Conducting polymer-based electrochemical biosensor for the detection of acetylthiocholine and pesticide via acetylcholinesterase.

A voltammetric biosensor for acetylthiocholine (ATCh) and paraoxon detection was successfully developed. To achieve this goal, polypyrrole (PPy) was synthesized onto the platinum (Pt) electrode surface in 0.30 M oxalic acid solution containing 25 mM pyrrole. PPy-coated Pt (Pt/PPy) electrode surface was covered with chitosan (Chi) (Pt/PPy/Chi). The acetylcholinesterase (AChE) enzyme was immobilized on the Pt/PPy/Chi electrode surface to build a voltammetric biosensor (Pt/PPy/Chi/AChE). The storage stability of the biosensor was determined to be 72% even after 60 days. The operational stability was determined to be 94% after 20 consecutive measurements. For the biosensor, the linear range was determined to be 30-50 µM for ATCh and 0.46-1.84 nM for paraoxon. The limit of detection (LOD) was determined to be 0.45 µM for ATCh and 0.17 nM for paraoxon.

An ultrasensitive signal-on electrochemiluminescence biosensor based on Au nanoclusters for detecting acetylthiocholine.

For improving the sensitivity of the electrochemiluminescent (ECL) detection and extending the applications of luminophore, the development of coreactant accelerator is one of the important ways. In this work, Au nanoclusters (Au NCs) were chosen as the luminescent material, and thiocholine, which was in situ generated by enzymatic reaction, was found to serve as a coreactant accelerator for Au NC-S2O82- ECL system. Based on this discovery, a highly sensitive detection of acetylthiocholine (ATCl) was achieved using the acetylcholinesterase (AChE) biosensor. CeO2 nanowires (CeO2 NWs) were used to improve the stability of Au NCs on the glassy carbon electrode (GCE) due to the large specific surface area and good film-forming properties of CeO2 NWs. ATCl was catalyzed by acetylcholinesterase (AChE) to produce thiocholine, which served as the coreactant accelerator to improve the ECL signal of Au NC-S2O82- system. The biosensor obtained a low detection limit of 0.17 nM. The integration of thiocholine and Au NCs would provide a new ECL platform for bioanalysis. Graphical abstract ᅟ.

Microfluidic thread-based electrode system to detect glucose and acetylthiocholine.

A reusable and simple to fabricate electrochemical sensor for the detection of glucose and acetylthiocholine using thread-based electrodes and nylon thread is described. The fabrication of the device consisted of two steps. First, three nylon-based electrodes (reference, working, and counter) were painted with one layer of conductive inks (silver and carbon ink, or silver/silver chloride ink). The electrodes were taped onto parafilm, and a piece of white nylon thread was wrapped around each electrode connecting the three electrodes. For the glucose system, a PBS solution containing glucose oxidase (GOx) (10 mg/mL), and potassium ferricyanide (K3 [Fe(CN)6 ]) (10 mg/mL) as mediator, was dried onto the thread, and increasing concentrations of glucose (0-15 mM) was added to the thread and measured by cyclic voltammetry (CV). The current output from the glucose oxidation was proportional to the concentration of glucose. For the second system, a solution of acetylcholinesterase (AChE) (0.08 U/mL) in PBS was added to the nylon thread, and increasing concentrations of acetylthiocholine (ATC) (0-9.84 mg/mL) was added and measured by CV. The current output from the oxidation of thiocholine (produced by AChE reacting with ATC) was proportional to the concentrations of ATC added to the thread. From both systems, a graph of current output versus substrate concentration was produced and fitted with a linear regression line that gave R2 values of 0.985 (GOX /glucose) and 0.995 (AChE/ATC).

Controlled synthesis of polydopamine: A new strategy for highly sensitive fluorescence turn-on detection of acetylcholinesterase activity.

A new water soluble fluorescent coronene probe (CTCA) was synthesized and is shown to display strong fluorescence (with excitation/emission maxima at 313/450 nm) in aqueous solution. Dopamine was oxidized under air to form polydopamine (PDA) which quenches the fluorescence of CTCA. The enzyme acetylcholinesterase (AChE) is known catalyze the hydrolysis of acetylthiocholine to produce thiocholine. Thiocholine inhibits the polymerization of DA, and this leads to recovery in CTCA fluorescence. These findings form the basis for a new method for detection of AChE activity. The assay has a detection limit as low as 0.05 mU·mL-1 of AChE. It is highly selective, and other enzymes do no noticeably interfere. It was applied to the determination of AChE activity in (spiked) human serum, and of AChE inhibitors in (spiked) lake water samples. Graphical abstract Controlled synthesis of polydopamine for the highly sensitive and selective sensing of AChE activity is reported for the first time.

Design and Development of Acetylthiocholine Electrochemical Biosensor Based on Zinc Oxide-Cerium Oxide Nanohybrid Modified Platinum Electrode.

Acetylcholinesterase (AChE) enzyme has been predominantly used for the detection of pesticides and metal ions. But, these sensors respond to pesticides as well as metal ions at certain concentration, which results in poor selectivity. Hence in this work, the amount of thiocholine produced during AChE inhibition has been estimated to detect the residual activity of AChE enzyme in-turn to enhance the efficiency of the biosensor. In this context, Pt/ZnO-CeO2/AChE/Chitosan based biosensor has been developed for sensitive voltammetric quantification of thiocholine in AChE. The sensor exhibited enhanced electron transfer rate, good conductivity and biocompatibility. Both the intrinsic and extrinsic parameters were simultaneously optimized using second order polynomial regression to get the best conditions for ATCh determination. Under optimized experimental conditions, the redox peak current was linear over the concentration range of 0.1-1.5 mM with detection and quantification limit of 0.05 and 0.15 µM respectively and the sensitivity of 1.47 µA mM-1.

Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response.

Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the autoimmune disease myasthenia gravis. Pyridostigmine is also recommended by most Western armies for use as pretreatment under threat of chemical warfare, because of its protective effect against organophosphate poisoning. Because of this drug's quaternary ammonium group, which prevents its penetration through the blood-brain barrier, the symptoms associated with its routine use primarily reflect perturbations in peripheral nervous system functions. Unexpectedly, under a similar regimen, pyridostigmine administration during the Persian Gulf War resulted in a greater than threefold increase in the frequency of reported central nervous system symptoms. This increase was not due to enhanced absorption (or decreased elimination) of the drug, because the inhibition efficacy of serum butyryl-cholinesterase was not modified. Because previous animal studies have shown stress-induced disruption of the blood-brain barrier, an alternative possibility was that the stress situation associated with war allowed pyridostigmine penetration into the brain. Here we report that after mice were subjected to a forced swim protocol (shown previously to simulate stress), an increase in blood-brain barrier permeability reduced the pyridostigmine dose required to inhibit mouse brain AChE activity by 50% to less than 1/100th of the usual dose. Under these conditions, peripherally administered pyridostigmine increased the brain levels of c-fos oncogene and AChE mRNAs. Moreover, in vitro exposure to pyridostigmine increased both electrical excitability and c-fos mRNA levels in brain slices, demonstrating that the observed changes could be directly induced by pyridostigmine. These findings suggest that peripherally acting drugs administered under stress may reach the brain and affect centrally controlled functions.

Gold nanoparticle enlargement coupled with fluorescence quenching for highly sensitive detection of analytes.

We report a versatile and facile route for highly sensitive detection of analytes through coupling the enlargement of gold nanoparticles with fluorescence quenching. The fluorescence intensity of dye molecules (e.g., fluorescein or rhodamine B) significantly decreased with the increasing concentration of reducing agents, such as hydrogen peroxide and hydroquinone. The sensitivity for the detection of reducing agents was much higher than that of other methods based on the absorbance measurement of enlarged gold nanoparticles or quantum-dot-enzyme hybridization. We could successfully detect acetylthiocholine with the detection limit of several nanomolar concentration using an enzymatic reaction by acetylcholine esterase, a key route for the detection of toxic organophosphate compounds. The fluorescence quenching approach described in this report requires only a simple addition of fluorescence dye to the reaction solution without any chemical modification. The strategy of fluorescence quenching coupled with nanoparticle growth would provide a new horizon for the development of highly sensitive optical biosensors.

The use of cholinesterase as potential biomarker: In vitro characterization in the polychaete Capitella teleta.

The ecological relevance of polychaetes coupled with their easy culture and maintenance in the laboratory, has led them to become increasingly used in marine ecotoxicological studies, raising the need to validate frequently applied monitoring tools at various biological levels. The present study was aimed to characterize the cholinesterases (ChE) activity in the polychaete Capitella teleta, using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and four known inhibitors (eserine hemisulfate, BW284c51, iso-OMPA and chlorpyrifos-oxon). Results showed that most of the measured cholinesterase activity was acetylcholinesterase (AChE). Inhibition of enzyme kinetic experiments denoted that sensitivity of C. teleta's ChE to the organophosphorous metabolite chlorpyrifos-oxon (IC50=60.72 nM) was analogous to some fish species. This study highlights the relevance of ChE characterization before its use as a biomarker in ecotoxicology and biomonitoring studies.

Development of a portable biosensor for screening neurotoxic agents in water samples.

A high sensitive portable biosensor system capable of determining the presence of neurotoxic agents in water has been developed. The system consists of (i) a screen-printed electrode with acetylcholinesterase (AChE) immobilized on it, (ii) a self-developed portable potentiostat with an analog to digital (A/D) converter and a serial interface for transferring data to a portable PC and (iii) an own designed software, developed with Lab-Windows CVI, used to record and process the measurements. The system has been developed to perform high precision amperometrical measurements with low drifts, low noise and a good reproducibility. In the configuration depicted, the percentage of AChE inhibition is proportional to the content of neurotoxic agents in a sample. This type of measurement is performed by the steady-state method from the first steady current (by a phosphate buffer solution) and the second steady current (by an enzymatic reaction produced by the addition of acetylthiocholine chloride to the solution). Validation was performed by analyzing spiked water samples containing pesticides. The design is specially suited for screening purposes, does not need sample preconcentration, is totally autonomous and suitable for the field detection of neurotoxic agents in water.