Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.     

Microbiosensor for acetylcholine and choline based on electropolymerization/sol–gel derived composite membrane

Amperometric enzyme biosensors for the determination of acetylcholine (ACh) and choline (Ch) have been described. For the fabrication of the biosensors, N-acetylaniline (nAN) was first electropolymerized on a Pt electrode surface to be served as a permselective layer to reject interferences. Bovine serum albumin (BSA) and choline oxidase (CHOD) were co-immobilized in a zinc oxide (ZnO) sol–gel membrane on the above modified Pt electrode for a Ch sensor, or CHOD, acetylcholinesterase (AChE) and BSA immobilized together for an ACh/Ch sensor. The poly (N-acetylaniline) (pnAN) film was the first time used for an ACh/Ch sensor and found to have excellent anti-interference ability, and the BSA in the sol–gel can improve the stability and activity of the enzymes. Amperometric detection of ACh and Ch were realized at an applied potential of +0.6 V versus SCE. The resulting sensors were characterized by fast response, expanded linear range and low interference from endogenous electroactive species. Temperature and pH dependence and stability of the sensor were investigated. The optimal ACh/Ch sensor gave a linear response range of 1.0 × 10⁻⁶ to 1.5 × 10⁻³ M to ACh with a detection limit (S/N = 3) of 6.0 × 10⁻⁷ M and a linear response range up to 1.6 × 10⁻³ M to Ch with a detection limit of 5.0 × 10⁻⁷ M. The biosensor demonstrated a 95% response within less than 10 s.     

乙酰胆碱和胆碱化学发光生物传感器的研究

将具有分子识别功能的乙酰胆碱酯酶和胆碱氧化酶及进行换能反应的luminol和Cu^2^+分别固定在多孔玻璃和离子交换剂的柱中,组成流动注射式胆碱和乙酰胆碱化学发光传感器,让传感器分子识别反应和换能反应在各自最佳pH值下进行。这一新型生物传感器优化了发光量子产率,避免了在传感元件上直接发光所产生的散射干扰。测定乙酰胆碱和胆碱的线性范围达到1～1000pmol,检测限为500fmol,每次测定时间为2min,寿命为6个月,已用于鼠脑及人血清中乙酰胆碱和胆碱的测定。     

Determination of choline and acetylcholine levels in rat brain regions by liquid chromatography with electrochemical detection

Regional choline (Ch) and acetylcholine (ACh) in rat brain were clearly determined by high-performance liquid chromatography with electrochemical detection. The method is based on that of Potter et al.: the hydrogen peroxide that is enzymatically produced from both compounds is measured and a successful improvement of the method, particularly for purification, is described. Recoveries were 96.1 +/- 1.4% for Ch and 95.6 +/- 2.2% for ACh and amounts as low as 10 pmol could be determined. Prior to measuring the compounds, a newly developed magnetic field microwave instrument (10 kW) was utilized for the rapid inactivation of brain enzymes. The levels of Ch and ACh in brain regions were compared with those reported elsewhere.     

高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

 建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。     

Simultaneous determination of choline and acetylcholine based on a trienzyme chemiluminometric biosensor in a single line flow injection system.

A detector for the simultaneous determination of choline (Ch) and acetylcholine (ACh) based on a sensitive trienzyme chemiluminometric biosensor in a single line flow injection (FI) system is described. Immobilized choline oxidase (ChOx), immobilized peroxidase (POx), immobilized acetylcholinesterase, and coimmobilized ChOx/POx were packed, in turn, in a transparent ETFE tube (1 mm i.d., 75 cm) and the tube was placed in front of a photomultipier tube as a flow cell. Two-peak response was obtained by one injection of the sample solution. The first and second peaks were dependent on the concentrations of Ch and ACh, respectively. The influence of some experimental parameters such as flow rate, amounts of immobilized enzymes on the behavior of the sensor was studied in order to optimize the sensitivity, sample throughput and resolution. Calibration curves were linear at 1 - 1000 nM for Ch and 3 - 3000 nM for ACh. The sample throughput was 25/h without carryover. The FI system was applied to the simultaneous determination of Ch and ACh in rabbit brain tissue homogenates.     

Assays for serum cholinesterase activity by capillary electrophoresis and an amperometric flow injection choline biosensor

A capillary electrophoresis method and a durable choline biosensor were developed for measuring serum cholinesterase (EC 3.1.1.8) activity, a useful clinical index for liver function. The former is based on separation of benzoate and benzoylcholine (the artificial substrate of cholinesterase) in an uncoated fused-silica capillary. The migration time of benzoylcholine and benzoate was 1.3 min and 5.5 min, respectively. By the peak areas of A₂₃₃ signals, the linear dynamic ranges for both analytes were 0.01–50.0 mM, and the relative standard deviations of 1.0 mM benzoylcholine and benzoate were less than 4% and 6%, respectively.The FIA-choline sensor was constructed with the working electrode of the flow cell covered with a natural chitinous membrane purified from Taiwanese soldier crab, Mictyris brevidactylus. The biomembrane served as the supporting material for enzyme immobilization (choline oxidase, EC 1.1.3.17), and also prevented protein adsorption on the electrode surface. The calibration curve was linear between 0.05 and 5.0 mM (r = 0.999). The relative standard deviations for 1.0 mM choline (n = 7) were less than 3%, and the activity of the bioactive membrane lasted for about 2 months. The analytical results of both methods correlated well (r = 0.940).     

Amperometric determination of phosphatidyl choline in serum with use of immobilized phospholipase d and choline oxidase

Phospholipase D (EC 3.1.4.4.) and choline oxidase (EC 1.1.99.1.) are immobilized together on a hydrophobic agarose gel and used to convert the phospholipid to betaine and hydrogen peroxide, which is measured amperometrically at + 0.60 V vs. SCE. The response time of the sensor is 2 min, and the calibration curve for 0–3 g l^-1 of phosphatidyl choline is linear. Different methods of insolubilizing the enzymes are compared.     

Ultrasensitive and accelerated detection of ciguatoxin by capillary electrophoresis via on-line sandwich immunoassay with rotating magnetic field and nanoparticles signal enhancement

A sensitive and rapid on-line immunoassay for the determination of ciguatoxin CTX3C was developed based on a capillary mixing system, which was integrated with capillary electrophoresis (CE) separation and electrochemical (EC) detection. In the sandwich immunoassay system, anti-CTX3C-functionalized magnetic nanoparticles were used as immunosensing probes, and horseradish peroxidase (HRP) and anti-CTX3C antibody were bound onto the surface of gold nanoparticles (AuNPs) and used as recognition elements. Online formation of immunocomplex was realized in capillary inlet end with an external rotating magnetic field. Compared with classical HPLC-MS and ELISA, the assay adopting AuNPs as multienzyme carriers and online sandwich immunoassay format with rotating magnetic field exhibited higher sensitivity and shorter assay time. The linear range of the assay for CTX3C was from 0.6 to 150 ng/L with a correlation coefficient of 0.9948 (n = 2), and the detection limit (S/N = 3) was 0.09 ng/L. The developed assay showed satisfying reproducibility and stability, and it was successfully applied for the quantification of CTX3C in fish samples.     

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 novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease

Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine‐d₄ as a surrogate substrate of ACh and measure product choline‐d₄ via liquid chromatography–tandem mass spectrometry (LC–MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC–MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate‐based LC–MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE.     

Biocentri-voltammetric biosensor for acetylcholine and choline

We report on the determination of choline and acetylcholine via biocentrivoltammetry. This method combines centrifugation and voltammetry and is based on a carbon paste electrode modified with acetylcholinesterase and choline oxidase. The electrode was placed at the bottom of a biocentrivoltammetric cell. Acetylcholine and choline are accumulated on the enzyme electrode via centrifugative forces, upon which a direct voltammetric scan is applied. Reaction time, pH values, quantities of enzyme and centrifugation parameters were optimized. A linear response is obtained in the 0.07 to 10?μM concentration range of acetylcholine, and a limit of detection as low as 0.5?μM. The linear range is between 0.1 and 500?μM for choline. The method was applied to the determination of acetylcholine and choline in spiked serum samples.

Homogeneous chemiluminescent assays for free choline in human plasma and whole blood

Choline was oxidized in the presence of choline oxidase and the hydrogen peroxide generated was detected using a chemiluminescent acridinium-9-carboxamide. The dose response for choline (0-150 μM) was established in buffer and was validated for the quantification of choline in human plasma and whole blood. This homogeneous assay was performed in a 96-well microplate format and required minimal sample volume (4 μL) and short analysis time (<5 s per well). The new assay(s) correlated well (R > 0.98, plasma; R > 0.97, whole blood) with LC-MS/MS.     

On-column silver substrate synthesis and surface-enhanced Raman detection in capillary electrophoresis

A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A ∼50-μm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-μm inner diameter CE capillary window or in a flow cell consisting of a 250-μm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20× objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25–25 ppm concentrations, by using 1.4–3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.     

Intermolecular forces between acetylcholine and acetylcholinesterases studied with atomic force microscopy

With the aid of atomic force microscopy, the intermolecular forces between acetyleholinesterases (AChE) and its natural substrate acetylcholine (ACh) have been studied. Through force spectrum measurement based on imaging of AChE molecules it was found that the attraction force between individual molecule pairs of ACh and AChE was (10±1) pN just before the quaternary ammonium head of ACh got into contact with the negative end of AChE and the decaying distance of attraction was (4±1) nm from the surface of ACHE. The adhesion force between individual ACh and AChE molecule pairs was (25±2) pN, which had a decaying feature of fast-slow-fast (FSF). The attraction forces between AChE and choline (Ch), the quaternary ammonium moiety and hydrolysate of ACh molecule, were similar to those between AChE and ACh. The adhesion forces between AChE and Ch were (20±2) pN, a little weaker than that between ACh and ACHE. These results indicated that AChE had a steering role for the diffusion of ACh toward it and had r     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.     

Optical choline sensor based on a water-soluble fluorescent conjugated polymer and an enzyme-coupled assay

We report on a simple and sensitive water-soluble fluorescent conjugated polymer for use in a choline biosensor. Choline is oxidized by the enzyme choline oxidase (ChOx), and the hydrogen peroxide (H₂O₂) formed is used to oxidize catechol via catalysis by horseradish peroxidase. The product of oxidation acts as a quencher of the photoluminescence of a fluorescent conjugated polymer. The ratio of the fluorescence intensity of the system in the presence and absence of the choline, respectively, serves as the analytical information. It is proportional to the concentration of choline in the 0.1 μM to 20 μM concentration range. The detection limit for choline is 50 nM. The biosensor was successfully applied to the determination of choline in milk samples with satisfactory reproducibility and accuracy. This is the first biosensor where a ChOx/HRP enzyme-coupled assay is used in combination with a water-soluble conjugated polymer for the fluorescent detection of choline. In our opinion, it provides a common platform for further development of enzymatic biosensors based on fluorescent conjugated polymers.

Simultaneous determination of allantoin, choline and L-arginine in Rhizoma Dioscoreae by capillary electrophoresis

A rapid, easy and reproducible capillary electrophoresis (CE) method for the simultaneous determination of allantoin, choline and arginine in Rhizoma Dioscoreae was developed first time. Under the optimum condition, the three analytes could be well separated within 5 min in a 70 cm (60 cm effective length) x 75 microm i.d. capillary. The relative standard deviations for both migration time and peak height were less than 3.20%. The linear response range was 5.0-150, 0.9-100 and 1.0-200 microg/ml for arginine, choline and allantoin, respectively. The detection limit of three components was 2.0, 0.4 and 0.5 microg/ml for arginine, choline and allantoin, respectively. Contents of arginine, choline and allantoin in the crude drug of Rhizoma Dioscoreae could be easily determined by the proposed method with satisfactory results.     

Quantification of glycine betaine,choline and trimethylamine N-oxide in seawater particulates: Minimisation of seawater associated ion suppression

A liquid chromatography/mass spectrometry (LC/MS, electrospray ionisation) method has been developed for the quantification of nitrogenous osmolytes (N-osmolytes) in the particulate fraction of natural water samples. Full method validation demonstrates the validity of the method for measuring glycine betaine (GBT), choline and trimethylamine N-oxide (TMAO) in particulates from seawater. Limits of detection were calculated as 3.5, 1.2 and 5.9 pg injected onto column (equivalent to 1.5, 0.6 and 3.9 nmol per litre) for GBT, choline and TMAO respectively. Precision of the method was typically 3% for both GBT and choline and 6% for TMAO. Collection of the particulate fraction of natural samples was achieved via in-line filtration. Resulting chromatography and method sensitivity was assessed and compared for the use of both glass fibre and polycarbonate filters during sample collection. Ion suppression was shown to be a significant cause of reduced instrument response to N-osmolytes and was associated with the presence of seawater in the sample matrix.     

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.