A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.     

Measurement of acetylcholine and choline in brain by HPLC with electrochemical detection

A simple and rapid method for measuring acetylcholine and choline using high performance liquid chromatography (HPLC) with electrochemical detection is presented. Acetylcholine and choline were first separated using reverse-phase chromatography; acetylcholine was then hydrolyzed post-column to choline by acetylcholinesterase. Choline was oxidized enzymatically by choline oxidase to betaine and hydrogen peroxide, and the peroxide was detected electrochemically. Changes in methodology from previous procedures include a different mobile phase, controlled heating of chromatography column and post-column reaction coil, and a different extraction method for quaternary amines. The changes resulted in less inhibition of derivatizing enzymes by mobile phase, narrow and consistent elution of peaks, and a rapid and efficient extraction of quaternary amines. Measurement of acetylcholine and choline in brain tissue was found to be replicable, and the levels agreed with literature values.     

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.     

Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

Disposable, enzymatically modified printed film carbon electrodes for use in the high-performance liquid chromatographic–electrochemical detection of glucose or hydrogen peroxide from immobilized enzyme reactors

Disposable screen-printed, film carbon electrodes (PFCE) were modified with cast-coated Osmium–polyvinylpyrridine-wired horse radish peroxidase gel polymer (Os-gel-HRP) to enable the detection of the reduction at 0 mV of hydrogen peroxide (H₂O₂) derived from a post-column immobilized enzyme reactor (IMER) containing acetylcholinesterase and choline oxidase. In another series of experiments PFCE were initially modified with cast-coated Os-gel-HRP and then treated with glucose oxidase in bovine serum albumin (BSA) and cross-linked with glutaraldehyde to form a bi-layer glucose–Os-gel-HRP PFCE. This bi-layer glucose–Os-gel-HRP PFCE generated a reduction current at 0 mV to H₂O₂ derived from the reaction of glucose oxidase and glucose in solution. These enzyme-modified PFCE were housed in a radial flow cell and coupled with cation-exchange liquid chromatographic methods to temporally separate substrates in solution for the determination of acetylcholine (ACh) and choline (Ch) in the first experimental series, or glucose in the second experimental series. These two disposable enzyme-modified PFCE exhibited linear current vs. substrate relations, were durable, being usable for approximately 40 determinations, and were sufficiently sensitive to be employed in biological sampling. Both assays utilized the same HPLC equipment. The limit of detection for ACh was 16 fmol/10 μl and that for glucose was 12 μmol/7.5 μl. ACh and Ch were measured from a microdialysate from the frontal cortex of a rat. Glucose in human urine was determined using the bi-layer glucose oxidase–Os-gel-HRP PFCE.     

Application of Exchangeable Biochemical Reactors with Oxidase‐Catalase‐Co‐immobilizates and Immobilized Microorganisms in a Microfluidic Chip‐Calorimeter

Several methods for the quantitative detection of different compounds, e.g., L‐amino acids, sugars or alcohols in liquid media were developed by application of an automatic measuring unit including a fluid chip‐calorimeter FCC‐21. For this purpose, enzymes were immobilized covalently on the inner and outer surface of CPG (controlled porous glass)‐spherules with an outer diameter of 100 μm and filled into a micro flow‐through reaction chamber (V_R = 20 μL). The design of the measuring cell allows for easy insertion into the calorimeter device of a stored series of comfortably pre‐fabricated measuring cells. These cells can be filled with different enzyme immobilizates. Different oxidases were used and co‐immobilized with catalase for the improvement of the detection sensitivity. A signal amplification could be achieved up to a factor of 3.5 with this configuration. β‐D‐glucose, ethanol and L‐lysine could be detected in a range of 0.25–1.75 mM using glucose oxidase, alcohol oxidase and lysine oxidase. The group of oxidases in combination with the enzymatic catalysis of the intermediate H₂O₂ allows the quantitative detection of a large number of analytes. A good measurement and storage stability could be achieved for several weeks by this immobilization method. In addition to enzyme‐based detection reactions, it was shown that living microorganisms can be immobilized in the reaction chamber. Thus, the system can be used as a whole‐cell biosensor. The quantitative detection of phenol in the range of 10–100 μM could be performed using the actinomycete Rhodococcus sp. immobilized on glass beads by means of embedding into polymers.     

Determination of acetylcholine by on-line microdialysis coupled with pre- and post-microbore column enzyme reactors with electrochemical detection

A sensitive procedure consisting of a pre- and post-microbore column reactor sequence of a LC-electrochemical detection system coupled with on-line microdialysis system is described in the present study to measure endogenous acetylcholine concentration in freely moving rats. The pre-column packed, with immobilized choline oxidase and catalase, was used to remove choline, whereas the post-column, packed with immobilized acetylcholine oxidase and choline oxidase, was used to measure acetylcholine selectively. The detection limit of acetylcholine was found to be 5 fmol/μl (50 fmol/10 μl). The usefulness of the described methodology was evaluated by examining the change in the striatal acetylcholine concentration of freely moving rats after physostigmine (0.5 mg/kg, s.c.) administration.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Construction of an acetylcholinesterase-choline oxidase biosensor for aldicarb determination

In this study, acetylcholinesterase and choline oxidase were co-immobilized on poly(2-hydroxyethyl methacrylate) membranes and the change in oxygen consumption upon aldicarb introduction was measured. Immobilization of the enzymes was achieved either by entrapment or by surface attachment via a hybrid immobilization method including epichlorohydrin and Cibacron Blue F36A activation. Immobilized enzymes had a long-storage stability (only 15% activity decrease in 2 months in wet storage and no activity loss in dry storage). Aldicarb detection studies showed that a linear working range of 10-500 and 10-250 ppb aldicarb could be achieved by entrapped and surface immobilized enzymes, respectively. Enzymes immobilized on membrane surfaces responded to aldicarb presence more quickly than entrapped enzymes. Aldicarb concentrations as low as 23 and 12 ppb could be detected by entrapped and surface immobilized enzymes, respectively, in 25 min.     

Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H(2)O(2) produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10(-10) M of paraoxon and carbofuran can be detected.     

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

Summary Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutardialdehyde according to Morris, Campbell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O₂-uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent K_m-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same K_m-value.Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier.Dedicated to Professor Dr. Dr. U. Hagen on the occasion of his 60^th birthdayDAAD-Fellow from AustriaDAAD-Fellow from South-Korea     

Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe

A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H₂O₂) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H₂O₂. The enzyme-generated H₂O₂ reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity.     

Highly sensitive determination of hydrogen peroxide and glucose by fluorescence correlation spectroscopy

Background

Because H₂O₂ is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H₂O₂ is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H₂O₂ and glucose using fluorescence correlation spectroscopy (FCS).

Methodology/Principal Findings

FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H₂O₂ by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H₂O₂. Our developed system gave a linear calibration curve for H₂O₂ in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 µM with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM.

Conclusions/Significance

In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma.     

Determination of phosphatidylcholine in a flow injection system using immobilized enzyme reactors

Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.     

Determination of serum cholinesterase activity by liquid chromatography with electrochemical detection.

A sensitive enzymatic assay to measure cholinesterase activity in serum using liquid chromatography with electrochemical detection has been devised and used to examine cholinesterase inhibition in mice treated with diisopropyl phosphorofluoridate. Acetylcholine was used as substrate, and a postcolumn reactor containing immobilized choline oxidase converted the enzymatic product, choline, and the internal standard, ethylhomocholine, into the electrochemically active H2O2. The postcolumn reactor also contained acetylcholinesterase to allow the indirect detection of the substrate. Assay optimization included investigations of substrate concentration, buffer pH and ionic strength, enzyme concentration, incubation time, and reaction termination method. The optimized procedure is applicable to samples with activities of 0.11 to 269 mmol/ml/h. Intrasample coefficient of variation for mouse serum samples was 1.7% (n = 12), while intersample coefficient of variation was 8.0% (n = 5). The mean +/- SE serum cholinesterase activity found for controls and mice treated with diisopropyl phosphofluoridate (6.3 mg/kg, ip, 24 h prior) was 158.7 +/- 5.7 mumol/ml/h and 36.6 +/- 3.1 mumol/ml/h, respectively (P less than 0.001).     

Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, an enzyme that converts choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H₂O₂, a by-product of the reaction catalyzed by choline oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate peroxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H₂O₂ produced by choline oxidase in the transformed plants might have stimulated the expression of H₂O₂ scavenging enzymes, with resultant maintenance of the level of H₂O₂ within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H₂O₂, protected the PS II complex in transformed plants from damage due to light stress.     

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Macrokinetics and operational stability of immobilized glucose oxidase and catalase

Glucose oxidation by immobilized glucose oxidase (GlO) and catalase (Cat) has been investigated in batch and continuous reactions for operational studies. The macrokinetics of the process depend on coupled reaction steps and diffusion rates. The problem may be approximated by a simple pseudohomogeneous model taking into account both substrates of glucose oxidase and the intermediate reaction product H₂O₂. The effectiveness of both enzymes is enhanced in the coupled reaction path, the overall effectiveness nevertheless is very low. H₂O₂ causes the inactivation of both GlO and Cat. The rates of deactivation depend on the oxidation rates of glucose that give different quasistationary levels of H₂O₂ concentration. As a first approximation, the deactivation rates may be described by first-order reactions with respect to H₂O₂.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

The use of diaminobenzidine for spectrophotometric and acrylamide gel detection of sulfite oxidase and its applicability to hydrogen peroxide-generating enzymes

DAB, in the presence of HRP, sulfite oxidase and FBS, polymerizes to a product with an absorption difference maximum at 352 nm. This reaction has been used as a sensitive and specific spectrophotometric assay for sulfite oxidase. Incubation of acrylamide gels containing sulfite oxidase with assay mixtures produces an insoluble product which precipitates where the enzyme is localized. This stain is at least as sensitive as the amido black protein stain and its specificity is such that no band is seen in the absence of substrate, and that only one band, migrating identically to purified enzyme is seen in rat liver homogenates. This polymerization reaction has been applied to other H₂O₂-generating enzymes and can be used to demonstrate the presence of these enzymes in the midst of other oxidases. DAB polymerization provides a sensitive spectrophotometric assay which can be used with either ultraviolet or visible optics. The application of this procedure to modified enzymes is discussed.