Immobilization of acetylcholinesterase on one-dimensional gold nanoparticles for detection of organophosphorous insecticides

This paper reports a simple method for immobilization of acetylcholinesterase (AChE) on one-dimensional (1D) gold (Au) nanoparticles for detection of organophosphorous (OP) insecticides. 1D Au nanoparticles were prepared by electrodeposition in the pores of an alumina template which was subsequently removed by 2.0 M NaOH solution. They were characterized by XRD and FESEM. The immobilized AChE retained its biological activity and catalyzed the hydrolysis of acetylthiocholine to form thiocholine, which was su...     

Biosensors based on highly sensitive acetylcholinesterases for enhanced carbamate insecticides detection

This paper presents the construction of amperometric biosensors for the highly sensitive detection of carbamate insecticides based on the inhibition of acetylcholinesterase (AChE). This enzyme was immobilised by entrapment in an optimised sol-gel matrix on TCNQ-modified screen-printed electrodes. The enzyme activity was estimated by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine using TCNQ as mediator. Wild and genetically engineered AChEs from Drosophila melanogaster (Dm) were chosen for their high sensitivity towards insecticides, which substantially improves the LOD compared with cholinesterases from other sources. The wild type and three mutant enzymes were tested against three carbamate insecticides: carbaryl, carbofuran and pirimicard. The best LOD were obtained with the Y370A mutant for carbaryl (1 × 10⁻⁸ M), the E69W mutant for pirimicarb (2 × 10⁻⁸ M) and the I161V mutant for carbofuran (8 × 10⁻¹⁰ M). The biosensors were applied to the analysis of two potable water samples.     

A new chromogenic reagent for carbamate insecticides

JPC – Journal of Planar Chromatography – Modern TLC -     

Micellar electrokinetic chromatography with amperometric detection and off-line solid-phase extraction for analysis of carbamate insecticides

Six selected primary carbamate insecticides, methomyl, carbaryl, carbofuran, propoxur, isoprocarb, and promecarb, were hydrolyzed in alkaline solution, resulting in electroactive derivatives detectable at a platinum (Pt) electrode poised at +0.8 V vs Ag/AgCl (3 M NaCl). The Pt electrode was inserted into a small electrochemical cell and positioned close to the capillary outlet as an end-column detector to detect the carbamate derivatives after electrophoretic separation. Based on their predicted pK_a values and aqueous solubilities, micellar electrokinetic chromatography (MEKC) was optimized for baseline separation of the derivatives using 20 mM borate, pH 10.2 containing 20 mM sodium dodecyl sulfate as a running buffer. When combined with solid-phase extraction (SPE) on octadecyl silica, a preconcentration factor of 100-fold achieved detection to 0.5 μM methomyl and to 0.01 μM for the remaining five pesticides, significantly below the level regulated by government agencies of most countries. The SPE-MEKC method when applied to the separation and analysis of spiked river water and soil samples, yielded results with excellent reproducibility, recovery and selectivity.     

Effect of bromine oxidation on high-performance thin-layer chromatography multi-enzyme inhibition assay detection of organophosphates and carbamate insecticides

Following high-performance thin-layer chromatography, thiophosphate pesticides, which inhibit choline esterases, are detectable using a multi-enzyme inhibition assay (HPTLC-EI) based on rabbit liver esterase (RLE), Bacillus subtilis (BS2) esterase, or cutinase (from Fusarium solani pisi). Because choline esterase inhibition is more effective after conversion of thiophosphate thions into their corresponding oxons, a pre-oxidation step was added to the HPTLC-EI assay. Bromine vapour was found to be more effective than iodine or UV irradiation for oxidation. Following oxidation, the inhibitory strength of parathion, parathion-methyl, chlorpyrifos, chlorpyrifos-methyl, and malathion, expressed as HPTLC enzyme inhibition factors (f(i)), increased by approximately 2 orders of magnitude. In contrast, bromine oxidation of organophosphate and carbamate insecticides resulted in a slight reduction in their inhibition factors, due to partial bromination and degradation of the parent compounds, while bromine oxidation increased the inhibition factors for demeton-S-methyl and propoxur. Apple juice and water samples spiked with paraoxon (0.001 mg/L), parathion (0.05 mg/L), and chlorpyrifos (0.5 mg/L) were used to test the HPTLC-EI system, resulting in mean recoveries of 95-106% and 91-102% for RLE and cutinase, respectively.     

Application of electrochemical detection to the analysis of some residues of carbamate insecticides by high performance liquid chromatography

A new high-performance liquid chromatographic determination of residues of some carbamate insecticides with electrochemical detection after degradation of molecules as the phenols is described. Residues of carbamate insecticides in vegetables can be quantified with high sensitivity.     

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.     

Optimization of a reaction detector for analysis at the femtomol level of carbamate insecticides by HPLC

Summary For the determination of trace amounts of carbamate insecticides in vegetables the combination of liquid chromatography with post column chemical derivatization (chemical reaction detector) is used. In a two step reaction detector the carbamates are firstly saponified in 0.01 M sodium hydroxide at 80°C with a reaction time of 30sec. To this mixture OPA reagent is added to detect the methylamine generated in the saponification. Optimization strategies for this reaction are demonstrated. At a temperature of 80 °C a reaction time of 80 sec is sufficient for quantitative transformation of the methylamine. The peak dispersion can be reduced and hence the detection limit improved by decreasing the diameter of the tubes and by diminishing dilution through the addition of reagent. A 1∶24 ratio of reagent to column (reactor) effluent is possible with cyclone-type mixers. The quantitation of carbamates in different vegetables is demonstrated. The detection limit is 20ppb at a signal to noise ratio of 10∶1. Thesis B. Lillig, Saarbrücken, 1984     

Binding of acetylcholinesterase to multiwall carbon nanotube-cross-linked chitosan composite for flow-injection amperometric detection of an organophosphorous insecticide

A novel method for immobilization of acetylcholinesterase (AChE) by binding covalently to a cross-linked chitosan-multiwall carbon nanotube (MWNT) composite is described. In addition a sensitive, fast, cheap and automatizable flow injection detection of an organophosphorous insecticide was developed. The MWNTs were homogeneously distributed in the chitosan membrane which showed a homogeneous porous structure. The immobilized AChE could catalyze the hydrolysis of acetylthiocholine with a K(M)app value of 177 microM to form thiocholine, which was then oxidized to produce detectable signal in a linear range of 1.0-500 microM and fast response. MWNTs could catalyze the electrooxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of an organophosphorous insecticide on the enzymatic activity of AChE, using Sulfotep as a model compound, the conditions for the flow-injection detection of the insecticide were optimized. Both biocompatibility of chitosan and inherent conductive properties of MWNTs favored the detection of the insecticide from 1.5 to 80 microM along with good stability and reproducibility. 95 % reactivation from inhibited AChE could be regenerated by using 2-pyridinealdoxime methiodide within 15 min for 15 times. The detection of Sulfotep samples exhibited satisfactory results. The proposed flow-injection analysis device can be applied to automated determination and characterization of enzyme inhibitors.     

Rapid separation and determination of carbamate insecticides using isocratic elution pressurized capillary electrochromatography

An isocratic elution pressurized CEC (pCEC) system was used to separate and determine ten carbamate insecticides. It was found that introduction of the electrical field, supplementary pressure, and SDS in the proposed method greatly improved the speed, column efficiency, selectivity, and repeatability for separation and determination of carbamates. On a capillary column of 75 microm ID packed with 3 microm octadecyl silica, baseline separation and detection of ten analytes was performed by using a mobile phase consisting of 30% v/v ACN and 70% v/v of 5 mmol/L ammonium acetate (pH 6.5) containing 1 mmol/L SDS and 0.01% triethylamine (TEA). Under the optimum conditions ten carbamate insecticides could be completely separated within 20 min. For the real vegetable samples, an SPE procedure for the cleanup of matrices was carried out prior to pCEC analysis. The detection limits of 0.05-1.6 mg/kg for ten carbamates and mean recoveries of 51.3-109.2% for eight kinds of vegetable samples at different concentrations of carbamates with RSD less than 11.4% were obtained, respectively. The proposed method has been proved to be effective in the rapid analysis of carbamate residues in vegetables.     

高效液相色谱法测定粮食中氨基甲酸酯类杀虫剂及其代谢物残留量

采用高效液相色谱柱后衍生荧光检测法测定了粮食(大米、小麦、玉米、大豆)中9种氨基甲酸酯类杀虫剂及代谢物残留量.方法的最低检测限为0.01～0.03 mg/kg,加标平均回收率为70.7%～108.0%,相对标准偏差小于18%.     

Synthesis of an ethynyl carbamate and application for enantioselective cyclocarbolithiation

[reaction: see text] The intramolecular trans-cyclocarbolithiation of the alpha-lithiated 4-substituted 5-hexynyl carbamate (1S,4RS)-14 employing lithiodestannylation is presented. The 5-exo-dig cyclization products cis-/trans-16a were formed exclusively. The highly enantioenriched organotin precursor (S)-11 was synthesized via an asymmetric deprotonation of the corresponding alkyl carbamate 10 by the chiral complex sec-butyllithium/(-)-sparteine and subsequent substitution with tributyltin chloride.     

Development of an immunosensor for the detection of Francisella tularensis antibodies

Tularemia, also known as rabbit fever, is a highly infectious zoonotic disease caused by a non-motile and non-spore-forming Gram-negative coccoid rod bacterium, Francisella tularensis. It occurs naturally in lagomorphs (rabbits and hares), but many animals have been reported to be susceptible. Transmission to humans is mostly caused by inhalation of aerosolised bacteria, handling of infected animals, arthropod stings, and ingestion of contaminated foods and water. At present, pathogenic isolation, molecular detection, and serology are the most commonly used methods to confirm the diagnosis of tularemia. In this work, an electrochemical immunosensor for the detection of anti-F. tularensis antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic-group-terminated bipodal alkanethiol that is covalently linked to a lipopolysaccharide (LPS) that can be found in the outer membrane of the bacteria F. tularensis. The presence of anti-F. tularensis antibodies was measured using horseradish peroxidase-labelled protein A (HRP-protein A) from Staphylococcus aureus, and the developed immunosensor gave a stable quantitative response to different anti-F. tularensis FB11 antibody concentrations after 30 min with a limit of detection of 15 ng/mL, RSD of 9 %, n?=?3. The developed immunosensor was tested with serum from animals infected with tularemia and was compared to the results obtained using ELISA showing an excellent degree of correlation.     

Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.