Application of genetically engineered acetylcholinesterases in screen-printed amperometric biosensor for detection of organophosphorus insecticides (original) (raw)
Related papers
Analytical and Bioanalytical Chemistry, 2002
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 5.2×10–3 mg L–1 and 0.56×10–3 mg L–1, respectively.
Analytica Chimica Acta, 2002
The analytical performance of three acetylcholinesterase (AChE) screen-printed biosensors designed for the detection of pesticides are evaluated. Bioencapsulation of the enzyme in a sol-gel composite and immobilization by metal-chelate affinity were compared with the entrapment of the enzyme in a photopolymerisable polymer. A very low amount of enzyme ranging between 0.8 and 1.2 mIU was immobilized on the electrode surface in each approach. The sensors exhibited a storage stability of over 6 months when the enzyme was encapsulated in a polymer film. Pesticide concentrations in the range of 10 −8 to 10 −9 M paraoxon, dichlorvos and chlorpyrifos ethyl oxon could be detected according to each configuration by following an incubation time of 20 min.
Analytica Chimica Acta, 1994
and carbamic pesticides have been determined with an amperometric acetylcholinesterasebased 4-aminophenyl acetate biosensor. The glassy carbon enzyme membrane covered electrode poised at + 250 mV (vs. sodium chloride saturated calomel electrode) oxidizes the 4-aminophenol formed in the hydrolysis of 4aminophenyl acetate by acetylcholinesterase in the glutaraldehyde cross-linked layer. The activity of acetylcholinesterase is inhibited in the presence of pesticides. The decrease in activity of the enzyme is monitored by the 4-aminophenyl acetate sensor and is correlated to the concentration of pesticide present in solution. The influence of the acetylcholinesterase loading and the acetylcholinesterase to neutral protein (bovine serum albumin) ratio on the biosensor response was studied and the measuring conditions including pH, substrate concentration, and others were optimized. Detection liits of 4.0 and 13.0 nmol 1-t for paraoxon and carbaryl, respectively, were achieved with a 3-min preincubation time.
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.
Biosensors and Bioelectronics, 2004
Screen-printed carbon electrodes modified with the dialdehydes, glutaraldehyde and terephthaldicarboxaldehyde, and then polyethyleneimine have been utilized for production of pesticide biosensors based on acetylcholinesterase. To improve the extent of dialdehyde modification, the electrodes were NH 2-derivatized, initially by electrochemical reduction of 4-nitrobenzenediazonium to a nitroaryl radical permitting attachment to the carbon surface. Subsequent reduction of the 4-nitrobenzene yields a 4-aminobenzene modified carbon surface. Drosophila melanogaster acetylcholinesterase was immobilized either covalently onto dialdehyde modified electrodes or non-covalently onto polyethyleneimine modified electrodes. Internal diffusion limitations due to the dialdehyde and polyethyleneimine modifications increased the apparent K m of the immobilized enzyme. The thiocholine sensitivity was about 90% for dialdehyde modified electrodes and about 10% for polyethyleneimine modified electrodes as compared with non-modified carbon electrodes. The detection limit of the biosensors produced by non-covalent immobilization of acetylcholinesterase onto polyethyleneimine modified carbon electrodes was found to be about 10 −10 M for the organophosphate pesticide dichlorvos.
Biotechnology & Biotechnological Equipment, 2012
A flow-injection system with integrated amperometric biosensor featuring an easily replaceable immobilzed acetylcholinesterase (AChE) membrane was studied. The amperometric biosensor was constructed on the basis of site-specific immobilization of AChE on a hybrid polymer membrane with integrated multi-walled carbon nanotubes. Multistage modification of the membrane and immobilization of the enzyme was proved by Fourier transform infrared spectroscopy. The optimum flow-rate of the flowinjection analysis (FIA) system was 0.5 mL/min. It gave a linear response to acetylthiocholine chloride from 2 μM to 100 μM, with an average RSD of 3.0% (n = 6). The sensitivity of the constructed biosensor was 0.093 µA/µM•cm 2. The K m app value of the immobilized AChE was 1.15 mM and the linear correlation coefficient R 2 , 0.9949. The method had a low detection limit for three organophosphorus pesticides (OPs) in model pesticide solutions-paraoxon ethyl (0.9×10-12 M), monocroptophos (1.8×10-12 M) and dichlorvos (2.0×10-12 M). This indicated that the action of multi-walled nanotubes and controlled site-specific enzyme immobilization ensured high electrocatalytic activity and selectivity of the biosensor towards pesticides. It was found that the biosensor can be reused 15 operation cycles. After storage for 30 days the enzyme membrane retained over 80% of its initial response. The FIA system was used for detection of anti-cholinesterase activity of two binary OP mixtures. The results for paraoxon + monocroptophos and paraoxon + dichlorvos showed that the total inhibition activity was not simply additive, but was lower than the sum of the individual inhibition values. Moreover, the difference between the sum of the individual inhibition values and the real results for the mixture was bigger for the binary system paraoxon and dichlorvos (7-10%) compared with that for paraoxon and monocroptophos (5-7%). The developed biosensor system is an ideal tool for monitoring of organophosphate pesticides.
Journal of Sensors, 2011
A poly(3,4-ethylenedioxythiophene) (PEDOT) conducting ink is presented as a new electroactive material to be incorporated in acetylcholinesterase-(AChE-) based screen printed biosensors, acting not only as a conducting template but also as an electrochemical mediator for thiocholine oxidation. Two different strategies have been studied for the chemical synthesis of PEDOT: (a) a classical oxidative polymerisation and (b) a more innovative enzymatic polymerisation, giving a water-soluble PEDOT. The use of this water-soluble conducting polymer as mediator in screen-printed biosensors enables its deposition by printing like the rest of the layers. Highly sensitive acetylcholinesterase-(AChE-) based screen-printed biosensors have been constructed using both classical and enzymatic PEDOT, in combination with genetically modified AChE. These electrodes allow the measurement of thiocholine oxidation at potentials of 100 mV versus Ag/AgCl reference electrode through the mediation of PEDOT. Inhibition of thiocholine production in presence of CPO allow for detection of this pesticide in concentrations as low as 1·10 −10 M.
Biochemistry Research International, 2013
The exponentially growing population, with limited resources, has exerted an intense pressure on the agriculture sector. In order to achieve high productivity the use of pesticide has increased up to many folds. These pesticides contain organophosphorus (OP) toxic compounds which interfere with the proper functioning of enzyme acetylcholinesterase (AChE) and finally affect the central nervous system (CNS). So, there is a need for routine, continuous, on spot detection of OP compounds which are the main limitations associated with conventional analytical methods. AChE based enzymatic biosensors have been reported by researchers as the most promising tool for analysis of pesticide level to control toxicity and for environment conservation. The present review summarises AChE based biosensors by discussing their characteristic features in terms of fabrication, detection limit, linearity range, time of incubation, and storage stability. Use of nanoparticles in recently reported fabrication strategies has improved the efficiency of biosensors to a great extent making them more reliable and robust.
2015
Organophophorous pesticides are some of the most widely used insecticides in citrus fruit, tomato and apple cultures. Biosensors based on the inhibition of acetylcholinesterase have been used for detection of pesticides in different samples. Due to its permitted use in such cultures, the methodology proposed was applied in order to evaluate the occurrence of matrix effects in the electroanalytical determination of paraoxon residues directly in the samples from apple, tomato and orange, without pretreatment or clean up steps. The results show that the biosensor has the potential for monitoring of pesticides in foods.
Sensors and Actuators B: Chemical, 2014
A novel amperometric biosensor based on a conducting polymer using multi walled carbon nanotube modified electrode was developed for detection of organophosphorus pesticides. Acetylcholinesterase (AChE) was successfully immobilized by covalent linkage on the modified graphite electrode. Carbon nanotubes were functionalized by electrochemical treatment. A conducting polymer; poly(4-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzenamine) (poly(SNS-NH 2)) was synthesized via electropolymerization to examine its matrix properties for biomolecule immobilization. This strategy enhanced electron transfer rate at a lower potential (+100 mV vs. Ag reference) and catalyzed electrochemical oxidation of acetylthiocholine effectively. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle measurements and Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV) techniques were used to monitor changes in surface morphologies and electrochemical characterizations. The proposed biosensor design offered a fast response time (6 s), a wide linear range (0.05 mM and 8.00 mM) and a low detection limit (0.09 mM) with a high sensitivity (24.16 µA mM-1 cm-2) for acetylthiocholine. The inhibition responses of paraoxon, parathion and chlorfenvinphos on the enzymatic activity of AChE were detected. The fabricated biosensor was tested for the detection of pesticides in fortified tap water samples. The results were found to be in good agreement with the ones determined by HPLC/DAD technique.