Detection of organophosphorus insecticides with immobilized acetylcholinesterase – comparative study of two enzyme sensors

Two-enzyme systems based on acetylcholinesterase (AChE) - a mono-enzyme system based on AChE, with p-aminophenyl acetate as substrate, and a bi-enzyme system based on AChE and tyrosinase, with phenyl acetate as substrate - have been studied for detection of organophosphate insecticides. The analytical performance and detection limits for determination of the pesticides were compared for the two AChE configurations. The enzyme loading, pH, and applied potential of the bi-enzyme system were optimised. When phenyl acetate was used as substrate for AChE activity the phenol generated by enzymatic hydrolysis was determined with a second enzyme, tyrosinase. Amperometric measurements were performed at 100 mV and -150 mV relative to the Ag/AgCl reference electrode for the mono-enzyme and bi-enzyme systems. Screen-printed sensors were used to detect the organophosphorus pesticides paraoxon and chlorpyrifos ethyl oxon; the detection limits achieved with phenyl acetate as substrate were 5.2x10(-3) mg L(-1) and 0.56x10(-3) mg L(-1), respectively.     

Biophysical aspects of cyclodextrin interaction with paraoxon

Cyclodextrins are torus‐shaped polymers of glucose that can bind organophosphorous compounds such as nerve agents and pesticides. We demonstrate here that cyclodextrin can bind up to two paraoxon molecules with a K_av of 6775 M^‐1. Molecular modeling shows that the paraoxon appears to bind in polar opposite orientation and have an average binding energy of ?89 Kcals/mol. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Surface-enhanced Raman scattering detection of cholinesterase inhibitors

A new sensitive surface-enhanced Raman scattering (SERS) assay for detection of cholinesterase inhibitors such as organophosphorous pesticides using silver colloidal nanoparticles was developed and optimized. Acetylcholinesterase (AChE) mediated the hydrolysis of acetylthiocholine to produce thiocholine, which interacted with the silver nanoparticles to give a specific SERS spectrum. Variation in enzyme activity due to inhibition was measured from changes in intensity of a characteristic peak (772 cm⁻¹) of the SERS spectrum that was directly correlated with the concentration of produced thiocholine. The method was demonstrated for the detection of paraoxon as reference AChE inhibitor. Limit of detection of paraoxon for 5 min incubation at 25 °C was 1.8 × 10⁻⁸ M. This assay can be utilized for the detection of trace amounts of any AChE inhibitor.     

Disposable potentiometric enzyme sensor for direct determination of organophosphorus insecticides

A potentiometric disposable enzyme sensor for the direct and fast determination of organophosphorus (OP) insecticides was developed by using an organophosphorus hydrolase (OPH) immobilized on an ion-selective electrode. The disposable screen-printed transducer was based on double matrix membrane technology which allows easy mass production. The potentiometric device consisted of a H(+)-sensitive electrode with integrated Ag/AgCl reference electrode. The electrodes were prepared with N,N-dioctadecylmethylamine as H(+)-sensitive ionophore and pH calibration resulted in slopes of 55 mV decade-1 over a pH range from 11 to 6. OPH was isolated from recombinant Escherichia coli DH5 alpha and immobilized within poly(carbamoyl sulfonate) prepolymer on the surface of the H(+)-sensitive electrode without any further fixation membrane. OPH catalyzes the hydrolytic cleavage of OP compounds which releases protons in a concentration proportional to hydrolyzed substrate. Sensor performance was investigated with regard to enzyme load, concentration, pH and temperature of the measuring buffer using paraoxon as analyte. Best sensitivity and response time were obtained with sensors prepared with 250 U of OPH and measuring at 37 degrees C in 1.0 mM HEPES buffer, pH 9.3, containing 100 mM NaCl. The enzyme sensor exhibited a linear calibration range of 0.01-0.15 mM chlorpyrifos, 0.05-0.35 mM diazinon, 0.05-0.4 mM paraoxon and 0.007-0.05 mM parathion, respectively. For all these analytes response times to reach 95% of maximum change in potential did not exceed 5 min. Sensors stored under dry conditions at 4 degrees C still showed 60% of initial hydrolytic rate after 70 d. The sensors even when stored dry were ready for measurements after 5 min incubation in measuring buffer. A range of putative interfering substances did not influence sensor response, and suitability of measuring OPs in soil extracts was ascertained.     

Method for measuring endogenous 3-O-methyldopa in urine and plasma.

The present report describes a method using column liquid chromatography with electrochemical detection for assaying concentrations of 3-O-methyldopa in urine and plasma. The technique combines a one-step sample preparation scheme with post-column flow-through electrodes in series, allowing adequate chromatographic separation of 3-O-methyldopa from other endogenous substances in urine. The validity of the method was confirmed by markedly decreased urinary 3-O-methyldopa levels after administration of an inhibitor of catechol-O-methyltransferase to rats, radioactivity in chromatographic fractions corresponding to 3-O-methyldopa in urine of rats undergoing infusion of [3H]-L-DOPA, and correlations between excretion rates of 3-O-methyldopa and catechols in humans. In healthy humans, urinary excretion of 3-O-methyldopa averaged 974 +/- 707 (S.D.) nmol per day, and plasma levels of 3-O-methyldopa averaged 89 +/- 32 nmol/l. The method should be useful in studies about the metabolism of endogenous and exogenous DOPA.     

Boric acid inhibits adenosine diphosphate-ribosyl cyclase non-competitively

Adenosine diphosphate-ribosyl cyclase (ADP-ribosyl cyclase) is a ubiquitous enzyme in eukaryotes that converts NAD+ to cyclic-ADP-ribose (cADPR) and nicotinamide. A quantitative assay for cADPR was developed using capillary electrophoresis to separate NAD+, cADPR, ADP-ribose, and ADP with UV detection (254 nm). Using this assay, the apparent Km and Vmax for Aplysia ADP-ribosyl cyclase were determined to be 1.24+/-0.05 mM and 131.8+/-2.0 microM/min, respectively. Boric acid inhibited ADP-ribosyl cyclase non-competitively with a Ki of 40.5+/-0.5 mM. Boric acid binding to cADPR, determined by electrospray ionization mass spectrometry, was characterized by an apparent binding constant, KA, of 655+/-99 L/mol at pH 10.3.     

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.     

A Disposable Biosensor for Organophosphorus Nerve Agents Based on Carbon Nanotubes Modified Thick Film Strip Electrode

A disposable biosensor based on acetylcholinesterase‐functionalized acid purified multi‐wall carbon nanotubes (CNTs) modified thick film strip electrode for organophosphorus (OP) insecticides was developed. The degree of inhibition of the enzyme acteylcholinesterase (AChE) by OP compounds was determined by measuring the electrooxidation current of the thiocholine generated by the AChE catalyzed hydrolysis of acteylthiocholine (ATCh). The large surface area and electro‐catalytic activity of carbon nanotubes lowered the overpotential for thiocholine oxidation to 200 mV (vs. Ag/AgCl) without the use of mediating redox species and enzyme immobilization by physical adsorption. The biosensor detected as low as 0.5 nM (0.145 ppb) of the model organophosphate nerve agent paraoxon with good precision, electrode to electrode reproducibility and stability. Analysis of real water sample using the sensor demonstrated the feasibility of the application of the sensor for on site monitoring of OP compounds.     

Diazonium-functionalized horseradish peroxidase immobilized via addressable electrodeposition: direct electron transfer and electrochemical detection

A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 +/- 0.7 s-1 and a DeltaEp of 8 mV (v = 150 mV/s). The large ks and low DeltaEp are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.     

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76?U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080?±?0.256 and 0.0003?±?0.018?mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010?±?0.173?mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003?mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178?mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.     

Synthesis and bioactivity of new phosphorylated R,R'-substituted sulfoximines

R,R'-disubstituted sulfoximines were phosphorylated with O,O-diethylchloro phosphate and phosphorothionate to obtain new organophosphorus compounds. After purification they were characterized by GC-MS and (1)H-NMR. The toxicity of the synthesized O,O-diethyl N-(R,R'-disubstituted sulfoximine) phosphoro-amidothionates was assayed on Musca domestica. It was found that the methyl phenyl derivative was the most toxic compound, followed by the dipropyl and dibutyl derivatives. The dihexyl compound was the less toxic of all the assayed compounds, being one hundred times less toxic than a paraoxon standard The anticholinesterasic activity of the corresponding phosphoramidates was assayed on homogenates of house flies' heads, giving values similar to paraoxon for the methyl phenyl derivative.     

AChE biosensor based on zinc oxide sol-gel for the detection of pesticides

Zinc oxide has been used as a matrix for immobilization of acetylcholinesterase (AChE) and detection of the pesticide paraoxon. The immobilized enzyme retained its enzymatic activity up to three months when stored in phosphate buffered saline (pH 7.4) at 4 °C. An amperometric biosensor for the detection of paraoxon was designed. The biosensor detected paraoxon in the range 0.035-1.38 ppm and can be used to detect other AChE inhibiting organophosphate pesticides.     

Kinetic study of cytochrome P450 3A4 activity on warfarin by capillary electrophoresis with fluorescence detection

The use of capillary electrophoresis (CE) for the determination of cytochrome P450 3A4 (CYP3A4) activity with R-warfarin as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, 10-hydroxywarfarin, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 60 cm effective length); 50 mM sodium phosphate buffer (pH 6.5); 23 kV (90 microA) applied voltage; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 37 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of 10-hydroxywarfarin from R-warfarin in the presence of CYP3A4 were calculated to be 166 +/- 12 microM and 713 +/- 14 pmol/min/nmol (or 91.4 pmol/min/mg) CYP3A4, respectively.     

Flow-injection spectrophotometric determination of paraoxon by its inhibitory effect on the enzyme acetylcholinesterase.

A spectrophotometric enzymatic flow injection (FI) system for the determination of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method was based on the determination of the acetic acid formed by the enzymatic reaction of the acetylcholinesterase, immobilized on glass beads, with the substrate acetylcholine. The acetic acid formed permeates through a PTFE membrane and is received by a solution (pH 7.0) containing the acid-base indicator Bromocresol Purple (B.C.P.), leading to a pH change and therefore to a color change. The variation of the absorbance of the solution is detected spectrophotometrically at 400 nm. The determination of paraoxon is related to its inhibitory action on the enzyme. Therefore the analytical signal is the difference between the signal that corresponds to the free and the one that corresponds to the inhibited enzyme, considering a fixed acetylcholine concentration. The correlation between the peak height and paraoxon concentration at a given acetylcholine concentration is linear in the range from 5.0 x 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 (r = 0.998) of paraoxon, with a relative estimated standard deviation (R.S.D.) of +/- 1.7% (n = 10) considering a solution containing 5.0 x 10(-6) mol L-1 of paraoxon and a solution containing 5.0 x 10(-3) mol L-1 of acetylcholine. Therefore, the quantitative limit detection is about 2.5 x 10(-7) of paraoxon (3 sigma). A 1,1'-trimethylene-bis(4-formylpyridinium bromide)dioxime (TMB-4) solution was used to reactivate the enzyme.     

An HPLC assay for rat liver ferrochelatase activity

A rapid, reliable, sensitive and reproducible HPLC method was developed for the assay of ferrochelatase activity in rat liver. The assay was carried out aerobically with Zn2+ and mesoporphyrin or protoporphyrin IX as substrates. Zn-porphyrins formed were extracted with dimethyl sulphoxide/methanol (30:70, v/v) containing Zn-deuteroporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for mesoporphyrin was 5.9 microM, for protoporphyrin IX 8.8 microM and for zinc 6.0 microM. The specific activities were 33.1 +/- 5.0 nmol Zn-mesoporphyrin or 13.4 +/- 2.0 nmol Zn-protoporphyrin formed per hour per mg of protein for mitochondria and 12.3 +/- 2.2 nmol Zn-mesoporphyrin or 4.6 +/- 0.9 nmol Zn-protoporphyrin per hour per mg of protein for liver homogenate.     

Determination of ornithine conjugates of some carboxylic acids in birds by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the determination of ornithine conjugation of some carboxylic acids in vitro has been developed. The ornithine conjugates of benzoic acid, p-nitrobenzoic acid, furancarboxylic acid and phenylacetic acid in an incubation mixture with kidney mitochondria were well separated on a reversed-phase C18 column using a mixture of 10 mM ammonium acetate buffer and methanol as the mobile phase. In addition, by varying the pH of the mobile phase and utilizing the absorption wavelengths (nm) of the conjugates it was possible to resolve and specifically detect each conjugate. The calibration curves were linear in the range of 0.2-16 micrograms/ml for all compounds and the detection limits were about 50 ng/ml except for the ornithine conjugate of phenyl acetic acid (S/N = 2). The ornithine conjugation of some carboxylic acids with chicken kidney mitochondria were determined by this assay method. The activity of ornithine conjugation of benzoic acid, furancarboxylic acid, p-nitrobenzoic acid and phenylacetic acid were 14.5, 5.5, 0.5 and 6.9 nmol/mg of protein, respectively. Moreover, the ornithine conjugation and the glycine conjugation of benzoic acid were examined in birds and rodents. The ornithine conjugation was observed only in chicken (14.5 nmol/mg of protein) and mallard (0.99 nmol/mg of protein).     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor

This paper presents an analysis method for organophosphorus insecticides based on AChE biosensors coupled with a preconcentration and oxidation on a solid phase column. Three organic solvents, acetonitrile (ACN), ethanol and methanol were tested for their influence on AChE activity, insecticide inhibition and their ability to elute the adsorbed insecticides. Our results showed that ACN in a concentration of 5% (v/v) had the less negative effect on biosensor analysis and was the most appropriate organic solvent for the column elution. The presence of the organic solvent in the incubation media of the biosensor was found to induce a reduction of the inhibition percentages. The inhibition of the biosensors was performed in phosphate buffer with 5% (v/v) ACN, while the initial and remaining response of the biosensors were measured in PBS. In these conditions, the LODs of paraoxon and dichlorvos were measured with or without a preconcentration step. The LODs of the AChE biosensor without sample preconcentration were 8 × 10⁻⁸ M for paraoxon and 1 × 10⁻⁷ M dichlorvos and the LOD obtained after the preconcentration step were 2.5 × 10⁻⁸ M for paraoxon and 2.5 × 10⁻⁸ M for dichlorvos. Moreover, the use of the column allowed the heterogeneous oxidation of organophosphorus insecticides for improved LOD.     

CE coupled with amperometric detection using a boron-doped diamond microelectrode: validation of a method for endogenous norepinephrine analysis in tissue

The level of endogenous norepinephrine (NE) in several tissue types was determined by CE with amperometric detection. We report herein on the method validation by HPLC using both amperometric and coulometric detection (CD). Keys to the method were the use of a diamond microelectrode for detection and off-line SPE for sample preparation. The run buffer was a 250 mM borate solution adjusted to pH 8.8 with potassium hydroxide. The diamond microelectrode exhibited a low and stable background current, and a low peak-to-peak noise < or =0.65 pA at the detection potential of +0.86 V versus Ag/AgCl. For standard solutions, the detector signal (i.e., oxidation current) changed linearly with the NE concentration (r(2) = 0.999) between 60 and 1000 nmol/L with an estimated LOD of 51 nmol/L (S/N = 3) and a response variability of 4.5% (RSD, n = 5). An Oasis MCX sorbent was used for SPE and the procedure produced an NE recovery of 95.1 +/- 5.6% (n = 6) from tissue homogenates. NE levels in the spleen, small intestine, and heart of a normotensive rat were found to be in the range of 0.77-0.97, 0.22-0.32, and 0.29-0.45 microg/g tissue (n = 3), respectively.     

His6-OPH enzyme-based bio-hybrid material for organophosphate detection

In this work, we report on the development of a bio-sensing film for the detection of organophosphorous compounds using sol–gel technology. A novel sol–gel immobilization method employing tetraethoxysilane/3-glycidoxypropyltrimethoxysilane/water hybrid material was developed and used to immobilize the hexahistidine-tagged organophosphorous hydrolase enzyme (His₆-OPH). Bio-sensing layers with encapsulated His₆-OPH of various structures (water/silane, precursor ratios) have been prepared. The optimal (P = 5:1, R = 188) bio-sensing layers retained 90% of the initial enzyme activity. Furthermore, the bio-sensing layer prepared by this method was able to maintain its activity at or above 80% of its initial activity for 2 weeks. The bio-hybrid film also showed excellent reusability and improved activity at neutral pH in comparison to the same enzyme in solution.