Comparison of pesticide sensitivity by electrochemical test based on acetylcholinesterase biosensor

Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method has been devised for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor. Because of the excellent biocompatibility and good stability of chitosan matrix, it prevented leakage of the AChE from electrode. Multiwall carbon nanotube (MWNT) promoted electron transfer reaction at a lower potential and catalyzed the electro-oxidation of thiocholine, thus amplifying the sensitivity and amperometric response of the biosensor. Four pesticides of carbaryl, malathion, dimethoate and monocrotophos were selected to discuss their inhibition efficiencies to AChE. The inhibition curves were similar to Michealis-Menten and the Michealis-Menten constants (Km) were calculated to be 0.96 microM, 1.78 microM, 1.97 microM and 4.28 microM, respectively. Ninety-five percent reactivation of the inhibited AChE could be regenerated using pralidoxime iodide within 8 min. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, low cost and simplified procedures, which is a promising new tool for comparison of pesticide sensitivity and for selection of the most efficient enzyme inhibitors.     

Sensitive detection of organophosphorus pesticides using a needle type amperometric acetylcholinesterase-based bioelectrode. Thiocholine electrochemistry and immobilised enzyme inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl(sat) was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm(-2) of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 microM for dipterex and 0.4 microM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.     

Ultra-sensitive biosensor based on mesocellular silica foam for organophosphorous pesticide detection

A sensitive amperometric acetylcholinesterase (AChE) biosensor was fabricated based on mesocellular silica foam (MSF), which functioned as both an enzyme immobilization matrix and a solid phase extraction (SPE) material for the preconcentration of target molecules. The hydrophilic interface, the good mechanical/chemical stability, and the suitable pore dimension of MSF provided the entrapped AChE a good environment to well maintain its bioactivity at basic condition. The AChE immobilized in MSF showed improved catalytic ability for the hydrolysis of acetylthiocholine, as evidenced by the increasing of the oxidation current of thiocholine, the enzymatic catalytic hydrolysis production of acetylthiocholine. In addition, the MSF with large surface area showed a modest adsorption capacity for monocrotophos, a model organophosphate used in this study, via the hydrogen bond or physical adsorption interaction. The combination of the SPE and the good enzyme immobilization ability in MSF significantly promoted the sensitivity of the biosensor, and the limit of detection has lowered to 0.05 ng/mL. The biosensor exhibited accuracy, good reproducibility, and acceptable stability when used for garlic samples analysis. The strategy may provide a new method to fabricate highly sensitive biosensors for the detection of ultra-trace organophosphorous pesticide infield.     

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 (0.1mM). 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.     

Oxidative desorption of thiocholine assembled on core-shell Fe3O4/AuNPs magnetic nanocomposites for highly sensitive determination of acetylcholinesterase activity: an exposure biomarker of organophosphates

Acetylcholinesterase (AChE) activity is a well established biomarker for biomonitoring of exposures to organophosphates (OPs) pesticides and chemical nerve agents. In this work, we described a novel electrochemical oxidative desorption-process of thiocholine, the product of enzymatic reaction, for rapid and highly sensitive determination of AChE activity in human serum. This principle is based on self-assembling of produced thiocholine onto core-shell Fe(3)O(4)/Au nanoparticles (Fe(3)O(4)/AuNPs) magnetic nanocomposites and its oxidation at electrode surface. Fe(3)O(4) magnetic core is not only used for magnetic separation from sample solutions, but also carrying more AuNPs due to its large surface-to-volume ratio. The core-shell Fe(3)O(4)/AuNPs nanocomposites were characterized by UV-Vis spectroscopy, field-emission scanning electron microscopy (FE-SEM) and electrochemical measurements. A linear relationship was obtained between the AChE activity and its concentration from 0.05 to 5.0 mU mL(-1) with a detection limit of 0.02 mU mL(-1). The method showed good results for characterization of AChE spiked human serum and detection of OP exposures from 0.05 to 20 nM, with detection limit of 0.02 nM. This new oxidative desorption assay thus provides a sensitive and quantitative tool for biomonitoring of the exposure to OP pesticides and nerve agents.     

Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface

In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs-GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000ngmL(-1) and from 2 to 15mugmL(-1), with a detection limit of 0.3ngmL(-1). The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.     

Sensitive Detection of Organophosphorus Pesticides Using a Needle Type Amperometric Acetylcholinesterase-based Bioelectrode. Thiocholine Electrochemistry and Immobilised Enzyme Inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl sat was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm ^?2 of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 μM for dipterex and 0.4 μM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.     

A simple, label-free AuNPs-based colorimetric ultrasensitive detection of nerve agents and highly toxic organophosphate pesticide

Here, a simple label-free colorimetric sensing method for organophosphate (OP) nerve agents and pesticide based on catalytic reaction of acetylcholine esterase (AChE) and the aggregation of lipoic acid (LA) capped AuNPs has been established, which is highly sensitive with a limit of detection (LOD) lowered to pM level. In this method, only the AChE hydrolysis product of acetylthiocholine (ATCh), i.e., cationic thiocholine (TCh) can induce the aggregation of LA capped AuNPs along with a distinct color change from red to steel-blue. When OPs as enzyme inhibitors exist, the generation of TCh can be suppressed and the color change of LA capped AuNPs is gradually diminished according to different concentrations of OPs. The feasibility of this method has been demonstrated by sensitive measurement of OP nerve agents and pesticide in a spiked fruit sample with reliable results. This distinct and rapid colorimetric response enables us to readily probe OPs without more technical demand.     

Determination of organophosphorous and carbamate insecticides by flow injection analysis.

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.     

Palladium and platinum ions interfere with the measurement of erythrocyte vesiculation by inhibiting the acetylcholinesterase activity of the released spectrin-depleted microvesicles

Palladium (Pd(2+)) and platinum (Pt(2+)) ions were found to inhibit erythrocyte membrane-bound acetylcholinesterase (AChE) with Ki values of 6.0 and 6.5 microg/ml, respectively. Lineweaver-Burke plots revealed that the inhibition of erythrocyte AChE by both metal ions was competitive in nature. Binding studies using alkaline phosphatase as a reporting enzyme confirmed that both metal ions indeed did bind to the enzyme molecules. In the process of red cell vesiculation, membrane-bound AChE is shed along with vesicles. The measurement of AChE activities in the medium containing vesiculated RBC could potentially be served as an index of vesiculation. Inhibition of AChE activities by both metal ions can thus constitute a potential source of error in vesiculation measurement. To illustrate these effects, a simulated vesiculation system, using green tea polyphenol in the presence (25 microg/ml) or absence of Pd(2+) ion was simultaneously examined by the electronmicrography and the AChE method. We observed vesiculation under the experimental condition in Pd(2+)-free controls that was associated with a time-dependent increase in AChE activity were barely detected in the Pd(2+)-spiked specimen because of the masking effect exerted by the metal ions themselves.     

Label-free detection of bacterial RNA using polydiacetylene-based biochip

A novel acetylcholinesterase (AChE)/choline oxidase (ChOx) bienzyme amperometric acetylcholine biosensor based on gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNTs) has been successfully developed by self-assembly process in combination of sol-gel technique. A thiolated aqueous silica sol containing MWCNTs and ChOx was first dropped on the surface of a cleaned Pt electrode, and then AuNPs were assembled with the thiolated sol-gel network. Finally, the alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and AChE was repeated to assemble different layers of PDDA-AChE on the electrode for optimizing AChE loading. Among the resulting biosensors, the biosensor based on two layers of PDDA-AChE multilayer films showed the best performance. It exhibited a wide linear range, high sensitivity and fast amperometric response, which were 0.005-0.4mM, 3.395 μA/mM, and within 15s, respectively. The biosensor showed long-term stability and acceptable reproducibility. More importantly, this study could provide a simple and effective multienzyme immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.     

Design of acetylcholinesterases for biosensor applications

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.     

Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30°C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1-50nM and 1.5-40nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95-100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4°C).     

Acetylcholinesterase with mesoporous silica: Covalent immobilization,physiochemical characterization,and its application in food for pesticide detection

Toxic contamination of commonly consumed food products and water due to food chain vulnerability via agricultural products and commodities is a serious health hazard. This study reports on Santa Barbara Amorphous (SBA-15), a type of mesoporous silica nanoparticles, for efficient and stable acetylcholinesterase (AChE) adhesion toward detection of toxic pesticides. AChE was immobilized to the inert framework of mesoporous materials viz. SBA-15 with a proficient hydrolytic response toward acetylthiocholine. The immobilized system acts as a biosensor for the detection of pesticides, which are organophosphorus compounds in food. Both the SBA-15 and immobilized SBA-15 were characterized to give an insight on the physiochemical and morphological modification properties. The enzyme activity was accessed by Ellman’s spectrophotometric bioassay for bare and enzyme-immobilized SBA-15 that resulted in promising enzymatic activity with the counterpart. Enzyme stability was also studied, which exhibited that immobilized AChE retained its catalytic activity up to 60 days and retained 80% of the hydrolytic activity even at 37°C. On the basis of the success of immobilized enzyme (covalent) being inhibited by acetylthiocholine, the sensor was administered for the inhibition by monocrotophos and dimethoate that are used widely as pesticides in agricultural. The inhibitory concentration (IC₅₀) value was found to be 2.5 ppb for monocrotophos and 1.5 ppb for dimethoate inhibiting immobilized AChE. This was verified using cyclic voltammetry, an electrochemical analysis thus proving that the SBA-15@AChE complex could be used as a sensitive and highly stable sensor for detecting the concentration of hazardous pesticide compounds.     

Metabolites from Microsphaeropsis olivacea, an endophytic fungus of Pilgerodendron uviferum

Seven compounds belonging to different structural skeletons were isolated from Microsphaeropsis olivacea grown in liquid and solid media. The enalin derivative 7-hydroxy-2,4-dimethyl-3(2H)-benzofuranone is reported for the first time, while additional spectroscopic information is provided for the acetates of botrallin and ulocladol. The activity of the isolated compounds was assessed towards the enzyme acetylcholinesterase (AChE) and their cytotoxicity against human lung fibroblasts. Graphislactone A and botrallin presented a moderate activity towards AChE, with IC50 of 8.1 and 6.1 microg/ml (27 and 19 microM, respectively). Under the same experimental conditions, the IC50 of the standard inhibitor galanthamine was 3 microg/ml. The cytotoxicity of both compounds was > 1000 and 330 microM, respectively. None of the compounds was promising as antibacterial or antifungic against phytopathogenic fungi and bacteria. Botrallin and graphislactone A were detected in the liquid potato-dextrose and yeast extract/malt extract/dextrose as well as on a solid substrate (rice). Butyrolactone I was obtained from the fungus growing on solid medium.     

Biosensor based on acetylcholinesterase immobilized onto layered double hydroxides for flow injection/amperometric detection of organophosphate pesticides

We developed a highly sensitive flow injection/amperometric biosensor for the detection of organophosphate pesticides (OPs) using layered double hydroxides (LDHs) as the immobilization matrix of acetylcholinesterase (AChE). LDHs provided a biocompatible microenvironment to keep the bioactivity of AChE, due to the intrinsic properties of LDHs (such as a regular structure, good mechanical, chemical and thermal stabilities, and swelling properties). By integrating the flow injection analysis (FIA) with amperometric detection, the resulting AChE-LDHs modified electrode greatly catalyzed the oxidation of the enzymatically generated thiocholine product, and facilitated the detection automation, thus increasing the detection sensitivity. The analytical conditions for the FIA/amperometric detection of OPs were optimized by using methyl parathion (MP) as a model. The inhibition of MP was proportional to its concentration ranging from 0.005 to 0.3μgmL(-1) and 0.3 to 4.0μgmL(-1) with a detection limit 0.6ngmL(-1) (S/N=3). The developed biosensor exhibited good reproducibility and acceptable stability.     

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.     

Effects of the organophosphate insecticide,monocrotophos, on acetylcholinesterase activity in the nile tilapia fish (Oreochromis niloticus) brain

The neurotoxic effects of monocrotophos on the brain of the nile tilapia fish (Oreochromis niloticus) were examined, using a static bioassay under laboratory conditions. By probit analysis the 96 h LC₅₀ value of monocrotophos was 4.9 mg/l. After 96 h exposure to acute levels of monocrotophos, the brain acetylcholinesterase (AChE) activity decreased progressively as the concentration of monocrotophos increased. In addition, four weeks following transfer to toxicant-free water after exposure to 1 mg monocrotophos, nile tilapia fish brain regained 95% of control AChE activity. The results indicate that inhibition of AChE activity in fish exposed to monocrotophos may serve as an indicator of hazard due to application of this chemical in the natural environment.Special issue dedicated to Dr. Robert Balazs.