Acetylcholine Receptor-Based Biosensor

Abstract

The acetylcholine (ACh) receptor protein isolated from Torpedo electric organ was incorporated into asolectin liposomes and immobilized noncovalently on the surface of a Planar Interdigitated Capacitive sensor to produce an ACh receptor-based biosensor. Capacitance of the biosensor, which was stable in buffered saline, increased rapidly when ACh was added. The response was dosedependent and specific for ACh, and the biosensor did not respond to six other neurotransmitters. Impure receptor preparations containing ACh esterase produced a smaller signal in response to ACh but showed enhanced response after inhibition of ACh esterase with diisopropylfluorophosphate.

The competitive antagonist d-tubocurare, and the noncompetitive antagonist amantadine, inhibited the response of the biosensor to ACh, but a time-dependent recovery occurred upon perfusion with ACh. The irreversible antagonist α-neurotoxin inhibited the response and there was no recovery following perfusion with ACh. Thus, the pharmacologic profile of the biosensor in response to drugs closely resembled that of the nicotinic ACh receptor.     

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.     

An Enzyme Sensor For Determination of Acetylcholine and Choline In Rat Brain Extracts

Abstract

A choline enzyme sensor, recently developed by the authors, was used for choline and acetylcholine determination in rat brain extracts, using choline oxidase immobilized on cellulose triacetate membranes, and acetylcholinesterase in homogeneous solution. the method proved useful for assay of the acetylcholine content in a commercial pharmaceutical formulation used in ophthalmology.     

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.     

Acetylcholine Sensor Based on Ion Sensitive Field Effect Transistor and Acetylcholine Receptor

Abstract

A new type of acetylcholine sensor was made with an Ion Sensitive Field Effect Transistor (ISFET) and acetylcholine receptor. The acetylcholine receptor was fixed on a polyvinylbutyral membrane which covered the ISFET gate. When acetylcholine was injected into this system, the differential gate output voltage gradually Shifted to the positive side and reached a constant value. This response was due to the positive charge of acetylcholine. A linear relationship was obtained between the initial rate of the differential gate output voltage change and the logarithmic value of the acetylcholine concentration. Acetylcholine was fixed in the range 0.1-10μM. When the acetylcholine receptor was immobilized with the lipid membrane, the response was amplified with both the positive charge of acetylcholine and sodium ion flux through the acetylcholine receptor's channel. Therefore, the difference in the differential voltage between the acetylcholine receptor-ISFET systems with and without the lipid membrane was caused by sodium ion flux through the acetylcholine receptor's channel.     

Immobilized enzymes in clinical and biochemical analysis : Applications to the Simultaneous Determination of Acetylcholine and Choline and to Determination of Lipids

Uses of immobilized enzyme mini-columns in flow-injection systems are described Simultaneous determination of ? × 10^?5 M choline and acetylcholine is achieved by using acetylcholinesterase and choline oxidase columns. A home-made amperometric detector is used to detec the hydrogen peroxide produced enzymatically. An ion-exchange column is used on-line to remove interferences at the amperometric detector during analysis of blood and brain samples. Immobilization of the lipid enzymes phospholipase-C and -D is described. These enzymes are used for the determination of phospholipids. Total phospholipids (1– mM) are determined with a combination of phospholipase-D, lipase and glycerol-3-phosphate oxidase. All the methods described are simple and reproducible and the immobilized enzymes show good stability.     

Screen‐printed Electrode Modified with ZnFe2O4 Nanoparticles for Detection of Acetylcholine

In this paper a sensor to detect acetylcholine on the basis of ZnFe₂O₄ nanoparticles modified screen printed electrode (SPE) is reported. In the range 0.08–500.0 μM, with the detection limit of 0.024±0.001 μM, acquired anodic peak currents where shown to be linearly dependent on acetylcholine concentrations by differential pulse voltammetry (DPV). It was proven that acetylcholine oxidation at adjusted electrode surface takes place at 100 mV potential less positive compared to an unadjusted screen printed electrode. The electron transfer coefficient (α=0.51) and diffusion coefficient (D=9.3×10^?6 cm²/s) of acetylcholine oxidation were determined too. In addition, this original sensor possesses numerous benefits such as reproducibility, high stability and rapid response (20 s).     

Production of Hemicellulose- and Cellulose-Degrading enzymes by various strains ofSclerotium Rolfsii

A number of wild-type isolates ofSclerotium rolfsii were screened for their capacity to produce lignocellulolytic enzymes when grown on a cellulose-based medium.S. rolfsii proved to be an efficient producer of hemicellulolytic enzymes under the conditions selected for this screening, although there was a great variability in enzyme activities formed by the different isolates. In addition to xylanase and mannanase, which were produced in remarkably high levels, a number of accessory enzymes, which are important for the complete degradation of substituted hemicelluloses and include a-arabinosidase, acetyl esterase, and a-galactosidase, are formed byS. rolfsii. Efficient production of xylanase and mannanase was achieved when cellulose-based media were used for growth. Under these conditions, enhanced levels of endoglucanase were formed as well. Formation of xylanase and mannanase could be more specifically induced when using xylan or mannan as growth substrates, although the enzyme activities thus obtained were significantly lower compared to cultivations on cellulose as main inducing substrate.

     

Enzyme Electrode Sensor for Hydrogen Peroxide

Abstract

An enzymatic sensor was constructed from an oxygen amperometric sensor on whose surface a dialysis membrane containing covalently bonded catalase was set. Immobilization of the enzyme on the dialysis membrane surface was achieved with 2,4-dichloro-6-methoxy-s-triazine, a derivative of cyanuric chloride, which unlike others of its monosubstituted derivatives ensures some advantages. The time of measurement is less than 12 sec, for the kinetic method of the initial slope and one minute for the steady-state method. The sensor responds linearly to hydrogen peroxide in the concentration range 10^?3 - 10^?5 moles/1. The utilization of this sensor in intermittent and continuous operation in a laboratory enzymatic minireactor is discussed.     

Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material

A new type of amperometric biosensor based on immobilised acetylcholine esterase was designed and constructed. The enzyme was immobilised on a flow-through working electrode, which was prepared from reticulated vitreous carbon (RVC) or from a composite material consisting of RVC and superporous agarose. The sensor was operated in FIA mode using acetylthiocholine as a substrate. The sensor responded to inhibitors such as paraoxon-10(-9) mol was detected by the sensor in a non-optimised configuration. The practical lifetime of the sensor was at least 1 month.     

Bioactive constituents from Croton sparsiflorus Morong

Whole plant extracts of Croton sparsiflorus in methanol have shown significant enzyme inhibition and antioxidant activities. Bioassay-guided isolation of chloroform fraction at pH 3 resulted in the identification of crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was purified from the chloroform fraction at pH 9. The structures of the compounds were confirmed through spectral analyses (EI-MS, ¹H and ¹³C NMR). The isolated compounds 1–3 exhibited remarkable enzyme inhibition activity with IC₅₀ values 27.01 ± 1.1, 22.26 ± 1.0 and 18.02 ± 1.3 μM in xanthine oxidase and 48.42 ± 1.5, 48.05 ± 1.4 and 7.42 ± 1.0 μM in acetylcholine esterase assays, respectively. These compounds also showed potent radical scavenging and reducing properties in DPPH and FRAP assays, respectively. The present results suggest the validity of the traditional uses of C. sparsiflorus in rheumatism and gout. Furthermore, the isolated noraporphine alkaloids can be useful in the treatment of neurodegenerative diseases.     

A new flavonoid from the leaves of Atalantia monophylla (L.) DC

A new flavonoid, atalantraflavone (1) as well as eight known compounds including atalantoflavone (2), racemoflavone (3), 5,4′-dihydroxy-(3″,4″-dihydro-3″,4″-dihydroxy)-2″,2″-dimethylpyrano-(5″,6″:7,8)-flavone (4), lupalbigenin (5), anabellamide (6), citrusinine I (7), p-hydroxybenzaldehyde (8), and frideline (9), were isolated from the leaves of Atalantia monophylla (L.) DC. Focusing on Alzheimer’s disease, acetylcholine esterase (AChE) inhibition and antioxidant activity were evaluated using the modified Ellman’s method and the ABTS scavenging assay, respectively. It was found that isoflavonoid 5, lupalbigenin, showed 79% inhibition to AChE and was 1.4-fold stronger than the tacrine standard. In addition, acridone 7, citrusinine I, displayed 90.68% antioxidant activity.     

Enzyme Sensor for the Detection of Herbicides Inhibiting Acetolactate Synthase

Abstract

An enzyme sensor for the detection of sulfonylurea herbicides inhibiting acetolactate synthase II (ALS) was developed using an oxygen electrode. ALS, which has an oxygen consumption side reaction, was entrapped in the matrix of PVA-SbQ polymer, and the enzyme membrane was attached to the electrode. The inhibition of side oxygen reaction of ALS II is measured as decreased consumption of O₂ monitored by an oxygen electrode. Preliminary results show that 10^?6 M herbicide concentration can be determined by this method.     

A novel amperometric biosensor based on single walled carbon nanotubes with acetylcholine esterase for the detection of carbaryl pesticide in water

Amperometric biosensor is fabricated for the detection of carbaryl based on single walled carbon nanotubes (SWCNTs) and acetylcholine esterase (AchE). The dispersion of SWCNTs in positively charged polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA), possibly takes place due to weak supramolecular interaction between them, which then binds electrostatically to the negatively charged AchE at pH 7.4 using layer-by-layer (LbL) self-assembly technique. The optical intensity of UV/vis spectra increased with the number of layers, indicating the build up of a multilayer coating on the electrode. The activity of acetylcholine esterase on modified electrode of 3 mm in diameter was found to be 0.2 U. The biosensor showed good sensitivity and stability towards the monitoring of carbaryl pesticides in water with the detection limit of 10⁻¹² g L⁻¹ and recovery of 99.8 ± 2.7% to 10⁻¹⁰ g L⁻¹. This protocol can be used for the immobilization of other enzymes to fabricate a range of biosensors.     

Long‐Term Stabilization of the Activity of Ascorbate Oxidase Adsorbed on a Porous Carbon Material by Polymaleimidostyrene

Abstract

The biological catalysts adsorbed on the carbon surface gradually lost their activities. In order to prolong stabilization of the enzyme activity, polymaleimidostyrene (PMS) was used to preserve enzyme conformation. Sulfhydryl and amino groups of enzyme or biological tissue can be covalently bound to PMS. By using the carbon material coated by PMS, the stability of L‐ascorbic acid sensor response using purified enzyme was significantly improved, and its initial response was not decreased at all for 150 days, although the purified enzymes adsorbed to the carbon without PMS lost their activity 22.5% after 10 days.     

Kinetic Resolution of Cyclic Sulfoxides by Enzyme Promoted Hydrolysis of 2-Alkoxycarbonyl-3,6-Dihydro-2H-Thiapyran-1-Oxides

Abstract

Among many methods of preparation of optically active sulfoxides, those applying enzymes are becoming more and more popular¹. In continuation of our recent investigations on the enzyme promoted hydrolysis of sulfinyldicarboxylates² we turned our attention to cyclic six-membered sulfoxides 1. These compounds are easily available by the cycloaddition reaction of butadienes with appropriate sulfines obtained in situ from the corresponding esters (or their O-silyl enolethers) with thionyl chloride in the presence of a base³. Three compounds shown in the scheme were chosen for our investigations. It should be noted that compounds lb and lc are obtained as single diastereoisomers.     

Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Abstract

Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase. Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell. This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles. The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes. The apparatus is generally applicable to other enzymatic reactions which yield electroactive products.     

Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer

Choline and acetylcholine sensors were prepared by using choline oxidase and acetylcholinesterase, entrapped in photocross- linkable poly(vinyl alcohol) bearing styrylpyridinium (PVA-SbQ). The measurements were based on the detection of hydrogen peroxide liberated by an enzyme reaction (choline oxidase) or two sequential enzyme reactions (acetylcholine esterase and choline oxidase). The determination range for choline was 2.5-2-150 αmol 1^-1 and for acetylcholine 20-2-750 αmol 1^-1. The response times were 2-2-4 min. The immobilized enzyme membranes stored in a dry state were very stable and no loss of activity was observed after storage for 60 days.     

Amperometric Enzyme‐Based Biosensor for Direct Detection of Formaldehyde in the Gas Phase: Dependence on Electrolyte Composition

An enzymatic sensor detecting the analyte formaldehyde directly from the gas phase is under investigation. In contrast to existing systems, it enables the quantification of the analyte without prior sampling or accumulation and thus can be used as an online system to monitor the formaldehyde concentration in ambient air. The amperometric sensor depends on the enzymatic conversion of the analyte using formaldehyde dehydrogenase from P. putida [EC. 1.2.1.46] as the recognition element. It shows a linear response curve up to 15 ppm, with a detection limit of 0.03 pm (S/N=3). In order to optimize the sensor performance the electrolyte composition within the sensor was varied with respect to pH value, buffer concentration and the addition of Ca²⁺ and Mg²⁺ ions. To elucidate the influence of the mediator and the enzyme on the sensor performance the stability and activity of the electrochemical mediator and the enzyme alone was examined separately in these different electrolytes.     

Kinetics from indirectly detected hyperpolarized NMR spectroscopy by using spatially selective coherence transfers

An important recent development in NMR spectroscopy is the advent of ex situ dynamic nuclear polarization (DNP) approaches, which are capable of yielding liquid‐state sensitivities that exceed considerably those afforded by the highest‐field spectrometers. This increase in sensitivity has triggered new research avenues, particularly concerning the in vivo monitoring of metabolism and disease by NMR spectroscopy. So far such gains have mainly materialized for experiments that focus on nonprotonated, low‐γ nuclei; targets favored by relatively long relaxation times T₁, which enable them to withstand the transfer from the cryogenic hyperpolarizer to the reacting centers of interest. Recent studies have also shown that transferring this hyperpolarization to protons by indirectly detected methods could successfully give rise to ¹H NMR spectra of hyperpolarized compounds with a high sensitivity. The present study demonstrates that, when merged with spatially encoded methods, indirectly detected ¹H NMR spectroscopy can also be exploited as time‐resolved hyperpolarized spectroscopy. A methodology is thus introduced that can successfully deliver a series of hyperpolarized ¹H NMR spectra over a minutes‐long timescale. The principles and opportunities presented by this approach are exemplified by following the in vitro phosphorylation of choline by choline kinase, a potential metabolic marker of cancer; and by tracking acetylcholine’s hydrolysis by acetylcholine esterase, an important enzyme partaking in synaptic transmission and neuronal degradation.