover, the response time was as short as ≈ 30 s as a result of the direct coating performed.

Use of an Electrodeposited Avidin Film for the Preparation of Lactate and Choline Sensors

Avidin, a glycoprotein found in chicken egg white, has a strong affinity to biotin. Its specific binding to biotin is practically irreversible, due to its extremely large binding constant, 1X1015 M1.13 For this reason, avidin and biotinylated enzymes have been widely used in analytical science4, biochemistry5 and histology6 fields. We recently reported that an avidin film can be prepared on the surface of a Pt electrode by an electrodeposition technique, and that biotinylated glucose oxidase is immobilized to an avidin film to construct highperformance glucose sensors.? 8 In this paper we describe the preparation and properties of lactate and Choline sensors based on electrodeposited avidin films and biotinylated enzymes, in order to establish the wide applicability of electrodeposited avidin films for enzyme sensor fabrication.

Direct biosensor coating with a photopolymerised microlayer through a controllable step-procedure

Biosensors are based on the intimate association of a transducer and of a sensing layer. The latter can be a preformed membrane further connected to the transducer, or a thin film directly deposited on its surface. As the stability is a key parameter to be considered, a polymer with high potentialities for this purpose was chosen to form a direct surface coating: a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). Choline oxidase was entrapped in this photo-cross-linkable gel for making an enzyme electrode through controllable steps. The influence of the ratio PVA-SbQ/enzyme was studied and the stability of the resulting modified electrodes was determined. After deposition of minute volumes (10–45 μl) including no more than one unit of choline oxidase and 0.3 mg of polymer, an efficient choline sensor was obtained. It exhibited a fast response time lower than 30 s, a low detection limit of 2.5 nM choline, a wide linear range extending up toca. 10–4 M and good stability, both operational and on storage. This method appears promising for making miniaturized biosensors.

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.

Micro-choline sensor for acetylcholinesterase determination

A micro-choline sensor was developed using a carbon fibre working electrode for the determination of acetylcholinesterase activity in rat serum samples. The mechanism is based on the measurement of hydrogen peroxide generated from choline which is produced by the enzymatic hydrolysis of acetylcholine. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzyme is entrapped in a PVA-SbQ photocross-linkable polymer [poly(vinyl alcohol) containing styrylpyridinium] and covered with Nafion (perfluorosulphonated membrane), which has permselectivity characteristics, so that the access of absorbate, a common interfering species, to the electrode surface is blocked. Using this sensor, the esterase activity was determined. The slope values showed a fast response time between 1 and 3 min and a high enzyme activity in the serum samples. The calibration graph for the choline sensor was linear between 0.05 and 5.0 mM. The operational and long-term stability of the sensor demonstrated good quality performance. The procedure for preparing this type of coated electrode indicates some attractive prospects for its use especially for the determination of organophosphorus pesticides in human serum.

Nafion‐coated carbon fiber for acetylcholine and choline sensors

AbstractA major improvement in the selectivity of carbon fiber microelectrodes for acetylcholine (Ach) and choline (Ch) as cationic amines is described. The electrodes are coated with Nafion, a perfluorosulfonated polymer. This coating is practically impermeable to ascorbic acid (AA) and anionic biogenic amine metabolites. Nafion‐coated fiber was characterized by cyclic voltammetry (CV). Selectivity and the dynamic range of these coated electrodes were evaluated using amperometric determination at a fixed potential of 1.2 V. The esterase activity was determined using choline sensor, and the relative slope values showed a fast response between 1 and 3 minutes. Calibration plots for Ach and choline showed a linearity range between 0.1 and 3 mM, with an increase of two orders of magnitude better than the previously used PVA‐SbQ membrane. No response was obtained between 0.1 and 0.3 mM of AA conc. The procedure, in preparing this type of coated electrodes, indicates some attractive perspectives for its use, especially for in vivo monitoring.

Anticholinesterase activity measurement by a choline biosensor: application in water analysis

A choline amperometric biosensor was assembled and used to measure the anticholinesterase activity due to compounds (which have the property to inhibit cholinesterase enzymes) present in water samples. This parameter can be used as a ‘toxicological index’, defined as the amount of compound which causes a certain percentage of cholinesterase inhibition equivalent to a known amount of a reference compound causing the same percentage inhibition. The organophosphorus insecticide Paraoxon, which has proved to be a strong inhibitor of cholinesterase enzymes, was chosen as the reference compound. The analysis was carried out by monitoring the decrease of cholinesterase activity in the presence of a pesticide and a substrate specific for the enzyme whose reaction produces choline. The decrease in choline production was measured by the choline sensor and correlated to the concentration of anticholinesterase compound present in the solution. Parameters such as buffer, pH, temperature and incubation time were optimized. The rate constant K i was calculated experimentally for Paraoxon and used in the anticholinesterase activity measurements at different fixed incubation times. The probe was calibrated with different standard solutions of Paraoxon. The effect of Paraoxon and heavy metals on the choline probe was evaluated. This probe was then used for the determination of anticholinesterase activity of some organophosphorus pesticides, and heavy metals in spiked waters. Samples were also analysed by liquid/liquid extraction and GC determination. Results seem to correlate with acute toxicity expressed as LD 50 (oral, rat). Analysis of water samples from different sources in central Italy were analysed for total anticholinesterase activity (TAA) and compared with a reference procedure.

Determination of organophosphorus insecticides with a choline electrochemical biosensor

Organophosphorus insecticides have been determined with an amperometric hydrogen peroxide based choline biosensor. This class of pesticides inhibits cholinesterase enzymes which in the presence of their substrates produce choline. The decrease in activity of these enzymes is monitored by the choline sensor and is correlated to the concentration of pesticide present in solution. Pesticides such as paraoxon, parathion, methyl paraoxon and methyl parathion have been coupled with acetylcholine and butyrylcholine esterases. Results showed that although the enzyme activity of acetylcholinesterase (AChE) was higher than butyrylcholinesterase (BuChE), the lower specificity of BuChE resulted in a higher enzyme inhibition. The detection limit was less than 1 ppb with an incubation time of 120 min and 2 ppb when the incubation time was 30 min. This method has been applied to the analysis of pesticides in water samples. Results, compared with those obtained with a different analytical procedure (liquid/liquid extraction and GLC determination), demonstrated that our method is a good analytical choice to measure the total anticholinesterase activity in water samples.

Rapid and sensitive discriminating determination of acetylcholinesterase activity in amniotic fluid with a choline sensor

A simple method for the separate determination of acetylcholinesterase and butyrylcholinesterase activities in amniotic fluid is reported. This determination is performed with an enzyme electrode involving an immobilized choline oxidase membrane associated with the amperometric detection of hydrogen peroxide. Acetylcholine or butyrylcholine, in the presence of samples containing acetylcholinesterase or butyrylcholinesterase are specifically hydrolyzed, the formation of choline being detected vs time by the sensor with no need for a selective inhibitor. The dynamic linear ranges for acetylcholinesterase and butyrylcholinesterase are respectively 100 μU to 10 mU and 30 μU to 3 mU per ml sample.

Determination of Organophosphorus and Carbamic Pesticides with a Choline and Acetylcholine Electrochemical Biosensor

Abstract Pesticides as paraoxon and aldicarb have been determined with an amperometric hydrogen peroxide based choline sensor. These pesticides inhibit the enzyme acetylcholinesterase which in presence of its substrate, acetylcholine, produces choline. When these pesticides are in presence of acetylcholinesterase, the activity of this enzyme decreases; this causes a decrease of choline production which is monitored by a choline sensor and correlated to the concentration of pesticide in solution. Two different procedures were followed: one with both the enzymes acetylcholinesterase and choline oxidase immobilized, the second one with the acetylcholinesterase in solution and the choline oxidase immobilized. Parameters as pH, buffer, enzyme concentration, substrate concentration and reaction and incubation times were optimized. Results showed that these compounds can be detected in the range 10 – 100 ppb. The use of the enzyme in solution gave the best results with a detection limit of 2 ppb pesticide.

Determination of serum cholinesterase activity and dibucaine numbers by an amperometric choline sensor

Serum cholinesterase activity and the dibucaine numbers have been determined by using a hydrogen peroxide electrode and the enzyme choline oxidase immobilized on a nylon net. The analysis procedure is extremely simple and very fast allowing 30 cholinesterase determinations per hour. Both cholinesterase activity and dibucaine number measurements could be performed in 5 min and by using serum samples of only 10 μl. When used in sera the probe showed no interference from electroactive compounds present in blood, and also showed good stability and reproducibility. These features make this sensor appropriate for continuous extracorporeal circuit blood monitoring of succinylcholine during surgery.

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.