RESUMO
The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants--LR, YR, and RR--were studied. The corresponding K (I) for each enzyme-MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR. The sensitivity of the enzymes increased in the following order: mutant PP2A < mutant PP1 < natural PP2A. The best limit of detection obtained was 21.2 pM MC-LR using the most sensible enzyme. Methanol, ethanol, and acetonitrile up to 2 % (v/v) may be used in inhibition measurements. An artificial neural network (ANN) was used to discriminate two MC variants--LR and YR--using the differences in inhibition percentages measured with mutant PP1 and natural PP2A. The ANN is able to analyze mixtures with concentrations ranging from 8 to 98 pM MC-LR and 31 to 373 pM MC-YR.
Assuntos
Inibidores Enzimáticos/análise , Microcistinas/análise , Peptídeos Cíclicos/análise , Proteína Fosfatase 1/química , Proteína Fosfatase 2/química , Animais , Clonagem Molecular , Inibidores Enzimáticos/química , Escherichia coli , Humanos , Isoenzimas/química , Isoenzimas/genética , Cinética , Limite de Detecção , Toxinas Marinhas , Microcistinas/química , Mutação , Redes Neurais de Computação , Peptídeos Cíclicos/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SolventesRESUMO
The exponential development of biosensors as powerful analytical tools in the last four decades mainly relies on the high sensitivity and selectivity offered when detecting the target analyte. The transducer and the biological receptor are the bases of the biosensor development. Nevertheless, the bioreceptor immobilisation is also important, playing a key role in the retention of the biological activity, and thus affecting the sensitivity. Parameters such as shelf-life and surface regeneration also depend on the biomolecule immobilisation. Researchers are focusing their efforts towards random and oriented immobilisation procedures. Adsorption, entrapment, cross-linking and electrostatic interactions provide randomly immobilised biomolecules, sometimes partially hindering their biological activity. Covalent binding and affinity interactions may enable oriented biomolecule immobilisations, providing controlled, reproducible and highly active modified surfaces. This paper reviews the main immobilisation strategies used in the biosensors development, putting special emphasis on our contribution to mild and oriented immobilisation techniques.
Assuntos
Técnicas Biossensoriais/métodos , Adsorção , Animais , Reagentes de Ligações Cruzadas/metabolismo , Polímeros/metabolismo , Eletricidade EstáticaRESUMO
The use of an acetylcholinesterase inhibition assay for the detection of dichlorvos in durum wheat samples by a simplified extraction procedure is reported. After an incubation step, the residual activity was determined with an amperometric biosensor using a portable potentiostat. The use of electric eel and recombinant acetylcholinesterase was compared. The effect of the matrix extract was evaluated by using various sample:solvent ratios, 1:2.5, 1:5, 1:10, and 1:20. The optimal extraction ratio, considering the electrochemical interferences and the effect on enzyme activity and bioavailability of the pesticide, was 1:10. Calibrations were performed in buffer and durum wheat extract. The calculated detection limits in buffer solution were 10 ng/ ml and 0.045 ng/ml for electric eel and recombinant acetylcholinesterase, respectively, whereas operating in the matrix extract they increased up to 45 ng/ml and 0.07 ng/ml, corresponding to 0.45 mg/kg (extraction ratio 1:10) and 0.07 mg/kg in samples. These characteristics allowed the detection of contaminated samples at the maximum residue limit, which is 2 mg/kg and well below. Fortified samples of durum wheat were obtained with both dichlorvos and the commercial product Didivane, which contains dichlorvos as an active molecule. At all the tested levels, the occurrence of contaminant was detected with an average recovery of 75%. The total assay time, including the extraction step, was 30 min. Because several extractions as well as most of the assay steps can be run simultaneously, the throughput for one operator is 12 determinations per hour.
Assuntos
Técnicas Biossensoriais/métodos , Diclorvós/análise , Contaminação de Alimentos/análise , Inseticidas/análise , Triticum/química , Acetilcolinesterase , Qualidade de Produtos para o Consumidor , Eletroquímica/métodos , Concentração Máxima Permitida , Sensibilidade e Especificidade , Solventes , Fatores de TempoRESUMO
An ultra-sensitive electrochemical biosensor was successfully developed for rapid detection of pirimiphos-methyl in olive oil, based of genetically-engineered acetylcholinesterase (AChE) immobilization into electrospun chitosan/poly (vinyl alcohol) blend nanofibers. Due to their unique properties such as spatial structure, high porosity, and large surface area, the use of nanofibers allowed improving the biosensor response by two folds. The developed biosensor showed a good performance for detecting pirimiphos-methyl, with a limit of detection of 0.2nM, a concentration much lower than the maximum residue limit allowed set by international regulations (164nM). The biosensor was used for the detection of pirimiphos-methyl in olive oil samples after a simple liquid-liquid extraction, and the recovery rates were close to 100%.
Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais/métodos , Quitosana/química , Membranas Artificiais , Azeite de Oliva/química , Compostos Organotiofosforados/análise , Álcool de Polivinil/química , Acetilcolinesterase/metabolismo , Acetiltiocolina/química , Animais , Eletricidade , Electrophorus , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Contaminação de Alimentos/análise , Nanofibras/químicaRESUMO
An ultra-sensitive screen-printed biosensor was successfully developed for phosmet detection in olive oil, based on a genetically-engineered acetylcholinesterase (AChE) immobilized in a azide-unit water-pendant polyvinyl alcohol (PVA-AWP)/Fe-Ni alloy nanocomposite. Fe-Ni not only allowed amplifying the response current but also lowering the applied potential from 80 mV to 30 mV vs Ag/AgCl. The biosensor showed a very good analytical performance for phosmet detection, with a detection limit of 0.1 nM. This detection limit is lower than the allowable concentrations set by international regulations. In addition to the good reproducibility, operational and storage stability, the developed biosensor was successfully used for the determination of phosmet in olive oil samples without any laborious pre-treatment. The phosmet recovery rate was about 96% after a simple liquid-liquid extraction.
Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Ferro/química , Nanocompostos/química , Níquel/química , Azeite de Oliva/análise , Fosmet/análise , Álcool de Polivinil/química , Acetilcolinesterase/genética , Ligas/química , Enzimas Imobilizadas/genética , Limite de Detecção , Extração Líquido-Líquido , Reprodutibilidade dos TestesRESUMO
Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensor's capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.
Assuntos
Acetilcolinesterase , Técnicas Biossensoriais/métodos , Inseticidas/análise , Malation/análogos & derivados , Redes Neurais de Computação , Acetilcolinesterase/genética , Animais , Técnicas Biossensoriais/estatística & dados numéricos , Carbofurano/análise , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Malation/análise , Mutação , Paraoxon/análise , Proteínas Recombinantes/genéticaRESUMO
A novel microbial sensor system that uses luminous bacteria was developed for the determination of both glucose and toxic compounds. The sensor system consisted of a membrane with luminous bacteria immobilized upon it and a photomultiplier. Measurements were based on the in vivo intensity of the light emitted by the bacteria, as this is affected by their environment. A linear relationship was observed between increased luminescence and concentrations of glucose between 0.05 mM and 0.55 mM. The relative standard deviation was 10% for 0.55 mM glucose (n = 10). Toxic compounds such as benzalkonium chloride, sodium dodecyl sulphate and chromium(VI) were also detected by measuring the decrease in luminescence in their presence.
Assuntos
Bactérias , Técnicas Biossensoriais , Compostos de Benzalcônio/análise , Cromo/análise , Glucose/análise , Medições Luminescentes , Dodecilsulfato de Sódio/análiseRESUMO
The immobilization of enzymes onto transducer support is a mature technology and has been successfully implemented to improve biocatalytic processes for diverse applications. However, there exists still need to design more sophisticated and specialized strategies to enhance the functional properties of the biosensors. In this work, a biosensor platform based on innovative fabrication strategy was designed, and employed for the detection of organophosphate (OP) in natural waters. The biosensor was prepared by incorporating acetylcholinesterase enzyme (AChE) to the graphite paste modified with tetracyanoquinodimethane (TCNQ) mediator, along with the use of a macroalgae (Cladaphropsis membranous) as a functional immobilization support. The novel immobilization design resulted in a synergic effect, and led to enhanced stability and sensitivity of the biosensor. The designed biosensor was used to analyze methyl parathion OP insecticide in water samples collected from a demonstrably contaminated lake of São Luis Island, Maranhão, Northeast of Brazil. Water analysis revealed that the aquatic ecosystem was polluted by sub-ppm concentrations of the OP insecticide, and a good correlation was found between values obtained through biosensor and GC-MS techniques. Our results demonstrated that macroalgae-biosensor could be used as a low-cost and sensitive screening method to detect target analyte.
Assuntos
Técnicas Biossensoriais/métodos , Inseticidas/análise , Metil Paration/análise , Alga Marinha/metabolismo , Poluentes Químicos da Água/análise , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Brasil , Electrophorus , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Grafite/química , Humanos , Inseticidas/metabolismo , Limite de Detecção , Metil Paration/metabolismo , Nitrilas/química , Alga Marinha/química , Poluentes Químicos da Água/metabolismoRESUMO
This work describes the use of layered double hydroxides (LDHs) for the immobilisation of acetylcholinesterase (AChE) on insulator/semiconductor solid supports. Different LDHs have been synthesised by a co-precipitation method. Afterwards, biohybrid materials based on AChE-LDH mixtures have been produced using wild and recombinant enzymes. Spectroscopic techniques have confirmed the LDH phase identity and the links created between the LDH and AChE. Spectrophotometric assays have demonstrated that most of the biohybrid materials are functional and stable. Several configurations have been used for AChE immobilisation. The highest catalytic responses have been observed when using wild enzyme and immobilising AChE-LDH mixtures on LDHs previously deposited on the solid supports. LDHs have been demonstrated to be suitable host matrices for AChE immobilisation on electrodes for the subsequent development of electrochemical biosensors.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Hidróxidos/química , Precipitação Química , Cloretos/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , EspectrofotometriaRESUMO
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.
Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais/instrumentação , Diclorvós/análise , Eletrônica/instrumentação , Análise de Injeção de Fluxo/instrumentação , Paraoxon/análogos & derivados , Praguicidas/análise , Biomimética/instrumentação , Biomimética/métodos , Técnicas Biossensoriais/métodos , Misturas Complexas/análise , Diclorvós/química , Eletroquímica/instrumentação , Eletroquímica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Paraoxon/análise , Paraoxon/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , LínguaRESUMO
Five different clones of antibodies developed against the aflatoxin M(1) were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM(1) in milk, in a wide linear working range (4-250 ng/L). The IC(50) value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM(1)), showing an excellent percentage of recovery, close to 100%.
Assuntos
Aflatoxina M1/análise , Ensaio de Imunoadsorção Enzimática/métodos , Magnetismo , Nanopartículas/química , Animais , Leite/químicaRESUMO
This paper presents the construction of an amperometric biosensor for the highly sensitive detection of the organophosphorus insecticide dichlorvos, based on the inhibition of acetylcholinesterase (AChE). The sensitivity of three AChEs from different sources were tested and compared: AChEs from Electric eel (Ee) and genetically engineered (B394) and wild type (B1) from Drosophila melanogaster (Dm). The enzymes were immobilized by entrapment in a photocrosslinkable PVA-SbQ polymer on a screen printed graphite electrode. The enzyme activity was estimated amperometrically at 100mV versus Ag/AgCl by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine substrate using cobalt phthalocyanine as electron mediator. The pesticide was measured in the presence of 5% acetonitrile without loss of enzyme activity. The best sensitivity was achieved with the Dm mutant B394 with a detection limit of 7x10(-11)M as compared to 1x10(-8)M with the B1 Dm and 6x10(-7)M with the Ee. The B394 biosensor was used to quantify dichlorvos in a sample of skin apple after extraction with acetonitrile.
Assuntos
Técnicas Biossensoriais , Inibidores da Colinesterase/análise , Diclorvós/análise , Eletroquímica/métodos , Malus/química , Resíduos de Praguicidas/análise , Acetilcolinesterase/metabolismo , Calibragem , Sensibilidade e EspecificidadeRESUMO
A new biosensor for detection of phenols, based on tyrosinase immobilization with alumina sol-gel on Sonogel-Carbon transducer, has been developed. The electrode was prepared using high energy ultrasounds directly applied to the precursors. The alumina sol-gel provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenols are oxidized by tyrosinase biosensor to form a detectable product, which was determined at -300 mV vs. Ag/AgCl reference electrode. For phenol, the sensor exhibited a fast response which resulted from the porous structure and high enzyme loading of the sol-gel matrix. The linear range was from 5 x 10(-7)M to 3 x 10(-5)M, with a detection limit of 3 x 10(-7)M. The stability of the biosensor was also evaluated.
Assuntos
Óxido de Alumínio/química , Técnicas Biossensoriais/métodos , Carbono/química , Enzimas Imobilizadas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Fenóis/química , Agaricales/enzimologia , Géis , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soluções , TransdutoresRESUMO
Determination of the organophosphorus pesticides paraoxon, chlorpyrifos oxon, and malaoxon has been performed by a method based on inhibition of acetylcholinesterase (AChE) and amperometric detection in a flow-injection system with enzymes obtained from the electric eel (eeAChE) and Drosophila melanogaster (dmAChE) and immobilized on the surface of platinum electrode within a layer of poly(vinyl alcohol) bearing styrylpyridinium groups. dmAChE is more sensitive than eeAChE to inhibition by chlorpyrifos oxon and paraoxon. The sensitivity difference was largest for chlorpyrifos oxon (detection limit approx. 17 times lower), and practically none for malaoxon. Determination of the analytes in spiked river water samples by use of the dmAChE biosensor resulted in recoveries from 50 to 90 % for chlorpyrifos oxon at levels of 20 to 40 nmol L(-1), 50 to 100 % for paraoxon at 0.6 to 0.8 micro mol L(-1), and 140 to 190 % for malaoxon at 0.6 to 1.2 micro mol L(-1).
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Clorpirifos/análise , Inibidores da Colinesterase/análise , Eletroquímica/métodos , Enzimas Imobilizadas/antagonistas & inibidores , Análise de Injeção de Fluxo/métodos , Inseticidas/análise , Animais , Técnicas Biossensoriais , Clorpirifos/farmacologia , Inibidores da Colinesterase/farmacologia , Drosophila melanogaster , Inseticidas/farmacologia , TorpedoRESUMO
Ethanol is commonly used to extract and dissolve insecticides acting as inhibitors of acetylcholinesterase (EC 3.1.1.7). Here, experiments were undertaken to investigate the influence of solvent on the reaction and inhibition of the enzyme from Drosophila melanogaster. Ethanol (up to 20% by volume) is shown to induce a dramatic reduction of the affinity of acetylcholinesterase for the acetylthiocholine iodide substrate and all the edrophonium chloride, paraoxon ethyl and propidium diiodide inhibitors, with little influence on the rate constants. Taken together, these results point to a main perturbation of active-center related components involved in the formation and/or stability of Michaelis complexes. Inactivation and ligand-stabilization studies of acetylcholinesterase activity further indicate the occurrence of specific "conformational scrambling" at catalytic and regulatory sites. It is proposed that ethanol affects the enzyme reactivity by modifying the conformation of the aromatic gorge containing the active centre and hence, interactions involved in the molecular recognition of substrates and ligands.
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Drosophila melanogaster/enzimologia , Etanol/farmacologia , Acetiltiocolina/metabolismo , Animais , Interações Medicamentosas , Edrofônio/farmacologia , Inseticidas/farmacologia , Ligantes , Paraoxon/análogos & derivados , Paraoxon/farmacologia , Propídio/farmacologiaRESUMO
A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.
Assuntos
Acetilcolinesterase , Enzimas Imobilizadas , Paraoxon/análise , Acetilcolinesterase/metabolismo , Reatores Biológicos , Calibragem , Inibidores da Colinesterase/análise , Enzimas Imobilizadas/metabolismo , Inseticidas/análise , Cinética , Medições Luminescentes , Sensibilidade e EspecificidadeRESUMO
Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.