Immobilization Of Acetylcholinesterase Research Articles

Aptamer-functionalized magnetic nanoparticles conjugated organic framework for immobilization of acetylcholinesterase and its application in inhibitors screening

Construction of new enzyme reactor based on aptamer functionalized magnetic nanoparticles conjugated organic framework (COF) for acetylcholinesterase immobilization has been an enabling endeavor in this work. The aptamer against acetylcholinesterase was selected through a method based on capillary electrophoresis in one round. A new magnetic COF material rich of carboxyl groups was firstly synthesized, and its surface was then modified with the selected aptamer through covalently linking. Acetylcholinesterase was immobilized to fabricate the enzyme reactor Fe3O4@COF-Apt-AChE through the high affinity and specificity with its binding aptamer. The as constructed enzyme reactor was comprehensively characterized and the key factors that affected its catalysis efficiency were investigated in detail. Owing to the surface modification of the magnetic COF materials by aptamer for acetylcholinesterase immobilization, the immobilized enzyme exhibited improved substrates affinity. What’s more, good reusability (more than 8 times) and prolonged stability (enzyme activity still kept at 90% after 42 days) were also achieved. Finally, the enzyme reactor could be applied in AChE inhibitors screening, which expanded its application capability. The proposed protocol not only paves a new way for fabrication of novel aptamer functionalized magnetic COF materials as enzyme reactors, but also indicates a broadened application of the integration of aptamer and its enzyme.

Electrochemical Biosensor Based on Polyelectrolyte Complexes with Dendrimer for the Determination of Reversible Inhibitors of Acetylcholinesterase

An acetylcholinesterase biosensor has been developed based upon a fourth generation poly(amidoamine) dendrimer with terminal amino groups. The immobilization of acetylcholinesterase was performed by physical adsorption onto the polyelectrolyte complex consisting of dendrimer and poly(styrene sulfonate) or deoxyribonucleic acid as counter ions. The efficiency of immobilization was evaluated by surface plasmon resonance that indicated a twofold increase of the quantity of enzyme on the surface in comparison with similar biosensors with no dendrimer. This property can be attributed to higher roughness of the underlying surface and its possible porosity caused by the star-shaped dendrimer molecules. The influence of the polyelectrolytes used on the biosensor performance as well as kinetic characteristics of enzymatic hydrolysis of thiocholine were assessed. The calibration curves of reversible inhibitors used in the therapy of neurodegenerative diseases (Huperzine A and galantamine) were ascribed with a four-parametrical logistic model. The 15% inhibition levels varied from 9.4 to 65 nM Huperzine A and 90 to 107 nM galantamine depending on the content of the polyelectrolyte complex. The biosensors were tested on example artificial and real urine samples and provided recoveries from 95 to 110%.

An Acetylcholinesterase Inhibition-Based Biosensor for Aflatoxin B1 Detection Using Sodium Alginate as an Immobilization Matrix.

In this study, we investigated a novel aflatoxin biosensor based on acetylcholinesterase (AChE) inhibition by aflatoxin B1 (AFB1) and developed electrochemical biosensors based on a sodium alginate biopolymer as a new matrix for acetylcholinesterase immobilization. Electrochemical impedance spectroscopy was performed as a convenient transduction method to evaluate the AChE activity through the oxidation of the metabolic product, thiocholine. Satisfactory analytical performances in terms of high sensitivity, good repeatability, and long-term storage stability were obtained with a linear dynamic range from 0.1 to 100 ng/mL and a low detection limit of 0.1 ng/mL, which is below the recommended level of AFB1 (2 µg/L). The suitability of the proposed method was evaluated using the samples of rice supplemented with AFB1 (0.5 ng/mL). The selectivity of the AChE-biosensor for aflatoxins relative to other sets of toxic substances (OTA, AFM 1) was also investigated.

Self-reduction bimetallic nanoparticles on ultrathin MXene nanosheets as functional platform for pesticide sensing

Two-dimensional (2D) transition metal carbides and nitrides, named MXene, appear promising application prospects in sensor filed. Metal nanoparticles, especially bimetallic nanoparticles, are the superior nanocatalyst, which process excellent features due to the high specific surface area and synergistic catalytic capacity. Using ultrathin MXene nanosheets as the natural reducing agent and support, we prepare the shape-controlled Au-Pd bimetallic nanoparticles via a self-reduction process at room temperature in a short time, which can well enhance the catalytic performance and are benefit for the acetylcholinesterase immobilization. Based on their desired properties, we propose a disposable electrochemical biosensor for the detection of organophosphorus pesticide using the multi-dimensional nanocomposites (MXene/Au-Pd) as the functional platform. Under the optimized conditions, our fabricated biosensor exhibits a favorable linear relationship with the concentration of paraoxon from 0.1 to 1000 μg L−1, with a low detection limit of 1.75 ng L−1. Furthermore, the biosensor can be applied for paraoxon detection in pear and cucumber samples, providing an effective and useful avenue for the applicability of novel 2D nanomaterials in biosensing field.

Acetylcholine esterase enzyme doped multiwalled carbon nanotubes for the detection of organophosphorus pesticide using cyclic voltammetry

Use of immobilized acetylcholine esterase (AChE) for detecting organophosphorus pesticides in water sources and body fluids can bring down the detection costs dramatically. In the present study, AChE was directly doped on multiwalled carbon nanotube (MWCNT) surface modified with carboxylic groups through amide bond and used for organophosphorus pesticide detection. Amide bond formation between MWCNTs and the enzyme molecules avoid use of any intermediate membranes, cross-linkers or binding materials. This strategy overcomes the hindrance to electron transfer posed by membranes or cross-linkers and increases the sensitivity of detection. MWCNTs carrying carboxyl groups were deposited on glassy carbon electrode and were subsequently immobilized with AChE. The activity of AChE was monitored by cyclic voltammetry after immobilization. Scanning electron microscopy and atomic force microscopy were used to characterize the electrode surface. FT-IR spectra were taken to characterize enzyme-MWCNT complex. Under optimized parameters, the electrode showed linear range between 10 and 50 nM, which is promising for detection of trace amounts of the pesticide. The lower and higher detection limits of the sensor are 0.1 nM and 500 nM respectively. The stability and reusability of the electrode were determined. Finally, successful detection of organophosphorus compounds in real samples established it as reliable for sensor applications.

Nonhydrolytic sol–gel synthesized oligosiloxane resin reinforced thiol-ene photocured coatings for the immobilization of acetylcholinesterase

Acetylcholinesterase (AChE), which is responsible for the hydrolysis of neurotransmitter acetylcholine, is a critical enzyme for the nervous system and also a biomarker for organophosphorous pesticide detection. The immobilization of AChE is an active area of research and recently the use of sol–gel-derived materials for enzyme immobilization has gained a lot of attraction. In this work, AChE was covalently immobilized onto a photocured substrate which was reinforced with an oligosiloxane resin. The oligosiloxane resin was designed to have both vinyl and epoxide groups and prepared via nonhydrolytic sol–gel technique. The strategy employed in this study offered a platform that has good mechanical and thermal properties and also suitable for modification. Thus, AChE was also immobilized onto these substrates after amine modification of the epoxy groups and followed by glutaraldehyde activation. Over 80% enzyme immobilization yield was achieved. At certain pH values (5.5 and 8.5) and under relatively higher temperatures (above 40 °C) the immobilized enzymes were found to have higher catalytic activity than the free enzyme. Furthermore, by immobilization the reuse and the storage stability of the enzyme was improved and the stability of the immobilized enzyme against the inhibitory effects of certain metal cations was enhanced.

Electrochemical Acetylcholinesterase Biosensor Based on Polylactide–Nanosilver Composite for the Determination of Anti-dementia Drugs

A new acetylcholinesterase biosensor has been developed for the determination of anticholinesterase drugs applied for neurodegenerative disease treatment. For this purpose, silver nanosendrites were deposited by potentiostatic electrolysis on a glassy carbon electrode covered with oligolactides cross-linked with p-tert-butylthiacliax[4]arene core in the cone, partial cone, and 1,3-alternate configurations. The roles of macrocycle configuration and electrolysis conditions on the silver depostion were characterized and optimal conditions selected for the subsequent immobilization of acetylcholinesterase. Silver nanoparticles provide higher response at low working potential (0.05 V) due to the electrostatic accumulation of silver ions and prevention of their leaching after reoxidation. The biosensor allows the determination of 10−12 – 10−7 M donepezil, berberine, and huperzine A within 20-30 s by the relative decay of the current related to the oxidation of thiocholine formed in enzymatic reaction. The reversible inhibition of immobilized acetylcholinesterase with huperzine A was quantified for the first time. The developed biosensor was employed for the analysis of spiked urine samples.

Voltammetric Biosensor Based on Nitrogen-doped Ordered Mesoporous Carbon for Detection of Organophosphorus Pesticides in Vegetables

Background: Pesticides residues in agricultural products have posed a serious threat to food safety and human health, so it is necessary to develop a rapid and accurate method to detect pesticide in the environment. N-OMC with excellent electroconductivity, high biocompatibility and the functional amino group that can be covalently attached to the enzyme can be applied to construct a sensitive and stable acetylcholinesterase biosensor for rapid and accurate detection of organophosphorus pesticides with the help of L-cysteine self-assembled monolayer and AuNPs. Methods: Transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and nitrogen adsorption measurements are used to characterize materials. Electrochemical impedance spectroscopy and cyclic voltammetry are used to study the surface features of modified electrodes. Differential pulse voltammetric is used to measure the peak current of modified electrodes. GC-MS is applied to verify the reliability of the prepared biosensor for organophosphorus pesticides detection. Results: N-OMC was synthesized and applied to constructed stable and sensitive acetylcholinesterase biosensors. The combination of N-OMC, L-cysteine self-assembled monolayer and AuNPs to modify the electrode surface has greatly improved the conductivity of biosensor and provided a stable platform for acetylcholinesterase immobilization. The linear detection range of paraoxon was from 3 to 24 nM with a lower detection limit of 0.02 nM. Conclusion: The biosensor exhibited satisfactory reproducibility, repeatability and stability, and was successfully employed to determine the paraoxon in vegetables as well as tap water samples, providing a promising tool for rapid and sensitive detection of organophosphorus pesticides in agricultural products.

Metallic Transition Metal Dichalcogenide Nanosheets as an Effective and Biocompatible Transducer for Electrochemical Detection of Pesticide.

Owing to their large specific surface, favorable electrical conductivity, and excellent electrocatalytic capabilities, two-dimensional transition metal dichalcogenides have received considerable attention in the field of biosensors. On the basis of these properties, we developed a portable and disposable enzyme-based biosensor for paraoxon detection using a metallic MoS2 nanosheets modified screen-printed electrode (SPE). The exfoliated ultrathin metallic MoS2 nanosheets can accelerate the electron transfer on electrode surface and contribute to the immobilization of acetylcholinesterase (AChE) via the cross-linking of glutaraldehyde. Electrodeposited gold nanoparticles (AuNPs) on SPE were used to immobilize MoS2 nanosheets through the interaction between Au atoms on AuNPs and S atoms on MoS2. Using acetylcholine as the substrate, AChE can catalyze the formation of electroactive thiocholine and further generate the redox current. In the presence of paraoxon, the activity of AChE can be inhibited, making the related electrochemical signals weaken. Under the optimized conditions, this electrochemical biosensor exhibited a favorable linear relationship with the concentration of paraoxon from 1.0 to 1000 μg L-1, with the detection limit of 0.013 μg L-1. Furthermore, this developed biosensor was successfully applied to detect paraoxon in pretreated apple and pakchoi samples, which can provide a reliable method for the rapid analysis of organophosphorus pesticides in agricultural products.

Highly Sensitive Acetylcholinesterase Biosensor Based on Shaped Controlled ZnO Nanostructure for Paraoxon Pesticide Detection

Zinc oxide (ZnO) is an attractive material that finds applications in areas ranging from transistors, varistors, solar cells, photocatalysts and biosensors. Here we report the electrodeposition of ZnO nanostructured thin films in the presence of various capping agents like potassium chloride, ammonium fluoride and ethylenediamine to form different morphologies of ZnO. These ZnO nanostructures were used as biosensing platforms for the immobilization of acetylcholinesterase (AChE) and detection of the pesticide paraoxon. The electrodeposition of ZnO nanostructures was performed on carbon paper and carbon fabric substrate by using a zinc nitrate (Zn(NO3)2) precursor at 70 °C temperature with an applied potential of -1.0 V and 30 min reaction time. Many different morphologies of ZnO such as nanorods, nanoneedles, nanoplatelets, nanoflowers were successfully synthesized and characterized using scanning electron microscopy (SEM). Further the ZnO nanostructures were characterized by X-ray diffraction (XRD), UV-Vis spectroscopy, Raman spectroscopy and electrochemical impedance spectroscopy (EIS). An amperometric current response for the detection of paraoxon was recorded. The biosensor shows a detection limit to paraoxon in the range of 1nM–5µM . The developed biosensor shows high sensitivity, stability and low cost. [PS[E1]Detection limit should be 1 nM. Let us not write a range. It can be confusing. [PS[E2]You have written the abstract very well. Good job!

Protein Surface Structural Recognition in Inactive Areas: A New Immobilization Strategy for Acetylcholinesterase.

This work reported a new method of design for the immobilization of acetylcholinesterase (AChE) based on its molecular structure to improve its sensitivity and stability. The immobilization binding site on the surface of AChE was determined using MOLCAD's multi-channel functionality. Then, 11 molecules ((+)-catechin, (-)-epicatechin, (-)-gallocatechin, hesperetin, naringenin, quercetin, taxifolin, (-)-epicatechin gallate, flupirtine, atropine, and hyoscyamine) were selected from the ZINC database (about 50 000 molecules) as candidate affinity ligands for AChE. The fluorescence results showed that the binding constant Kb between AChE and the ligands ranged from 0.01344 × 104 to 4.689 × 104 M-1 and there was one independent class of binding site for the ligands on AChE. The AChE-ligand binding free energy ranged from -12.14 to -26.65 kJ mol-1. Naringenin, hesperetin, and quercetin were the three most potent immobilized affinity ligands. In addition, it was confirmed that the binding between the immobilized ligands only occurred at a single site, located in an inactive area on the surface of AChE, and did not affect the enzymatic activity as shown through a competition experiment and enzyme assay. This method based on protein surface structural recognition with high sensitivity and stability can be used as a generic approach for design of the enzyme immobilization and biosensor development.

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid-based nanofiber membrane.

In this study, polyacrylic acid-based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid-based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid-based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1mM Mn2+, Ni2+, Cu2+, Zn2+, Mg2+, Ca2+ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis-Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis-Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE-based biosensors.

Immobilization of acetylcholinesterase on functionalized SBA-15 mesoporous molecular sieve for detection of organophosphorus and carbamate pesticide

The immobilization of acetylcholinesterase (AChE) on amino functionalized SBA-15 mesoporous sieves (NH2-SBA-15) was explored for detection of organophosphorus and carbamate pesticides. Several methods have been studied and the “adsorption-crosslinking” is found to be the best one. Firstly, AChE and bovine serum albumin (BSA) were adsorbed in turn on the surface of NH2-SBA-15 by electrostatic adsorption, and then glutaraldehyde was added for cross-linking the amino group of AChE. The NH2-SBA-15 showed around 95% immobilization efficiency and the specific activity of immobilized AChE (AChE-NH2-SBA-15) reached 130% relative to free AChE under optimal immobilization conditions. It was found AChE-NH2-SBA-15 retained 94.0% and 82.8% of its initial activity after 60days at 4°C and 25°C, respectively. It remained 55.0% of its initial activity after 6 times recycling. During the detection of pesticide, the AChE-NH2-SBA-15 showed wider linear range and much lower limit of detection compared with free AChE. The current method showed good recovery (93.8%-109.3%) and low RSD (<5%) for both standard solution and real vegetable samples of Carbaryl and Trichlorfon. It was believed that AChE-NH2-SBA-15 could be exploited as a fast, sensitive and low-cost biocatalyst towards the detection of pesticides residues which could be stored at room temperature for a long time.

Polymer–Clay Nanocomposite-Based Acetylcholine esterase Biosensor for Organophosphorous Pesticide Detection

In this study, a nanocomposite of chitosan (CH)–montmorillonite (MT) clay was prepared via uniform dispersion of MT particles in a homogenous CH solution and deposited onto indium tin oxide (ITO) coated glass substrate. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy, Fourier transform infrared spectroscopy, and electrochemical techniques were used for the characterization of the CH–MT nanocomposite. The XRD and TEM results confirmed the exfoliation and intercalation of the MT clay into CH matrix. This CH–MT nanocomposite was employed for the fabrication of enzymatic biosensor via immobilization of acetylcholine esterase (AChE) for organophosphorous (chlorpyriphos) detection. It was found that the diffusion coefficient, electron transfer rate, surface concentration were increased after the immobilization of AChE onto CH–MT/ITO electrode. This AChE/CH–MT/ITO bio-electrode showed the linearity range from 0.5 to 1000 µg L−1 with an LOD of 0.448 µg L−1 and sensitivity of 69.6 µA mM−1 cm2. The apparent Michaelis–Menten constant (\(K_{\text{m}}^{\text{app}}\)) was obtained as 0.219 mM as a function of chlorpyriphos. This prepared bioelectrode can be an excellent platform for chlorpyriphos detection.

Efficient determination and pesticide control by means of immobilization of acetylcholinesterase

Supports involving tetrazole (o/m/p-F-Tet-1H) were prepared to detect pesticides. This novel te-trazole including fluorine in different positions was attached to nanoparticles (2AEPS-(o/m/p-F-Tet-1H)) by a condensation method. Primarily, the tetrazole derivatives were characterized by 1H-NMR,13C-NMR and LC-MS. Then, nanoparticles were prepared by a condensation method in non-aqueous medium and characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, gel permeation chromatography. The enzymatic properties of immobilized acetylcholinesterase (AChE) were investigated for the determination of phosmet. This research is the first example.

Palladium-copper nanowires-based biosensor for the ultrasensitive detection of organophosphate pesticides

A highly sensitive acetylcholinesterase (AChE) electrochemical biosensor for the quantitative determination of organophosphate pesticides (OPs) in vegetables and fruits based on palladium-copper nanowires (Pd-Cu NWs) was reported. AChE immobilized on the modified electrode could catalyze hydrolysis of acetylthiocholine chloride (ATCl), generating an irreversible oxidation peak. When exposed to the OPs, the activity of the AChE was inhibited and the current significantly decreased. The detection mechanism is based on the inhibition of AChE. The Pd-Cu NWs not only provide a large active surface area (0.268 ± 0.01) cm2 for the immobilization of AChE, which was approximately 3.8 times higher than the bare glass carbon electrode, but also exhibit excellent electro-catalytic activity and remarkable electron mobility. The biosensor modified with Pd-Cu NWs displayed a good affinity to ATCl and catalyzed hydrolysis of ATCl, with a low Michaelis–Menten constant (KM) of 50.56 μM. Under optimized conditions, the AChE-Cs/Pd-Cu NWs/GCE biosensor detected malathion with wide linear ranges of 5–1000 ppt and 500–3000 ppb, and the low detection limit was 1.5 ppt (4.5 pM). In addition, the OPs biosensor has been applied to the analysis of malathion in commercial vegetable and fruit samples, with excellent recoveries in the range of 98.5%–113.5%. This work provides a simple, sensitive and effective platform for biosensors and exhibits future potential in practical application for the OPs assay.

Application of pristine and doped SnO2 nanoparticles as a matrix for agro-hazardous material (organophosphate) detection

With an increasing focus on applied research, series of single/composite materials are being investigated for device development to detect several hazardous, dangerous, and toxic molecules. Here, we report a preliminary attempt of an electrochemical sensor fabricated using pristine Ni and Cr–doped nano tin oxide material (SnO2) as a tool to detect agro-hazardous material, i.e. Organophosphate (OP, chlorpyrifos). The nanomaterial was synthesized using the solution method. Nickel and chromium were used as dopant during synthesis. The synthesized material was calcined at 1000 °C and characterized for morphological, structural, and elemental analysis that showed the formation of agglomerated nanosized particles of crystalline nature. Screen-printed films of powder obtained were used as a matrix for working electrodes in a cyclic voltammogram (CV) at various concentrations of organophosphates (0.01 to 100 ppm). The CV curves were obtained before and after the immobilization of acetylcholinesterase (AChE) on the nanomaterial matrix. An interference study was also conducted with hydroquinone to ascertain the selectivity. The preliminary study indicated that such material can be used as suitable matrix for a device that can easily detect OP to a level of 10 ppb and thus contributes to progress in terms of desired device technology for the food and agricultural-industries.

Acetylcholinesterase Inhibitors Assay Using Colorimetric pH Sensitive Strips and Image Analysis by a Smartphone

Smartphones are widely spread and their usage does not require any trained personnel. Recently, smartphones were successfully used in analytical chemistry as a simple detection tool in some applications. This paper focuses on immobilization of acetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane. AChE degrades acetylcholine into choline and acetic acid which causes color change of acid-base indicator. Smartphone served as a tool for measurement of indicator color change from red to orange while inhibitors blocked this process. AChE inhibitors were measured with limits of detection, 149 nM and 22.3 nM for galanthamine and donepezil, respectively. Organic solvents were measured for method interferences. Measurement procedure was performed on 3D printed holder and digital photography was evaluated using red-green-blue (RGB) channels. The invented assay was validated to the standard Ellman's test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance.

Surface Controlled Electrochemical Sensing of Chlorpyrifos in Pinellia Ternate Based on a One Step Synthesis of Palladium-Reduced Graphene Nanocomposites

A novel acetylcholinesterase (AChE) biosensor for the detection of chlorpyrifos has been developed based on the immobilization of AChE on palladium-reduced graphene nanocomposites (rGO@Pd) modified glassy carbon electrode (GCE). The nanocomposites were synthesized by a one-step electrochemical co-deposition approach. Pd nanoparticles and rGO can be alternately attached onto the electrode surface, forming an interpenetrating network, which with excellent conductivity, catalysis, and biocompatibility offered an extremely hydrophilic surface for AChE adhesion. Under optimum conditions, the biosensor detects chlorpyrifos in two linear ranges, one is from 0.71 to 5.70 μM and the other is from 14.26 to 71.13 μM. The detection limit is 0.23 μM and the biosensor exhibits good performance such as reproducibility and stability, which makes it possible to provide a new and promising tool for the analysis of enzyme inhibitors.

Color Change of Phenol Red by Integrated Smart Phone Camera as a Tool for the Determination of Neurotoxic Compounds.

The use of a cell phone as a detection system is easy, simple and does not require trained personnel, which is in contrast to standard laboratory instruments. This paper deals with immobilization of acetylcholinesterase (AChE) in a gelatin matrix, and phenol red, as an indicator of AChE activity, is used in order to establish a method that is easily compatible with a camera device. AChE splits acetylcholine into choline and acetic acid, which changes the pH of a medium, resulting in a phenol red color change. The coloration changed in presence of an AChE inhibitor. Measurements were performed on 3D-printed, tube-shaped holder, and digital photography, with subsequent analysis of red-green-blue (RGB), served for assay purposes. Calibration of AChE inhibitors, tacrine and galantamine, was performed, with limit of detection equal to 1.1 nM and 1.28 µM, respectively. Interferences were also measured, resulting in a proof-of-method stability. The method was further successfully validated for the standard Ellman’s assay, and verified on murine plasma samples spiked with inhibitors.