Showing papers on "Acetylthiocholine published in 2023"

Acetylcholinesterase Activity Staining in Freshwater Planarians

Abstract: The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system. © 2023 Wiley Periodicals LLC.

Dual-mode fluorescence and colorimetric determination of acetylcholinesterase accomplished by BSA-MnO2 QDs with oxidase-mimetic activity

Abstract: It has been demonstrated that acetylcholinesterase (AChE) contributed significantly to the maintenance of normal cholinergic neurophysiology, and its abnormal expression has been linked to several diseases. Due to intrinsic self-confirmation and self-adjustment, multi-mode detection was able to provide more dependable and accurate results when compared to commonly used single-signal detection. Despite this, the problem of achieving multi-mode analysis of AChE has not been solved. In this study, we developed a dual-mode approach to determine AChE by multifunctional BSA-MnO2 QDs with oxidase-mimetic activity. A visible color signal was induced by BSA-MnO2 QDs which exhibit good oxidase-mimetic activity to oxidize 3, 3′, 5, 5′-tetramethylbenzidine (TMB) to blue oxide; by employing AChE to hydrolyze acetylthiocholine (ATCh), the generated thiocholine (TCh) can inhibit BSA-MnO2 QDs activity, that displayed an outstanding fluorescence signal but the oxidase-mimetic activity decreased. Following this principle, dual-mode determination of AChE with high sensitivity was achieved. By analyzing real samples, it was verified that the method demonstrated excellent interference tolerance and reliability. In addition to having excellent properties and practicability, the proffered dual-mode method was likely to be an effective method for detecting AChE with high efficiency and reliability.

An Amperometric Acetylcholinesterase Biosensor Based on Polyvinyl‐Alcohol‐Capped Silver Nanoparticles (AgNPs@PVA) for Detection of Organophosphate Pesticides

Abstract: In the present study polyvinyl alcohol (PVA) capped AgNPs ([email protected]) were used as nanocarrier for fabrication of an acetylcholinesterase (AChE) biosensor for organophosphate (OP) pesticide detection. AChE enzyme was immobilized via glutaraldehyde on drop casted [email protected] layer on glassy carbon electrode (GCE) for fabrication of the electrochemical biosensor. Through differential pulse voltammetry (DPV) the biosensor measured peak current at 0.6 V generated by AChE mediated hydrolysis of acetylthiocholine (ATCl) to thiocholine and its subsequent oxidation to dimer. By hindering AChE hydrolysis activity of the sensor probe by a model OP pesticide, malathion, the pesticide biosensor was developed as the inhibition effect was found to be proportional to the concentration of malathion. The fabricated biosensor exhibits linearity at range of 0.01 to 1 ng/mL of malathion at optimal conditions with detection limit (LOD) of 2.6 pg/mL. The fabricated AChE biosensor showed good selectivity for OP pesticides and exhibited good efficiency in real sample analysis.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae)

Abstract: A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.

An ultrasensitive “mix-and-detect” kind of fluorescent biosensor for malaoxon detection using the AChE-ATCh-Ag-GO system

Abstract: Malaoxon, a highly toxic metabolite of malathion, can lead to severe harm or death if ingested. This study introduces a rapid and innovative fluorescent biosensor that relies on acetylcholinesterase (AChE) inhibition for detecting malaoxon using Ag-GO nanohybrid. The synthesized nanomaterials (GO, Ag-GO) were evaluated with multiple characterization methods to confirm their elemental composition, morphology, and crystalline structure. The fabricated biosensor works by utilizing AChE to catalyze the substrate acetylthiocholine (ATCh), which generates positively charged thiocholine (TCh) and triggers citrate-coated AgNP aggregation on the GO sheet, leading to an increase in fluorescence emission at 423 nm. However, the presence of malaoxon inhibits the AChE action and reduces the production of TCh, resulting in a decrease in fluorescence emission intensity. This mechanism allows the biosensor to detect a wide range of malaoxon concentrations with excellent linearity and low LOD and LOQ values of 0.001 pM to 1000 pM, 0.9 fM, and 3 fM, respectively. The biosensor also demonstrated superior inhibitory efficacy towards malaoxon compared to other OP pesticides, indicating its resistance to external influences. In practical sample testing, the biosensor displayed recoveries of over 98% with extremely low RSD% values. Based on the results obtained from the study, it can be concluded that the developed biosensor has the potential to be used in various real-world applications for detecting malaoxon in food, and water samples, with high sensitivity, accuracy, and reliability.

Construction of a ratiometric fluorescent sensing platform based on near-infrared carbon dots for organophosphorus pesticides detection

Abstract: In this work, a convenient ratiometric fluorescent platform was designed to measure organophosphorus pesticides (OPs) based on acetylcholinesterase (AChE), acetylthiocholine (ATCh), manganese dioxide nanosheets (MnO2), near-infrared carbon dots (RCDs) and o-phenylenediamine (OPD). In this platform, a direct oxidation of OPD by MnO2 generated the luminescent product 2,3-diaminophenolazine (DAP) through intrinsic oxidase activity, while RCDs served as a fluorescent reference indicator. In the presence of AChE and ATCh, the enzymatic hydrolysate thiocholine (TCh) would reduce MnO2 nanosheets to Mn2+, leading to the quenching of DAP fluorescence. On the other hand, OPs can inhibit the catabolism of ATCh by AChE thus acting as a recognizer of OPs. According to these reactions, OPs were quantitatively analyzed by the intensity ratio of fluorescence emitted from RCDs and DAP (F560/F676). The constructed platform can detect OPs with the range of 0.2–0.6 μM with a detection limit of 4.3 nM. Figure A ratiometric fluorescent probe based on carbon dots was obtained and using it to determine the concentration of organophosphorus pesticides

An acetylcholinesterase-based biosensor for isoprocarb using a gold nanoparticles-polyaniline modified graphite pencil electrode

Abstract: An analysis tool for isoprocarb has been successfully developed as a biosensor system based on enzymatic inhibition of acetylcholinesterase (AChE) by isoprocarb. A gold nanoparticles-polyaniline modified graphite pencil electrode (AuNPs-PANI-GPE) was utilized to detect the change of thiocholine in the presence of isoprocarb. This electrode was prepared by two cyclic voltammetry steps, including the electro-polymerization of aniline on a graphite pencil and the electro-deposition of gold nanoparticles on the polyaniline surface. Characterization performed by SEM-EDX indicates that 8-80 nm size of gold nanoparticles could be deposited on the surface of polyaniline-modified graphite pencil (PANI-GPE). Electrochemical characterization using cyclic voltammetry suggested that the active surface area of the prepared electrode was 0.17019 cm2, which was about 4 times higher than (PANI-GPE) and 13 times higher than the unmodified GPE. Furthermore, an oxidation peak of thiocholine could be observed at the modified GPE at a potential of + 0.675 V (vs. Ag/AgCl), formed by an enzymatic reaction of AChE in the presence of acetylthiocholine. This peak current was found to linearly increase with acetylthiocholine concentrations, while in the presence of isoprocarb in a constant concentration of AChE and acetylthiocholine the peak linearly decreases. At the optimum condition of 0.1 M phosphate buffer solution pH 7.4 containing 0.1 M KCl; 100 mU/ml AChE; and 1 mM acetylthiocholine chloride in an inhibition and contact time of 25 and 15 min, respectively, a linear calibration curve of isoprocarb in the concentration range of 0.05-1.0 μM could be provided. Estimated limits of detection and quantifications of 0.1615 nM and 0.5382 nM, respectively, with a sensitivity of 1.7771 μA/μM.mm2 could be achieved. Furthermore, an excellent stability for 8 times measurements was observed with an RSD of 4.87%, suggesting that the developed tool is promising for the real detection of isoprocarb.