A novel, sensitive, reusable and low potential acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel

A novel, low potential and highly sensitive acetylcholinesterase (AChE) biosensor was developed based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotube composite gel thiocholine sensor. Composite gel promoted electron transfer reaction at a lower potential (+50 mV) and catalyzed electrochemical oxidation of thiocholine with high sensitivity. AChE was immobilized in sol-gel matrix that provides a good support for enzyme without any inhibition effect from the ionic liquid. The amount of immobilized enzyme and incubation time with chlorpyrifos were optimized. Chlorpyrifos could be determined in the range of 10(-8)-10(-6)M with a detection limit of 4 nM. Fast and efficient enzyme reactivation was obtained at low obidoxime concentration (0.1mM). Moreover, the biosensor exhibited a good stability and reproducibility and could be use for multiple determinations of pesticide with no loss of the enzyme activity.     

Acetylcholinesterase biosensor for carbaryl detection based on interdigitated array microelectrodes

In this study, an acetylcholinesterase (AChE) biosensor with superior accuracy and sensitivity was successfully developed based on interdigitated array microelectrodes (IAMs). IAMs have a series of parallel microband electrodes with alternating microbands connected together. Chitosan was used as the enzyme immobilization material, and AChE was used as the model enzyme for carbaryl detection to fabricate AChE biosensor. Electrochemical impedance spectroscopy was used in conjunction with the fabricated biosensor to detect pesticide residues. Based on the inhibition of pesticides on the AChE activity, using carbaryl as model compounds, the biosensor exhibited a wide range, low detection limit, and high stability. Moreover, the biosensor can also be used as a new promising tool for pesticide residue analysis.     

Controlled immobilization of acetylcholinesterase on improved hydrophobic gold nanoparticle/Prussian blue modified surface for ultra-trace organophosphate pesticide detection

An ultrasensitive amperometric acetylcholinesterase (AChE) biosensor was fabricated by controlled immobilization of AChE on gold nanoparticles/poly(dimethyldiallylammonium chloride) protected Prussian blue (Au-PDDA-PB) nanocomposite modified electrode surface for the detection of organophorous pesticide. The Au-PDDA-PB membrane served as an excellent matrix for the immobilization of enzyme, which not only enhanced electron transfer but also possessed a relatively large surface area. In addition, the surface hydrophilicity of the Au-PDDA-PB nanocomposite was finely controlled in the static water contact angle range of 25.6-78.1° by adjusting the ratio of gold nanoparticles to PDDA-PB. On an optimized hydrophobic surface, the AChE adopts an orientation with both good activity and stability, which has been proven by electrochemical methods. Benefit from the advantages of the Au-PDDA-PB nanocomposite and the good activity and stability of AChE, the biosensor shows significantly improved sensitivity to monocrotophos, a typical highly toxic organophorous pesticide, with wide linear range (1.0-1000 pg/mL and 1.0-10 ng/mL) and an ultra-low detection limit of 0.8 pg/mL. The biosensor exhibits accuracy, good reproducibility and stability. This strategy may therefore provide useful information for the controlled immobilization of protein and the design of highly sensitive biosensors.     

A novel and highly sensitive acetyl-cholinesterase biosensor modified with hollow gold nanospheres

In this work, a highly sensitive acetylcholinesterase (AChE) inhibition-based amperometric biosensor has been developed. Firstly, a glassy carbon electrode (GCE) was modified with chitosan (Chits). Then, hollow gold nanospheres (HGNs) were absorbed onto the surface of chitosan based on the strong affinity through electrostatic adsorption. After that, l-cysteine (l-cys) was assembled on HGNs through Au–S bond. The hollow gold nanospheres were prepared by using Co nanoparticles as sacrificial templates and characterized by scanning electron microscopy, transmission electron microscopy and ultraviolet spectra, respectively. Finally, AChE was immobilized with covalent binding via –COOH groups of l-cysteine onto the modified GCE. The AChE biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. Under optimum conditions, the inhibition rates of pesticides were proportional to their concentrations in the range of 0.1–150 and 0.1–200 μg L^?1 for chlorpyrifos and carbofuran, respectively, the detection limits were 0.06 μg L^?1 for chlorpyrifos and 0.08 μg L^?1 for carbofuran. Moreover, the biosensor exhibited a good stability and reproducibility and was suitable for trace detection of pesticide residues in vegetables and fruits.     

Immobilization of acetylcholinesterase on gold nanoparticles embedded in sol-gel film for amperometric detection of organophosphorous insecticide

A simple method to immobilize acetylcholinesterase (AChE) on silica sol-gel (SiSG) film assembling gold nanoparticles (AuNPs) was proposed, thus a sensitive, fast and stable amperometric sensor for quantitative determination of organophosphorous insecticide was developed. The large quantities of hydroxyl groups in the sol-gel composite provided a biocompatible microenvironment around enzyme molecule and stabilized its biological activity to a large extent. The immobilized AChE could catalyze the hydrolysis of acetylthiocholine chloride (ATCl) with a Kmapp value of 450 microM to form thiocholine, which was then oxidized to produce detectable single with a linear range of 10-1000 microM. AuNPs catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of organophosphorous insecticide on the enzymatic activity of AChE, using monocrotophos as a model compound, the conditions for detection of the insecticide were optimized. The inhibition of monocrotophos was proportional to its concentration ranging from 0.001 to 1 microg/ml and 2 to 15 microg/ml, with the correlation coefficients of 0.9930 and 0.9985, respectively. The detection limit was 0.6 ng/ml at a 10% inhibition. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.     

Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos

The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.     

Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster: application in amperometric biosensors for methamidophos pesticide detection

Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.     

Automated resolution of dichlorvos and methylparaoxon pesticide mixtures employing a Flow Injection system with an inhibition electronic tongue

An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface

In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs-GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000ngmL(-1) and from 2 to 15mugmL(-1), with a detection limit of 0.3ngmL(-1). The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.     

Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30°C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1-50nM and 1.5-40nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95-100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4°C).     

Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.     

Design of acetylcholinesterases for biosensor applications

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.     

Acetylcholinesterase with mesoporous silica: Covalent immobilization,physiochemical characterization,and its application in food for pesticide detection

Toxic contamination of commonly consumed food products and water due to food chain vulnerability via agricultural products and commodities is a serious health hazard. This study reports on Santa Barbara Amorphous (SBA-15), a type of mesoporous silica nanoparticles, for efficient and stable acetylcholinesterase (AChE) adhesion toward detection of toxic pesticides. AChE was immobilized to the inert framework of mesoporous materials viz. SBA-15 with a proficient hydrolytic response toward acetylthiocholine. The immobilized system acts as a biosensor for the detection of pesticides, which are organophosphorus compounds in food. Both the SBA-15 and immobilized SBA-15 were characterized to give an insight on the physiochemical and morphological modification properties. The enzyme activity was accessed by Ellman’s spectrophotometric bioassay for bare and enzyme-immobilized SBA-15 that resulted in promising enzymatic activity with the counterpart. Enzyme stability was also studied, which exhibited that immobilized AChE retained its catalytic activity up to 60 days and retained 80% of the hydrolytic activity even at 37°C. On the basis of the success of immobilized enzyme (covalent) being inhibited by acetylthiocholine, the sensor was administered for the inhibition by monocrotophos and dimethoate that are used widely as pesticides in agricultural. The inhibitory concentration (IC₅₀) value was found to be 2.5 ppb for monocrotophos and 1.5 ppb for dimethoate inhibiting immobilized AChE. This was verified using cyclic voltammetry, an electrochemical analysis thus proving that the SBA-15@AChE complex could be used as a sensitive and highly stable sensor for detecting the concentration of hazardous pesticide compounds.     

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.     

New performance of biosensor technology for Alzheimer's disease drugs: in vitro comparison of tacrine and 7-methoxytacrine

Two drugs were tested using electrochemical biosensor with immobilized acetylcholinesterase (AChE). The first was commercialized drug tacrine (known also as Cognex) used for treatment of cognitive manifestation of Alzheimer\'s disease (AD). The second one was its 7-methoxy derivate (7-MEOTA) that has not been marketed. We determined the IC50 (6.67+/-0.92)x10-7 M for tacrine and (1.66+/-1.43)x10-9 M for 7-MEOTA. In this in vitro study, 7-MEOTA acts as stronger inhibitor of AChE and in this way could be more favorable for treatment of cognitive manifestation of AD. Our study shows that biosensor technology could be used as a quick and cheap tool for testing of promising AChE inhibitors (AD drug candidates).     

An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.     

Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)(3)(2+) (bpy=2,2'-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 UmL(-1). Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC(50) of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K(i) were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.     

Biosensor based on acetylcholinesterase immobilized onto layered double hydroxides for flow injection/amperometric detection of organophosphate pesticides

We developed a highly sensitive flow injection/amperometric biosensor for the detection of organophosphate pesticides (OPs) using layered double hydroxides (LDHs) as the immobilization matrix of acetylcholinesterase (AChE). LDHs provided a biocompatible microenvironment to keep the bioactivity of AChE, due to the intrinsic properties of LDHs (such as a regular structure, good mechanical, chemical and thermal stabilities, and swelling properties). By integrating the flow injection analysis (FIA) with amperometric detection, the resulting AChE-LDHs modified electrode greatly catalyzed the oxidation of the enzymatically generated thiocholine product, and facilitated the detection automation, thus increasing the detection sensitivity. The analytical conditions for the FIA/amperometric detection of OPs were optimized by using methyl parathion (MP) as a model. The inhibition of MP was proportional to its concentration ranging from 0.005 to 0.3μgmL(-1) and 0.3 to 4.0μgmL(-1) with a detection limit 0.6ngmL(-1) (S/N=3). The developed biosensor exhibited good reproducibility and acceptable stability.     

An acetylcholinesterase biosensor based on graphene–gold nanocomposite and calcined layered double hydroxide

In this study, a novel acetylcholinesterase-based biosensor was fabricated. Acetylcholinesterase (AChE) was immobilized onto a glassy carbon electrode (GCE) with the aid of Cu–Mg–Al calcined layered double hydroxide (CLDH). CLDH can provide a bigger effective surface area for AChE loading, which could improve the precision and stability of AChE biosensor. However, the poor electroconductibility of CLDHs could lead to the low sensitivity of AChE biosensor. In order to effectively compensate the disadvantages of CLDHs, graphene–gold nanocomposites were used for improving the electron transfer rate. Thus, the graphene–gold nanocomposite (GN-AuNPs) was firstly modified onto the GCE, and then the prepared CLDH-AChE composite was immobilized onto the modified GCE to construct a sensitive AChE biosensor for pesticides detection. Relevant parameters were studied in detail and optimized, including the pH of the acetylthiocholine chloride (ATCl) solution, the amount of AChE immobilized on the biosensor and the inhibition time governing the analytical performance of the biosensor. The biosensor detected chlorpyrifos at concentrations ranging from 0.05 to 150 μg/L. The detection limit for chlorpyrifos was 0.05 μg/L.