Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors (original) (raw)
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Analytical and Bioanalytical Chemistry, 2003
Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7′,8,8′-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2×10−8 mol L−1 chloropyrifos-methyl, 5×10−8 mol L−1 coumaphos, and 8×10−9 mol L−1 carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.
Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination
Analytica Chimica Acta, 2000
A number of potentiometric biosensors based on cholinesterases from various sources have been developed and examined for the determination of Co-Ral and Trichlorfon to establish factors affecting the analytical performance of biosensors. Acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from horse serum were immobilised on the surface of antimony electrode and on the commercial membranes (nylon and cellulose nitrate) by treatment with glutaraldehyde in vapours and aqueous solution. The biosensors developed make it possible to determine Trichlorfon and Co-Ral with detection limits 0.01 and 0.02 mg l −1 , respectively.
Journal of Advanced Science, 2000
A disposable cholinesterase biosensor based on screen-printed electrodes (SPE) was assembled and used to assess the effect of miscible organic solvents on the acetylcholinesterase activity and on the inhibitory effect of organophosphorus pesticides on acetylcholinesterase activity. Acetonitrile, ethanol and DMSO were tested in a range of 0 to 30% mixed with phosphate buffer (0.1M, pH7). With 5% acetonitrile and 10% ethanol, an increase of the recorded current was observed. The addition of 0.2% polyethylenimine to the enzyme preparation, before immobilization, allowed the utilization of 15% acetonitrile without negative effect on the enzyme activity. An inhibition calibration curve was obtained using chlorpyrifosethyl-oxon, a compound widely used for agricultural purposes. The lowest detectable amount was 1 ppb following an incubation time of 10min. The use of 5% acetonitrile and 0.2% polyethylenimine did not interfere with the enzyme-inhibitors interactions. The second part is focused on the evaluation of the activity of several genetically modified acetylcholinesterases obtained from Drosophila melanogaster and their inhibition constant face to the methamediphos. The selection of one of them and its immobilization on a SPE allowed a detection of 1.4 ppb methamediphos.
Disposable potentiometric sensors for monitoring cholinesterase activity
Talanta, 2010
A highly sensitive disposable screen-printed butyrylcholine (BuCh) potentiometric sensor, based on heptakis (2,3,6-tri-o-methyl)--cyclodextrin (-CD) as ionophore, was developed for butyrylcholinesterase (BuChE) activity monitoring. The proposed sensors have been characterized and optimized according to the constituents of homemade printing carbon ink including -CD, anionic sites, and plasticizer. The fabricated sensor showed Nernstian responses from 10 −6 to 10 −2 mol L −1 with detection limit of 8 × 10 −7 mol L −1 , fast response time (1.6 s) and adequate shelf-life (6 months). Improved selectivity towards BuCh with minimal interference from choline (Ch) was achieved and the sensor was used for determination of 0.06-1.25 U mL −1 BuChE. The developed disposable sensors have been successfully applied for real-time intoxication monitoring through assaying cholinesterases (ChEs) activity in human serum. Determination of organophosphate pesticide was conducted by measuring their inhibition of BuChE with successful assaying of malathion in insecticide samples with high accuracy and precision.
Biosensors and Bioelectronics, 2014
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 0.1 V under low potential scan rate. The thiocholine sensitivity of the electrode was found to be 143 mA/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 1.1 ppb and that for carbofuran is 0.12 ppb. 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 1C 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.
Comprehensive Analytical Chemistry, 2007
This review is focussed towards the development of acetylcholinesterase enzymatic based biosensors for the quantification of trace concentrations of highly toxic pesticides via their inhibitory effect on the enzyme. Initial results were obtained using wild-type enzymes which have a broad spectrum of susceptibility to a variety of pesticides. The sensitivity and selectivity of the enzyme activity was improved by development and screening of a wide range of mutant enzymes. Optimal enzymes were then exploited within a range of sensor formats. A range of immobilisation techniques including adsorption based approaches, binding via proteins and entrapment within conducting polymers were all studied. The incorporation of stabilisers and co-factors were utilised to optimise electrode performance and stability-with both planar and microelectrode geometries being developed. Reproducible quantification of pesticides could be obtained at concentrations down to 10-17 M, representing a detection limit hitherto unavailable.
The use of polyethyleneimine for fabrication of potentiometric cholinesterase biosensors
Talanta, 2002
Potentiometric biosensors based on butyrylcholinesterase are developed by co-reticulation of the enzyme with glutaraldehyde on an electropolymerized polyethyleneimine film at the electrode surface. The BuChE-electrode was tested as biochemical sensor for detection of an organophosphorus pesticide, trichlorfon in liquid, the detection being based on the enzyme inhibition. The enzyme electrode showed a detection limit for trichlorfon below 10 − 7 M.
Analytica Chimica Acta, 2006
In the present paper, a comparative study using Co-phthalocyanine and Prussian Blue-modified screen-printed electrodes, has been performed. Both the electrodes have demonstrated an easiness of preparation together with high sensitivity towards thicoholine (LOD = 5 × 10 −7 and 5 × 10 −6 M for Co-phthalocyanine and Prussian Blue, respectively) with high potentialities for pesticide measurement. Prussian Blue-modified screen-printed electrodes were then selected for successive enzyme immobilization due to their higher operative stability demonstrated in previous works. AChE and BChE enzymes were used and inhibition effect of different pesticides was studied with both the enzymes. AChE-based biosensors have demonstrated a higher sensitivity towards aldicarb (50% inhibition with 50 ppb) and carbaryl (50% inhibition with 85 ppb) while BChE biosensors have shown a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with 1 ppb). Real samples were also tested in order to evaluate the matrix effect and recovery values comprised between 79 and 123% were obtained.
Analytica Chimica Acta, 2004
A comparison between several acetylcholinesterase (AChE) immobilization procedures on the 7,7,8,8-tetracyanoquinodimethane (TCNQ)modified graphite working electrodes is presented. The immobilization methods employed crosslinking with glutaraldehyde in presence of BSA protein and photopolymerization with poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). The main variations were related to the enzyme charge in each electrode and the enzyme conditioning and storage conditions after immobilization. Initially, the enzyme-substrate reaction was carried out and the following parameters were chrono-amperometrically and-coulometrically monitored: current intensities, time to stabilize the current response, and the mass transfer represented by the Coulomb charge. The screen-printed biosensors that presented best characteristics were then used to perform the inhibition assays and to verify the sensitivity against the following NMC insecticides: aldicarb, carbaryl, carbofuran, and methomyl. In general, diffusion of electrons into the sensitive layer, mass transfer, and time to stabilize the current were adequate in all cases. The Cottrell law was followed before the 1 min of enzyme-substrate reaction. Adequate reproducibility within electrochemical measurements was also observed, with relative standard deviations varying from 6.5 to 18.6%. AChE immobilization with glutaraldehyde allow to obtain robust and reproducible biosensors, but they need a much higher enzyme content (80 mUA per electrode) to achieve current values comparable to that constructed by immobilizing the AChE through photopolymerization with PVA-SbQ (0.7 to 1 mUA per electrode). The limits of detection were determined with a minimum 10% inhibition, and varied from 10 −9 to 8 × 10 −9 M (0.2 to 1.5 ppb) by employing the enzyme immobilization through photopolymerization with PVA-SbQ. In practice, this kind of immobilization procedure is much simpler and produces good results: fast response, adequate reproducibility, large pesticides working ranges, and excellent sensitivities to N-methylcarbamates (NMCs) which in general do not present enzyme inhibition power as elevated as for the organophosphate pesticides.