Detection Of Acetylcholinesterase Research Articles

Detection of acetylcholinesterase and butyrylcholinesterase in vitro and in vivo using a new fluorescent probe.

A new fluorescence probe OHPD that could specifically identify acetylcholinesterase/butyrylcholinesterase has been developed and successfully applied to imaging in vivo. Probe OHPD shows significant color change, high selectivity, high sensitivity, and low detection limit for the detection of cholinesterase. Moreover, the real-time imaging in situ indicated that endogenous cholinesterase was mainly present in the yolk sac of zebrafish.

A sensitive and selective fluorescent probe for acetylcholinesterase: Synthesis, performance, mechanism and application

The detection of acetylcholinesterase (AChE) activity is of great significance for studying the physiological functions of AChE and clinical diagnosis of pesticide poisoning. Herein, a small-molecule fluorescent probe BDFA was rationally designed and readily synthesized via a one-step reaction, which enables qualitative and quantitative detection of AChE. BDFA emits a slight fluorescence in an aqueous medium, while the fluorescence is significantly enhanced under the catalysis of AChE. Mechanism studies reveal that BDFA eliminates the N, N-dimethyl carbamate protective group in the presence of AChE and then spontaneously undergoes intramolecular cyclization conversion to generate an intense fluorescent product. Based on the above mechanism, BDFA exhibits a sensitive, selective, rapid and stable “turn-on” fluorescence response to AChE, without interference from pH, ions, thiols, amino acids and other enzymes. The fluorescence intensity of BDFA at 525 nm has a linear relationship with the AChE concentration in the range of 0.0045–1.0 U/mL, and the detection limit is 4.5 mU/mL. Moreover, BDFA is suitable for rapidly diagnosing AChE activity in blood samples, thus providing an efficient and convenient tool for diagnosing organophosphorus and carbamate pesticide poisoning. Compared with the reported AChE fluorescent probes, BDFA exhibits apparent advantages including simple synthesis, low detection limit and fast response speed.

A simple paper-based biosensor for on-site visual detection of organophosphate residues

In general, the laboratory method of analyzing pesticides in vegetables is complicated due to the high cost of equipment and chemicals. The process of analyzing pesticide residues generally requires expertise as well as a significant period of time. In this study, a paper-based biosensor was developed for the detection of acetylcholinesterase (AChE) inhibitors, particularly organophosphate pesticides. The paperbased biosensor was constructed based on the Ellman colorimetric assay by immobilizing AChE on cellulose paper with 2% alginate gel, 0.25% glutaraldehyde and the colorimetric reagent 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) in phosphate buffer (pH 8.0). As a substrate, acetylthiocholine chloride (ATChCl) was used. The results showed that the developed paperbased biosensor has been stable for 2 weeks with a detection limit of 0.03 mM of chlorpyrifos. The paper-based biosensor was applied to detect organophosphate pesticides in vegetables from the farmers’ market, Ratchaburi Province. It was found that the test results of the paper-based biosensor were similar to the commercial GT-test kit. The paper-based biosensor was 10 times faster than the GT-test kit in terms of testing time and the results were easy to identify due to the color-based indicator. As a result, a paper-based biosensor is rapid, portable and easy to use by the general population.

A New Strategy Using a Fluorescent Probe Combined with Polydopamine for Detecting the Activity of Acetylcholinesterase

A water-soluble and sensitive fluorescent probe N,N′-bis[tris-(2-aminoethyl)amine]-3,4,9,10-perylenetetracarboxylic diimide (PTRIS) was synthesized and, in combination with polydopamine (PDA), utilised in the detection of acetylcholinesterase (AChE) activity. PDA is spontaneously polymerized from dopamine (DA) in aerobic and alkaline solutions. The excellent absorption of PDA results in the aggregation of PTRIS around PDA as well as π–π stacking between them, which consequently quenched the fluorescence of PTRIS due to aggregation induced quenching (AIQ) in 9 min. The hydrolysis product of acetylthiocholine (ATCh) catalyzed by AChE, thiocholine (TCh), was proved to inhibit the polymerization of DA, therefore the free monomeric PTRIS retained its strong fluorescence intensity. The fluorescence was switched off and on depending on the activity of AChE. According to the change of fluorescence intensity at 550 nm, the detection limit of AChE was quantified as 0.02 mU mL−1. It was also proved that this probe possessed excellent selectivity for AChE. Tacrine and the organophosphate pesticide diazinon were further evaluated for inhibitor screening. The half-maximal inhibitory concentration value of tacrine and diazinon was calculated to be 1.4 and 1.6 μM respectively, revealing potential applications for inhibition and pesticide detecting.

Novel Carbon/PEDOT/PSS-Based Screen-Printed Biosensors for Acetylcholine Neurotransmitter and Acetylcholinesterase Detection in Human Serum.

New reliable and robust potentiometric ion-selective electrodes were fabricated using poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) as the solid contact between the sensing membrane and electrical substrate for an acetylcholine (ACh) bioassay. A film of PEDOT/PSS was deposited on a solid carbon screen-printed platform made from ceramic substrate. The selective materials used in the ion-selective electrode (ISE) sensor membrane were acetylcholinium tetraphenylborate (ACh/TPB/PEDOT/PSS-ISE) (sensor I) and triacetyl-β-cyclodextrin (β-CD/PEDOT/PSS-ISE) (sensor II). The sensors revealed clear enhanced Nernstian response with a cationic slope 56.4 ± 0.6 and 55.3 ± 1.1 mV/decade toward (ACh+) ions over the dynamic linear range 1.0 × 10−6–1 × 10−3 and 2.0 × 10−6–1.0 × 10−3 M at pH 5 with limits of detection 2.0 × 10−7 and 3.2 × 10−7 M for sensors I and II, respectively. The selectivity behavior of both sensors was also tested and the sensors showed a significant high selectivity toward ACh+ over different common organic and inorganic cations. The stability of the potential response for the solid-contact (SC)/ISEs was evaluated using a chronopotentiometric method and compared with that of electrodes prepared without adding the solid-contact material (PEDOT/PSS). Enhanced accuracy, excellent repeatability, good reproducibility, potential stability, and high selectivity and sensitivity were introduced by these cost-effective sensors. The sensors were also used to measure the activity of acetylcholinesterase (AChE). A linear plot between the initial rate of the hydrolysis of ACh+ substrate and enzyme activity held 5.0 × 10−3–5.2 IU L−1 of AChE enzyme. Application to acetylcholine determination in human serum was done and the results were compared with the standard colorimetric method.

Sensitive colorimetric sensor for point-of-care detection of acetylcholinesterase using cobalt oxyhydroxide nanoflakes.

Point-of-care monitoring of acetylcholinesterase (AChE) is of significant importance for pesticide poisoning and disease diagnosis because it plays a pivotal role in biological nerve conduction systems. Herein, we designed a colorimetric strategy for the facile and accurate detection of AChE based on tandem catalysis with a multi-enzyme system, which is constituted by cobalt oxyhydroxide nanoflakes (CoOOH NFs) and choline oxidase (CHO). In this sensor, AChE catalytically hydrolyzed acetylcholine (ACh) to produce choline, which was further efficiently oxidized by CHO to yield H2O2. CoOOH NFs, as a nanozyme, efficiently catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB with the help of H2O2, accompanied by an enhancement of absorbance intensity. The resulting intensity could be employed as the signal output of the CHO/CoOOH/ACh system in monitoring AChE. Under optimal conditions, the developed sensor possessed a sensitive response to AChE with a detection limit of 33 μU mL-1. Interestingly, the proposed platform was applied to fabricate a paper-based sensor for rapidly recognizing AChE by direct observation with the naked eyes. Combined with a smartphone and ImageJ software, we further developed an image-processing algorithm for the quantitative detection of AChE with highly promising results, which validated the outstanding potential of on-site application in clinical diagnostics and pesticide poisoning.

Biochemistry detection of acetylcholinesterase activity in Trypanosoma evansi and possible functional correlations

Several chemical and immunohistochemical techniques can be used for the detection of acetylcholinesterase (AChE) activity. In this experiment we aimed to detect AChE activity in Trypanosoma evansi. For this, the parasites were isolated from the blood of experimentally infected rats using a DEA-cellulose column. Enzymatic activity was determined in trypomastigote forms at 0, 0.2, 0.4, 0.8 and 1.2 mg/mL of protein concentrations by a standard biochemical protocol. At all concentrations tested, the study showed that T. evansi expresses the enzyme AChE and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.70 μmol of AcSCh/h. Therefore, we concluded that it is possible to biochemically detect AChE in T. evansi, an enzyme that may be associated with vital functions of the parasite and also can be related to chemotherapy treatments, as further discussed in this article.

Morphometric and Histochemical Characteristics of the Cluster Organization of Neurons in Rat Spinal Nerve Sensory Ganglia and the Cervicothoracic Ganglion of the Sympathetic Trunk

The topography of neurons in cervical spinal nerve sensory ganglia and the cervicothoracic ganglion in the sympathetic trunk were studied in adult rats (n = 12). The results showed that these organs are characterized by heterogeneity of neurons in terms of morphometric (stained with thionine by the Nissl method) and histochemical (detection of acetylcholinesterase and NADPH-diaphorase activities) properties. Common features to the organization of ganglia were seen, in terms of the cluster nature of neuron topography. Clusters formed on the heterogeneity principle and consisted of 3–4 to 9–10 neurons. A combined histochemical method used here allowed identification of the histofunctional properties of neurons within clusters in the ganglia studied, as detected using standard histological methods.

Immune detection of acetylcholinesterase in subcellular compartments of Trypanosoma evansi

Trypanosoma evansi is a worldwide distributed hemoparasite with a strong economic impact in veterinary activities. Despite widespread knowledge about the etiology of the disease caused by T. evansi, there are few detailed studies about the metabolism of this parasite. The aim of this study was to determine the presence of Acetylcholinesterase (AChE) in T. evansi through a strategy of subcellular localization and confocal microscopy. The localization of the AChE by differential and isopycnic centrifugation strategy showed that this enzyme has a predominant localization in the glycosome, similar to hexokinase, and it is not present in either the cytosol or the plasma membrane. This study shows novel data that help to understand the non-neuronal role of AChE in the Trypanosomatidae family.

Development of a Bioactive Paper Sensor for Detection of Neurotoxins Using Piezoelectric Inkjet Printing of Sol−Gel-Derived Bioinks

There is an increasing interest in new strategies to rapidly detect analytes of clinical and environmental interest without the need for sophisticated instrumentation. As an example, the detection of acetylcholinesterase (AChE) inhibitors such as neurotoxins and organophosphates has implications for neuroscience, drug assessment, pharmaceutical development, and environmental monitoring. Functionalization of surfaces with multiple reagents, including enzymes and chromogenic reagents, is a critical component for the effective development of "dipstick" or lateral flow biosensors. Herein, we describe a novel paper-based solid-phase biosensor that utilizes piezoelectric inkjet printing of biocompatible, enzyme-doped, sol-gel-based inks to create colorimetric sensor strips. For this purpose, polyvinylamine (PVAm, which captures anionic agents) was first printed and then AChE was overprinted by sandwiching the enzyme within two layers of biocompatible sol-gel-derived silica on paper. AChE inhibitors, including paraoxon and aflatoxin B1, were detected successfully using this sensor by measuring the residual activity of AChE on paper, using Ellman's colorimetric assay, with capture of the 5-thio-2-nitrobenzoate (TNB(-)) product on the PVAm layer. The assay provided good detection limits (paraoxon, approximately 100 nM; aflatoxin B1, approximately 30 nM) and rapid response times (<5 min). Detection could be achieved either by eye or using a digital camera and image analysis software, avoiding the need for expensive and sophisticated instrumentation. We demonstrate that the bioactive paper strip can be used either as a dipstick or a lateral flow-based biosensor. The use of sol-gel-based entrapment produced a sensor that retained enzyme activity and gave reproducible results after storage at 4 degrees C for at least 60 days, making the system suitable for storage and use in the field.

Serum Acetyl Cholinesterase as a Biomarker of Arsenic Induced Neurotoxicity in Sprague-Dawley Rats

Arsenic is an environmental toxicant, and one of the major mechanisms by which it exerts its toxic effect is through an impairment of cellular respiration by inhibition of various mitochondrial enzymes, and the uncoupling of oxidative phosphorylation. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Recent studies have pointed out that arsenic toxicity is associated with the formation of reactive oxygen species, which may cause severe injury/damage to the nervous system. The main objective of this study was to conduct biochemical analysis to determine the effect of arsenic trioxide on the activity of acetyl cholinesterase; a critical important nervous system enzyme that hydrolyzes the neurotransmitter acetylcholine. Four groups of six male rats each weighing an average 60 +/- 2 g were used in this study. Arsenic trioxide was intraperitoneally administered to the rats at the doses of 5, 10, 15, 20mg/kg body weight (BW), one dose per 24 hour given for five days. A control group was also made of 6 animals injected with distilled water without chemical. Following anaesthesia, blood specimens were immediately collected using heparinized syringes, and acetyl cholinesterase detection and quantification were performed in serum samples by spectrophotometry. Arsenic trioxide exposure significantly decreased the activity of cholinesterase in the Sprague-Dawley rats. Acetyl cholinesterase activities of 6895 +/- 822, 5697 +/- 468, 5069 +/- 624, 4054 +/- 980, and 3158 +/- 648 U/L were recorded for 0, 5, 10, 15, and 20 mg/kg, respectively; indicating a gradual decrease in acetyl cholinesterase activity with increasing doses of arsenic. These findings indicate that acetyl cholinesterase is a candidate biomarker for arsenic-induced neurotoxicity in Sprague-Dawley rats.

Defective cholinergic innervation in pyloric muscle of patients with hypertrophic pyloric stenosis

The etiology of hypertrophic pyloric stenosis (HPS) is not known. Many investigators have studied the myenteric plexus and muscle in HPS, but there are no reports of cholinergic nerve distribution in this condition. A histochemical technique for the detection of acetylcholinesterase (AChE) has been used to study cholinergic nerve distribution. Recently, it has been shown that the development and survival of cholinergic neurons in the peripheral and central nervous system depend on the presence of nerve growth factor (NGF) and its receptor (NGFR). We examined pyloric muscle from 18 patients with HPS and 10 controls using monoclonal antibody to NGFR and AChE histochemistry. Myenteric plexus displayed strong NGFR and AChE reactivity. Quantitative assessment of immunoreactivity in nerve fibres within pyloric muscle demonstrated selective absence of NGFR and AChE-positive nerve fibres in the circular and longitudinal muscle of HPS, whereas these fibres were in abundance in the muscle of controls. These findings suggest that NGFR is an important neurotrophic factor in the maintenance of cholinergic neuronal function and that NGFR deficiency, resulting in defective cholinergic innervation of pyloric muscle, may have an important role in the pathogenesis of HPS.

Scanning electron microscopy and X-ray microprobe analysis in detection of acetylcholinesterase in cultured embryonal carcinoma cells.

Mouse F9 cells, induced by retinoic acid and dibutyryl cyclic adenosine monophosphate (cAMP) to differentiate into neural-type cells, were incubated for localization of specific acetylcholinesterase (AChE) activity according to the Karnovsky-Roots method where the final enzymatic reaction product is crystalline cupric ferrocyanide and cuprous thiocholine iodide. By scanning electron microscopy (SEM) neural-type cells with long processes were seen. Most of these cells exhibited crystalline precipitates on their surface that in microprobe analysis contained copper, iron, and sulfur. These elements were also detected in some of the neural-type cells that had no visible surface precipitates. Thus, the X-ray analysis also revealed intracellular enzymatic activity. Undifferentiated rounded cells, devoid of AChE activity at the light microscope level, did not show any surface precipitates by SEM and lacked copper, iron, and sulfur emission peaks in the elementary analysis. These results demonstrate that elementary analysis of cytochemical enzymatic reaction products by SEM can be used in identifying cells.

Contribution to the Study of the Pineal Stalk Nerve Fibres in the Rat

Publisher Summary The problem whether the rat pineal gland is innervated via its stalk from the central nervous system has never been satisfactorily answered. Most authors admit that the gland is uniformly innervated by orthosympathetic fibres coming from the superior cervical ganglia, the only rare fibres seen in the stalk and coming from the epithalamus being aberrant and without any functional significance. However, some experiments carried out in the last few years pointed to the persistence of nerve fibers and of electrical reactivity in the pineal gland after bilateral superior cervical ganglionectomy. For these reasons and for describing the pathways of these fibers, a multi-aspect histological approach was undertaken in which serial paraffin sections of rat brains, especially stained to show nerve fibers, as well as semithin and ultrathin sections of the pineal complex were studied. From these experiments, it appeared that the stalk is composed of at least two types of nerve fibers: (1) afferent unmyelinated fibers originating in the superior cervical ganglia and directed to the deep pineal and the habenula and (2) Efferent myelinated and unmyelinated fibers coming from the habenular and posterior commissures and entering the superficial pineal, which lose their sheet and break up into many axonal branches ending in intercellular spaces with small bulbs containing clear synaptosomes. This morphological approach has been completed by a detection of acetylcholinesterase and catecholamines.