Oxidation Of Thiocholine Research Articles

Amperometric biosensing of organophosphate and organocarbamate pesticides utilizing polypyrrole entrapped acetylcholinesterase electrode

The work presented here describes a novel, easy and low-cost method of fabrication of a highly sensitive acetylcholinesterase biosensor and its application to detect organophosphate and organocarbamate pesticides. Acetylcholinesterase was electro-immobilized into a thick conducting layer of polypyrrole. Porcine skin gelatin and gluteraldehyde mixture was used for stabilizing the system. Acetylthiocholine chloride was used as the substrate. Polypyrrole catalyzed the electrochemical oxidation of thiocholine and promoted the electron transfer, thus lowering the oxidation potential and increasing the detection sensitivity. Electro oxidation of thiocholine in polypyrrole matrix occurred at 0.1V under low potential scan rate. The thiocholine sensitivity of the electrode was found to be 143mA/M. The sensor was applied to detect the sample organophosphate pesticide ethylparaoxon and organocarbamate pesticide carbofuran. The detection limit for paraoxon was found to be 1.1ppb and that for carbofuran is 0.12ppb. The sensor showed good intra and inter state precision with relative standard deviation (RSD) 0.742% and 6.56% respectively. Both dry and wet storage stability were studied. The sensor stored at 0°C in dry condition had a good storage stability retaining 70% of its original activity for 4 months. During wet storage, the activity decrease followed the same trend, however, the operational stability at the end of the storage period was found to be less compared to the dry storage case. The developed biosensor is as a promising new tool for analysis of cholinesterase inhibitors.

Development of acetylcholinesterase biosensor based on platinum–carbon aerogels composite for determination of organophosphorus pesticides

A fast, sensitive and stable acetylcholinesterase (AChE) biosensor was developed based on platinum–carbon aerogels (Pt–CAs) composite to detect organophosphorous pesticides (OPs). The Pt–CAs composite was prepared by the sol–gel polymerization and the ethylene glycol liquid-phase reduction method, then was firstly used to modify boron-doped diamond (BDD) electrode for AChE immobilization. The electron transfer resistance of the Pt–CAs/BDD electrode was smaller than that of the bare BDD electrode, indicating that the presence of Pt–CAs composite on the electrode surface could improve the reactive site, reduce the interfacial resistance, and make the electron transfer easier. In comparison with AChE/BDD electrode, the negative shift of peak potential and the increase of peak current upon the oxidation of thiocholine were obtained on AChE/Pt–CAs/BDD electrode. Using methamidophos as model compound, the conditions for OPs detection were optimized. Under the optimized conditions, the inhibition rate of methamidophos and monocrotophos were linearly proportional to their concentrations in the range of 10−11–10−6 M. The detection limits were found to be 3.1 × 10−13 M (0.05 ppt) for methamidophos and 2.7 × 10−12 M (0.6 ppt) for monocrotophos. The developed biosensor exhibited good reproducibility and acceptable stability.

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene–tetracyanoquinodimethane/ionic liquid conductive gels

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF–TCNQ–ionic liquid gel thiocholine sensor. The TTF–TCNQ–ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF–TCNQ–ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2μAmM−1cm−2 and a low detection limit equal to 7.6μM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol–gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26pM eserine and 0.3nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

Electrochemically reduced graphene oxide and Nafion nanocomposite for ultralow potential detection of organophosphate pesticide

Abstract The development of rapid and field monitoring tools is highly desirable for the sensitive detection of organophosphate pesticide (OP) residues in water and agriculture products without interference. A novel disposable acetylcholinesterase (AChE) biosensor with low detecting potential (+50 mV), good selectivity, and high sensitivity was developed based on electrochemically reduced graphene oxide and Nafion hybrid nanocomposite (Er-GRO–Nafion) modified electrode. The Er-GRO–Nafion nanocomposite with conductive and three-dimensional interpenetrating network owns excellent characters as electrode modification material. It effectively promoted the electron transfer rate of electrode interface, could catalyze the anodic oxidation of thiocholine at ultra-low potential, and facilitated the access of substrates to the active centers. The modified electrode was used for the electrochemical detection of dichlorvos after adsorbing AChE on the biocompatible matrix. By dexterously controlling the electrochemical reduction variables and optimizing the electrode preparation parameters, the proposed electrochemical biosensor showed high sensitivity, wide linear range (from 5.0 to 100 ng mL−1 and from 1.0 to 20 μg mL−1), low detection limit (2.0 ng mL−1), good stability and reproducibility. It also had high precision for the real sample analysis, which indicated the good perspective for field application.

Biosensor based on Prussian blue nanocubes/reduced graphene oxide nanocomposite for detection of organophosphorus pesticides

We demonstrate a facile procedure to efficiently prepare Prussian blue nanocubes/reduced graphene oxide (PBNCs/rGO) nanocomposite by directly mixing Fe(3+) and [Fe(CN)(6)]((3)-) in the presence of GO in polyethyleneimine aqueous solution, resulting in a novel acetylcholinesterase (AChE) biosensor for detection of organophosphorus pesticides (OPs). The obtained nanocomposite was characterized by X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) microanalysis. It was clearly observed that the nanosheet has been decorated with cubic PB nanoparticles and nearly all the nanoparticles are distributed uniformly only on the surface of the reduced GO. No isolated PB nanoparticles were observed, indicating the strong interaction between PB nanocubes and the reduced GO and the formation of PBNCs/rGO nanocomposite. The obtained PBNCs/rGO based AChE biosensor make the peak potential shift negatively to 220 mV. The over-potential decreases ∼460 mV compared to that on a bare electrode, suggesting that PBNCs/rGO has a high electrocatalytic activity towards the oxidation of thiocholine. The AChE biosensor shows rapid response and high sensitivity for detection of monocrotophos with a linear range from 1.0 to 600 ng mL(-1) and a detection limit of 0.1 ng mL(-1). These results suggest that the PBNCs/rGO hybrids nanocomposite exhibited high electrocatalytic activity towards the oxidation of thiocholine, which lead to the sensitive detection of OP pesticides.

A novel biosensor based on acetylecholinesterase/prussian blue–chitosan modified electrode for detection of carbaryl pesticides

A sensitive, fast and cheap sensor for quantitative determination of carbaryl pesticide using amperometric acetylcholinesterase (AChE) biosensor based on prussian blue (PB)–chitosan (CHIT) hybrid film was reported. This biosensor was fabricated by firstly electrodepositing PB–CHIT hybrid film on glassy carbon electrode (GCE) and subsequently assembling AChE on PB–CHIT hybrid film. The PB–CHIT hybrid film exhibited a good biocompatibility with AChE and effectively immobilized AChE on it. Nanosized PB uniformly distributed in the PB–CHIT hybrid films and showed a good electrocatalytic activity toward oxidation of thiocholine, which was a product from the hydrolysis of acetylthiocholine catalyzed by AChE. The oxidation potential of thiocholine was decreased from 0.68 V to 0.32 V and accordingly the sensitivity of biosensor was largely improved. The inhibition of carbaryl on the activity of AChE was proportional to carbaryl concentration in the ranges from 0.01 to 0.4 μM and from 1.0 to 5.0 μM with the correlation coefficients of 0.9996 and 0.9997, respectively. The detection limit was calculated to be about 3 nM. The biosensor provided a new promising tool for pesticide analysis.

Voltammetric Biosensor Based on Acetylcholinesterase and Different Immobilization Protocols: A Simple Tool for Toxic Organophosphate Assay

The present study is aimed at construction of biosensors consisting of a screen printed sensor with a platinum working electrode and immobilized acetylcholinesterase (AChE) as a biorecognition element. Several immobilization protocols such as precipitation with glutaraldehyde, immobilization onto spherical graphite microparticles, sorption, interception into gelatin, and sol-gel were used in order to construct biosensors suitable for practical performance. The activity of AChE was evaluated by means of voltammetric oxidation of thiocholine originating from acetylthiocholine. Square wave and cyclic voltammetry were used throughout the experiments. Suitability of the constructed biosensors was evaluated using the toxic organophosphate diisopropylfluorophosphate.

Acetylcholinesterase biosensor based on single-walled carbon nanotubes—Co phtalocyanine for organophosphorus pesticides detection

A simple and reliable technique has been developed for the construction of an amperometric acetylcholinesterase biosensor based on screen-printed carbon electrodes. For the first time, one-step modification using single-walled carbon nanotubes and Co phtalocyanine has been proposed to decrease the working potential and to increase the signal of thiocholine oxidation. The biosensor developed made it possible to detect 5–50ppb of paraoxon and 2–50ppb of malaoxon with detection limits of 3 and 2ppb, respectively (incubation 15min). The biosensor showed high reproducibility when measurements of the substrate and inhibitor were performed (R.S.D. about 1% and 2.5%, respectively). The reliability of the inhibition measurements was confirmed by testing spiked samples of sparkling and tape waters.

A novel, sensitive, reusable and low potential acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel

A novel, low potential and highly sensitive acetylcholinesterase (AChE) biosensor was developed based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotube composite gel thiocholine sensor. Composite gel promoted electron transfer reaction at a lower potential (+50 mV) and catalyzed electrochemical oxidation of thiocholine with high sensitivity. AChE was immobilized in sol–gel matrix that provides a good support for enzyme without any inhibition effect from the ionic liquid. The amount of immobilized enzyme and incubation time with chlorpyrifos were optimized. Chlorpyrifos could be determined in the range of 10 −8–10 −6 M with a detection limit of 4 nM. Fast and efficient enzyme reactivation was obtained at low obidoxime concentration (0.1 mM). Moreover, the biosensor exhibited a good stability and reproducibility and could be use for multiple determinations of pesticide with no loss of the enzyme activity.

Derivatization of SWCNTs with cobalt phthalocyanine residues and applications in screen printed electrodes for electrochemical detection of thiocholine

Single-walled carbon nanotubes (SWCNTs) derivatized with cobalt phthalocyanine (CoPh) were applied onto screen-printed graphite electrodes (SPEs) to be used for the low-potential electrochemical oxidation of thiocholine (TCh). Covalent attachment of CoPh to SWCNTs via stable sulfonamide bonds was confirmed by Raman/FT-IR spectroscopy and thermogravimetric analysis (TGA) coupled with FT-IR detection. The resulting modified SPE surfaces (CoPh-SWCNT-SPEs) were characterized by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) with the redox probe [F 3(CN) 6] 3−/4−. Detection of TCh was accomplished using cyclic voltammetry and amperometry; a lower overpotential (100 mV vs. Ag/AgCl pseudoreference electrode) was obtained using CoPh-SWCNT-SPEs as compared to unmodified SPEs and SPEs modified with non-functionalized SWCNTs (SWCNT-SPEs). The linear range for TCh detection was 0.077–0.45 mM, with a sensitivity of 5.11 × 10 −1 μA mM −1 and a limit of detection of 0.038 mM according to the 3 s/m definition.

Highly Sensitive Detection of Organophosphate Insecticides Using Biosensors Based on Genetically Engineered Acetylcholinesterase and Poly(3,4-Ethylenedioxythiophene)

A poly(3,4-ethylenedioxythiophene) (PEDOT) conducting ink is presented as a new electroactive material to be incorporated in acetylcholinesterase-(AChE-) based screen printed biosensors, acting not only as a conducting template but also as an electrochemical mediator for thiocholine oxidation. Two different strategies have been studied for the chemical synthesis of PEDOT: (a) a classical oxidative polymerisation and (b) a more innovative enzymatic polymerisation, giving a water-soluble PEDOT. The use of this water-soluble conducting polymer as mediator in screen-printed biosensors enables its deposition by printing like the rest of the layers. Highly sensitive acetylcholinesterase-(AChE-) based screen-printed biosensors have been constructed using both classical and enzymatic PEDOT, in combination with genetically modified AChE. These electrodes allow the measurement of thiocholine oxidation at potentials of 100 mV versus Ag/AgCl reference electrode through the mediation of PEDOT. Inhibition of thiocholine production in presence of CPO allow for detection of this pesticide in concentrations as low as 1·10−10 M.

Low potential thiocholine oxidation at carbon nanotube-ionic liquid gel sensor

Low-potential thiocholine (TCh) detection was achieved at ionic liquid–multi-walled carbon nanotube (IL–MWCNT) modified electrodes. Cyclic voltammetry and amperometric characteristics of eight different thiocholine sensors were compared. With an oxidation potential between 0 and +50 mV ( vs. Ag/AgCl) [EMIM][OTF]-, [EMIM][NTF 2]-, [EMIM][TCB]- and [BMIM][BF 4]–MWCNT gels offer a substantial decrease of the working potential comparing with other types of thiocholine sensors. Correlation between the physical properties and electrochemical behaviour of IL–MWCNT gels is also reported. Electrochemical impedance spectroscopy (EIS) and pH-dependence experiments suggest the existence of an electrostratic interaction between thiocholine and composite gels which favours the electrochemical reaction. The catalytical rate constant and the diffusion coefficient of thiocholine were calculated from chronoamperometric data. A very good sensitivity between 36 and 45 μA/mM cm 2 was achieved for acetylthiocholine detection using selected gel-modified sensors.

Screen-printed poly(3,4-ethylenedioxythiophene) (PEDOT): A new electrochemical mediator for acetylcholinesterase-based biosensors

This work describes the use of a PEDOT:PSS-based conductive polymer for designing AChE-based biosensors. The transducers were obtained directly by screen-printing a PEDOT:PSS suspension on the surface of thick film carbon electrodes. The obtained working electrodes showed a high conductivity when compared with electrodes modified with conventional mediators like cobalt phthalocyanine or tetracyanoquinodimethane. The PEDOT:PSS polymer was shown to be suitable for thiocholine oxidation, allowing the measurement of AChE activity at 100 mV vs Ag/AgCl. The high conductivity of PEDOT:PSS allowed the accurate detection of the organophosphate insecticide chlorpyrifos-oxon at concentrations as low as 4 × 10 −9 M, corresponding to an inhibition ratio of 5%.

Development of Au nanoparticles dispersed carbon nanotube-based biosensor for the detection of paraoxon

A disposable and sensitive biosensor has been fabricated for the detection of the organophosphorous (OP) compound paraoxon using an amperometric technique. For the measurements, gold nanoparticles dispersed on the outer surface of multiwalled carbon nanotubes (Au-MWNTs) has been used as the electrode material, as it possesses high electron transfer rates and provides large immobilization sites for the bioenzymes, which combines with the high electrocatalytic activity of MWNTs for thiocholine oxidation at low potential. Au-MWNTs have been synthesized by chemically reducing Au salt over functionalized MWNTs, and the same has been characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and high resolution transmission electron microscopic (HRTEM) techniques. The ability of the Au-MWNTs nanocomposite-based biosensor has been demonstrated to reliably measure the concentration of paraoxon in the nanomolar range.

Amperometric biosensor based on immobilized acetylcholinesterase on gold nanoparticles and silk fibroin modified platinum electrode for detection of methyl paraoxon, carbofuran and phoxim

A new sensitive and stable amperometric biosensor for the detection of methyl paraoxon, carbofuran and phoxim had been developed based on immobilization of acetylcholinasterase (AChE) on gold nanoparticles (AuNPs) and silk fibroin (SF) modified platinum electrode. The SF provided a biocompatible microenvironment around the enzyme molecule to stabilize its biological activity and effectively prevented it from leaking out of platinum electrode surface. In the presence of acetylthiocholine chloride (ATCl) as a substrate, AuNPs promoted electron transfer reaction at a lower potential and catalyzed the electrochemical oxidation of thiocholine (TCh), thus increasing detection sensitivity. The influences of pH, enzyme loading, temperature and inhibition time on the response of the fabricated biosensor were investigated. Under optimum conditions, the inhibition percentages of methyl paraoxon, carbofuran and phoxim were proportional to their concentrations in the range of 6 × 10 −11–5 × 10 −8 M, 2 × 10 −10–1 × 10 −7 M and 5 × 10 −9–2 × 10 −7 M, respectively. The detection limits were found to be 2 × 10 −11 M for methyl paraoxon, 1 × 10 −10 M for carbofuran and 2 × 10 −9 M for phoxim. Moreover, the fabricated biosensor had good reproducibility and acceptable stability. The biosensor is a promising new tool for pesticide analysis.

Electrochemical study of rat brain acetylcholinesterase inhibition by chlorofos: Kinetic aspects and analytical applications

An electrochemical sensor was applied for investigating the immobilized rat brain acetylcholinesterase inhibition by chlorofos. Two alternative routes were explored as response-generating reactions: (i) direct electrochemical oxidation of thiocholine produced upon acetylthiocholine enzymatic hydrolysis and (ii) reduction of the produced thiocholine with hexacyanoferrate (III), followed by hexacyanoferrate (II) electrochemical detection. The advantages of the direct way are simplicity and higher sensitivity compared to the indirect one, which however avoids the interferences because of the lower potential applied. Enzyme inhibition was identified as competitive, the K M app increasing from 1.31 to 1.43 mmol L −1 with chlorofos concentration in the range 0.2–1.0 mmol L −1 and the maximal rate of the enzyme reaction remaining constant ( I max = 579.30 ± 5.71 μA) in the presence of chlorofos. The inhibition constant was calculated using the Dixon method ( K I = 10.07 mmol L −1). The suppression of the acetylcholinesterase activity by the inhibitor, expressed as current decrease at a constant substrate concentration, was exploited for chlorofos quantification optimized by the design of experiments methodology. Optimal response was obtained for an acetylthiocholine concentration of 0.2 mmol L −1, at 26 °C and pH 7.

Variation of cholinesterase-based biosensor sensitivity to inhibition by organophosphate due to ionizing radiation.

A cholinesterase based biosensor was constructed in order to assess the effects of ionizing radiation on exposed AChE. Although the primary objective of the experiment was to investigate the effect of ionizing radiation on the activity of the biosensor, no changes in cholinesterase activity were observed. Current provided by oxidation of thiocholine previously created from acetylthiocholine by enzyme catalyzed reaction was in a range 395–455 nA. No significant influence of radiation on AChE activity was found, despite the current variation. However, a surprising phenomenon was observed when a model organophosphate paraoxon was assayed. Irradiated biosensors seem to be more susceptible to the inhibitory effects of paraoxon. Control biosensors provided a 94 ± 5 nA current after exposure to 1 ppm paraoxon. The biosensors irradiated by a 5 kGy radiation dose and exposed to paraoxon provided a current of 49 ± 6 nA. Irradiation by doses ranging from 5 mGy to 100 kGy were investigated and the mentioned effect was confirmed at doses above 50 Gy. After the first promising experiments, biosensors irradiated by 5 kGy were used for calibration on paraoxon and compared with the control biosensors. Limits of detection 2.5 and 3.8 ppb were achieved for irradiated and non-irradiated biosensors respectively. The overall impact of this effect is discussed.

Nanomaterials - Acetylcholinesterase Enzyme Matrices for Organophosphorus Pesticides Electrochemical Sensors: A Review

Acetylcholinesterase (AChE) is an important cholinesterase enzyme present in the synaptic clefts of living organisms. It maintains the levels of the neurotransmitter acetylcholine by catalyzing the hydrolysis reaction of acetylcholine to thiocholine. This catalytic activity of AChE is drastically inhibited by trace amounts of organophosphorus (OP) pesticides present in the environment. As a result, effective monitoring of OP pesticides in the environment is very desirable and has been done successfully in recent years with the use of nanomaterial-based AChE sensors. In such sensors, the enzyme AChE has been immobilized onto nanomaterials like multiwalled carbon nanotubes, gold nanoparticles, zirconia nanoparticles, cadmium sulphide nano particles or quantum dots. These nanomaterial matrices promote significant enhancements of OP pesticide determinations, with the thiocholine oxidation occurring at much lower oxidation potentials. Moreover, nanomaterial-based AChE sensors with rapid response, increased operational and long storage stability are extremely well suited for OP pesticide determination over a wide concentration range. In this review, the unique advantages of using nanomaterials as AChE immobilization matrices are discussed. Further, detection limits, sensitivities and correlation coefficients obtained using various electroanalytical techniques have also been compared with chromatographic techniques.

Nerve Agents Assay Using Cholinesterase Based Biosensor

AbstractElectrochemical biosensor based on electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) was performed for assay of nerve agents – tabun, sarin, soman, cyclosarin, and VX. The biosensor used AChE as biorecognition element. The presence of nerve agents was accompanied by a strong inhibition of AChE activity. Enzyme activity is easily measurable by electrochemical oxidation of thiocholine created from acetylthiocholine (ATChCl) by AChE‐catalyzed hydrolysis. The tested nerve agents were successfully assayed. The best limits of detection were achieved for sarin (5.88×10−10 M) and VX (8.51×10−10 M) after one‐minute assay. The biosensor was found long term stable at low as well as laboratory temperature.

Electrocatalytic oxidation of thiocholine at chemically modified cobalt hexacyanoferrate screen-printed electrodes

In this work, cobalt hexacyanoferrate (CoHCF) modified screen-printed electrodes were developed and characterised electrochemically. The effect of different cations was investigated showing a more reversible system with a well-defined peak around +0.35 V vs. Ag/AgCl when Na + was used as cation in the supporting electrolyte. The transfer coefficient ( α) and the apparent charge transfer rate constant ( k s) for electron transfer between the electrode and the CoHCF layer was calculated. The electrocatalytic behaviour of CoHCF towards oxidation of enzymatically generated thiocholine was thoroughly investigated. Cyclic voltammetry experiments performed in thiocholine solution showed the classical shape of a mediated redox system. The diffusion constant of thiocholine was estimated using chronoamperometry. Also, the catalytic rate constant k of thiocholine at CoHCF–SPE was determined. The current due to thiocholine mediated oxidation was then evaluated in batch amperometric mode at +0.5 V vs. Ag/AgCl obtaining a linear range (5 × 10 −7–1 × 10 −5 mol L −1) with a low detection limit and a high sensitivity equal to 5 × 10 −7 mol L −1 and 435 mA mol −1 L cm −2, respectively.