Study of Inhibition,Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles‐Based Acetylcholinesterase Biosensor

Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the ‘active site’ of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self‐assembling AChE to graphene nanosheet (GN)‐gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.     

Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

Au掺杂Fe3O4纳米粒子酶传感器的制备及其应用于有机磷农药检测的研究

合成了金掺杂的四氧化三铁纳米粒子(Au-Fe₃O₄), 以壳聚糖为交联剂, 制备了电流型乙酰胆碱酯酶(AChE)生物传感器, 并将其应用于有机磷农药(OPs)的检测. 实验表明, Au-Fe₃O₄纳米粒子具有良好的生物兼容性, 能够有效地促进电子传递, 修饰了Au-Fe₃O₄纳米粒子的酶传感器, 响应速度快, 检测灵敏度高, 稳定性好; 固定在传感器上的乙酰胆碱酯酶有良好的酶动力学响应, 其表观米氏常数( )为10.3 mmol/L. 利用有机磷农药对乙酰胆碱酯酶的抑制作用, 以硫代乙酰胆碱(ATCh)为底物, 对有机磷农药敌敌畏进行了检测, 检测限达到4.0×10^－13 mol/L.     

A New Bienzymatic Biosensor Based on Butyrylcholine Esterase‐Sulfhydryl Oxidase Enzymes

Abstract

Over the last decades, cholinesterase (ChE) biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. In this study, a new bienzymatic biosensor based on butyrylcholinesterase (BuChE) and sulfhydryl oxidase (SOX) enzymes was developed. This system makes use of the biocatalyzed hydrolysis of butyrylthiocholine to butyric acid and thiocholine that acts as a SOX substrate. Measurements were performed by following of the consumed oxygen level related to butyrylthiocholine concentration in the enzymatic reactions. Bienzymatic system was characterized and applied for detection of donepezil which is a reversible inhibitor of cholinesterase and belongs to a new class of cholinesterase inhibitors having an N‐benzylpiperidine and an indanone moiety, by following of the decrease in biosensor response as a result of competitive inhibition.     

An amperometric biosensor based on acetylcholinesterase immobilized onto iron oxide nanoparticles/multi-walled carbon nanotubes modified gold electrode for measurement of organophosphorus insecticides

An acetylcholinesterase (AChE) purified from maize seedlings was immobilized covalently onto iron oxide nanoparticles (Fe₃O₄NP) and carboxylated multi walled carbon nanotubes (c-MWCNT) modified Au electrode. An organophosphorus (OP) biosensor was fabricated using this AChE/Fe₃O₄/c-MWCNT/Au electrode as a working electrode, Ag/AgCl as standard and Pt wire as an auxiliary electrode connected through a potentiostat. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The properties of nanoparticles modified electrodes were studied by scanning electron microscopy (SEM), Fourier transform infrared (FTIR), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Fe₃O₄NP and c-MWCNT showed excellent electrocatalytic activity at low potential (+0.4 V). The optimum working conditions for the sensor were pH 7.5, 35 °C, 600 μM substrate concentration and 10 min for inhibition by pesticide. Under optimum conditions, the inhibition rates of OP pesticides were proportional to their concentrations in the range of 0.1–40 nM, 0.1–50 nM, 1–50 nM and 10–100 nM for malathion, chlorpyrifos, monocrotophos and endosulfan respectively. The detection limits were 0.1 nM for malathion and chlorpyrifos, 1 nM for monocrotophos and 10 nM for endosulfan. The biosensor exhibited good sensitivity (0.475 mA μM⁻¹), reusability (more than 50 times) and stability (2 months). The sensor was suitable for trace detection of OP pesticide residues in milk and water.     

Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A simple and practical approach to improve the sensitivity of acetylcholinesterase (AChE)-inhibited method has been developed for monitoring organophosphorous (OP) pesticide residues. In this work, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to detect AChE activity. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS facilitates rapid and high-throughput screening of OP pesticides. Here we describe a new method to obtain low detection limits via employing external reagents. Among candidate compounds, n-octylphosphonic acid (n-Octyl-PA) displays assistant effect to enhance AChE inhibition by OP pesticides. In presence of n-Octyl-PA, the percentages of AChE inhibition still kept correlation with OP pesticide concentrations. The detection limits were improved significantly even by 10²–10³ folds in comparison with conventional enzyme-inhibited methods. Different detection limits of OP pesticides with different toxicities were as low as 0.005 μg L⁻¹ for high toxic pesticides and 0.05 μg L⁻¹ for low toxic pesticides. Besides, the reliability of results from this method to analyze cowpea samples had been demonstrated by liquid-chromatography tandem mass spectrometry (LC–MS/MS). The application of this commercial available assistant agent shows great promise to detect OP compounds in complicated biological matrix and broadens the mind for high sensitivity detection of OP pesticide residues in agricultural products.     

Detection of Pesticides in the Environment Using Biosensors Based on Cholinesterases

Abstract

Amperometric biosensors based on acetylcholinesterase or butyrylcholinesterase were used for the kinetic determination of organophosphate and carbamate pesticides. The current of the biosensor I_ss was measured continuously with substrate; the addition of samples with pesticides resulted in the time decrease of the current dl/dt. The relative inhibition RI=(dl/dt)I_ss was used as the signal for evaluations. For several pesticides, different calibration curves (dependencies of RI on concentration c) were obtained depending on the affinity of the individual pesticide to the cholinesterase used. This affinity was described using the bimolecular inhibition constant k_i. The single calibration curve independent on the type of pesticide was obtained as the dependence of RI on the product k_ic, thus indicating the effects of both concentration and inhibiting properties on the response of the biosensor. The relative inhibition was used to characterise anticholinesterase toxicity of the sediments collected from the Morava River and its tributary streams. The influence of both point (large cities) and nonpoint (agriculture) sources of pollution was identified.     

Biosensors based on cholinesterase inhibition for insecticides, nerve agents and aflatoxin B1 detection (review)

The present review reports the research carried out during last 9 years on biosensors based on cholinesterase inhibition for nerve agents, organophosphorus and carbammic insecticides, and aflatoxin B₁ detection. Relative applications in environmental and food areas are also reported. Special attention is paid to the optimization of parameters such as enzyme immobilization, substrate concentration, and incubation time in the case of reversible inhibition by aflatoxin B₁ or irreversible inhibition by organophosphorus and carbamic insecticides, and nerve agents in order to optimize and improve the analytical performances of the biosensor. Evaluation of selectivity of the system is also discussed.     

Carbamate Insecticide Sensing Based on Acetylcholinesterase/Prussian Blue-Multi-Walled Carbon Nanotubes/Screen-Printed Electrodes

Prussian blue (PB)-multi-walled carbon nanotubes (MWCNTs) modified screen-printed electrodes (SPEs) were used to immobilize enzyme acetylcholinesterase (AChE) for carbamate insecticide sensing. The synthesized hybrid PB-MWCNTs had high stability at pH values in the range of 5–10 and presented a porous and homogeneous microenvironment to entrap enzyme molecules. The generated hybrids not only acted as carriers of acetyl cholinesterase, but also promoted electron-transfer reactions because of the synergistic effects between MWCNTs and PB. Under the optimal conditions, the response of the sensor was proportional to acetylthiocholine (ATCh) concentrations ranging from 0.1 mM to 0.6 mM, with a sensitivity of 21.97 µA · mM^?1 · cm^?2. The sensors were further used to detect pesticides, and the inhibition rate of pirimicarb was proportional to the logarithm of its concentration ranging from 1.0 × 10^?6 to 1.0 g · L^?1, with a limit of detection (LOD) equal to 5.32 × 10^?8 g · L^?1. In order to evaluate the performance of the detection system, the sensors were applied to determine pirimicarb in water samples and exhibited high sensitivity and good stability. The detection system is fast, simple for analysis of pirimicarb in environmental samples, and could provide a semi-automated analytical system through further improvement in biosensor arrays.     

Electrochemical sensor for the detection and presumptive identification of quinolone and tetracycline residues in milk

A novel application of an electrochemical biosensor is here employed as analytical method for the detection and presumptive identification of antimicrobial drug residues in milk. The measurement was based on carbon dioxide production rate in relation to inhibition of microbial grow (Escherichia coli ATCC 11303). In this pilot study quinolone and tetracycline residues have been taken into consideration because use of these last in livestock production has been identified as area of particular concern. The experimental approach and analytical method developed appear adequate for the purpose, and compared to older screening methods as, for example, the microbial inhibition assays and immunoassays, offers the advantages of (i) very short analysis time (about 120 min); (ii) smaller sample amount (approximately 0.5 mL); (iii) no sample treatment (iv) good precision; and (v) the possibility of following, in a continuous manner, the inhibition process. Moreover, sensitivity of electrochemical biosensor system is resulted very high considering that for all quinolones and tetracyclines investigated it has been possible detect a residue concentration below or equal to 25 μg L⁻¹. Under this point of view, it must be considered that the maximum residue limits fixed by UE for quinolones and tetracyclines in milk are, at present, all higher of this concentration.     

Dual amperometric-potentiometric biosensor detection system for monitoring organophosphorus neurotoxins

A dual-transducer flow-injection biosensor detection system for monitoring organophosphorus (OP) neurotoxins is described. Such simultaneous use of different physical transducers in connection to the same (organophosphorous hydrolase (OPH)) enzyme enhances the information content and provides discrimination between various subclasses of OP compounds. While the potentiometric biosensor responds favorably to all OP compounds, reflecting the pH changes associated with the OPH activity, the amperometric device displays well-defined signals only towards OP substrates (pesticides) liberating the oxidizable p-nitrophenol product. The potentiometric detection has been accomplished with a silicon-based pH-sensitive electrolyte-insulator-semiconductor (EIS) transducer, operated in the constant-capacitance (ConCap) mode. Both transducers are prepared by a thin-film fabrication technology, and respond rapidly and independently to sudden changes in the level of the corresponding OP compound, with no apparent cross reactivity. Relevant experimental variables were evaluated and optimized. Such development holds great promise for field screening of OP neurotoxins in connection to various defense and environmental scenarios. The multiple-transduction concept could be extended for increasing the information content of other ‘class-enzyme’ biosensor systems.     

A mediator-free screen-printed amperometric biosensor for screening of organophosphorus pesticides with flow-injection analysis (FIA) system

A mediator-free amperometric biosensor for screening organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system based on anticholinesterase activity of OPs to immobilized acetylcholinesterase enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al₂O₃ sol-gel matrix screen-printed on an integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al₂O₃-AChE biosensor detects the substrate at 0.25 V (versus Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al₂O₃-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples. A wide linear inhibition response for dichlorvos, typical OP, is observed in the range of 0.1-80 μM, corresponding to 7.91-84.94% inhibition for AChE. The detection limit for dichlorvos is achieved at 10 nM in the simulated seawater for 15 min inhibiting time, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.     

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.     

FET‐Based Biosensors for The Direct Detection of Organophosphate Neurotoxins

Recent world‐wide terrorist events associated with the threat of hazardous chemical agent proliferation, and outbreaks of chemical contamination in the food supply has demonstrated an urgent need for sensors that can directly detect the presence of dangerous chemical toxins. Such sensors must enable real‐time detection and accurate identification of different classes of pesticides (e.g., carbamates and organophosphates) but must especially discriminate between widely used organophosphate (OP) pesticides and G‐ and V‐type organophosphate chemical warfare nerve agents. Present field analytic sensors are bulky with limited specificity, require specially‐trained personnel, and, in some cases, depend upon lengthy analysis time and specialized facilities. Most bioanalytical based systems are biomimetic. These sensors utilize sensitive enzyme recognition elements that are the in‐vivo target of the neurotoxic agents which the sensor is attempting to detect. The strategy is well founded; if you want to detect cholinesterase toxins use cholinesterase receptors. However, this approach has multiple limitations. Cholinesterase receptors are sensitive to a wide range of non‐related compounds and require lengthy incubation time. Cholinesterase sensors are inherently inhibition mode and therefore require baseline testing followed by sample exposure, retest and comparison to baseline. Finally, due to the irreversible nature of enzyme‐ligand interactions, inhibition‐mode sensors cannot be reused without regeneration of enzyme activity, which in many cases is inefficient and time‐consuming. In 1996, we pioneered a new “kinetic” approach for the direct detection of OP neurotoxins based on agent hydrolysis by the enzyme organophosphate hydrolase (OPH; EC 3.1.8.2; phosphotriesterase) and further identified a novel multi‐enzyme strategy for discrimination between different classes of neurotoxins. The major advantage of this sensor strategy is it allows direct and continuous measurement of OP agents using a reversible biorecognition element. We also investigated incorporation of enzymes with variations in substrate specificity (e.g., native OPH, site‐directed mutants of OPH, and OPAA (EC 3.1.8.1), based upon preferential hydrolysis of P? O, P? F and P? S bonds to enable discrimination among chemically diverse OP compounds. Organophosphate hydrolase enzymes were integrated with several different transduction platforms including conventional pH electrodes, fluoride ion‐sensitive electrodes, and pH‐responsive fluorescent dyes. Detection limit for most systems was in the low ppm concentration range. This article reviews our integration of organophosphate hydrolase enzymes with pH sensitive field effect transistors (FETs) for OP detection.     

Ionic liquid-functionalized graphene for fabricating an amperometric acetylcholinesterase biosensor

This work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated by modifying a glassy carbon electrode with acetylcholinesterase (AChE) immobilized on ionic liquid-functionalized graphene (IL-G). The functionalized graphene sheets had good dispersibility and long-term stability in various solvents. The as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.77 mM. Furthermore, based on the inhibition by OPs of the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.0025 to 0.48 and 0.48 to 1.42 μg mL(-1) with a detection limit of 0.8 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for OPs detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.     

Development of an Amperometric Microbial Biosensor Based on Thiobacillus thioparus Cells for Sulfide and Its Application to Detection of Sulfate‐Reducing Bacteria

This paper describes a novel electrochemical microbial biosensor based on Thiobacillus thioparus cells for sulfide detection. The morphology and electrochemical properties of the proposed biosensor were characterized by SEM and cyclic voltammetry, respectively. Working conditions of the microbial biosensor were optimized to obtain good electrochemical performances. Under the optimum conditions, analytical performances were evaluated, and the results suggested that the microbial biosensor could be used for selective detection of sulfide. The microbial biosensor was then successfully applied in detection of sulfate‐reducing bacteria by oxidizing its characteristic metabolite, sulfide, which was accumulated in culture media during bacterial growth.     

Development of Quantum Dots Modified Acetylcholinesterase Biosensor for the Detection of Trichlorfon

Poly (N‐vinyl‐2‐pyrrolidone) (PVP)‐capped CdS quantum dots (QCdS‐PVP) was synthesized with CdCl₂ and Na₂S in the presence of PVP. QCdS‐PVP has been used for the immobilization and stabilization of the acetylcholinesterase (AChE). The electrocatalytic activity of QCdS‐PVP leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, and higher sensitivity and stability. The GCE/QCdS‐PVP/AChE biosensor was used for the detection of organophosphate pesticides (OPs), such as trichlorfon. The sensor performance, including pH and inhibition time, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 12 ppb trichlorfon with a 5‐min inhibition time.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

A sensitive, rapid and inexpensive way to assay pesticide toxicity based on electrochemical biosensor

We reported a rapid toxicity assay method using electrochemical biosensor for pesticides, Escherichia coli (E. coli) was taken as a model microorganism for test. In this method, we adopted ferricyanide instead of natural electron acceptor O₂, and then microbial oxidation was substantially accelerated. Toxicity assays measured the effect of toxic materials on the metabolic activity of microorganisms. The current signal of ferrocyanide produced from the metabolism was proven to be directly related to the toxicity, which could be amplified by ultramicroelectrode array (UMEA). The ratio of the electrochemical signals, recorded in the presence and absence of toxin, provided an index of inhibition. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. 3,5-Dichlorophenol (DCP) was taken as the reference toxicant, its IC50 was estimated to be 8.0 mg/L. Three pesticides were examined using this method. IC50 values of 6.5 mg/L for Ametryn, 22 mg/L for Fenamiphos and 5.7 mg/L for Endosulfan were determined and in line with EC50 values reported in the literature. Atomic force microscopy (AFM) was also used for morphology characterization of E. coli induced by three pesticides. These results confirmed the present electrochemical method used is reliable. In addition, the electrochemical method is a sensitive, rapid and inexpensive way for toxicity assays of pesticides.     

Cholinesterase-based dipstick assay for the detection of organophosphate and carbamate pesticides

A cholinesterase (ChE)-based dipstick-type assay for the class-specific detection of organophosphate (OP) and carbamate (CM) pesticides was developed. The principle of the assay is based on inhibition of the activity of a ChE by these two families of pesticides, which is dependent on the concentration of pesticides. The proposed assay system is composed of a test strip with an acetylcholinesterase (AChE)-coated membrane and an enzyme substrate solution. The assay protocol involves incubation of the enzyme-coated strip in the pesticide-containing sample solution followed by incubation of the sample-treated strip in a chromogenic enzyme substrate solution. The color intensity is estimated by the naked eye or a reflectometer. Of the membranes tested as the enzyme support, Hybond N⁺ was the most suitable. Among the compounds tested as the enzyme substrate, indophenyl acetate was the best. The detectable concentration range of the dipstick assay for the OP and CM pesticides was 10⁻⁶-10² and 10⁻⁶-10⁰ μg mL⁻¹, respectively. The sensitivity of the dipstick assay to the oxidized form of parathion (paraoxon) was higher than to parathion. The strip showed a large matrix effect with pesticide-spiked lettuce samples, whereas it showed a small matrix effect with pesticide-spiked rice samples.