RESUMEN
Due to their ability to form extremely heat resistant spores, anaerobic bacteria are responsible for frequent food spoilage. The development of rapid and specific methods for the detection and quantification of spore contamination is therefore of major interest. In this paper, we describe for the first time the selection of aptamers specific to spores of Geobacillus stearothermophilus (Gbs), which induce flat sour spoilage in vegetable cans. Eighteen Spore-SELEX cycles were performed including 4 counter-selections with 12 bacteria commonly found in cannery. To optimise candidate amplification, PCR in emulsion was performed, and high-throughput sequencing analysis was applied to follow candidate evolution. Sequencing of aptamers from cycle 18 revealed 43 overrepresented sequences whose copy number exceeds 0.15% of the total obtained sequences. Within this group, the A01 aptamer presented a much higher enrichment with a relative abundance of 17.71%. Affinity and specificity for Gbs spores of the 10 most abundant candidates at cycle 18 were confirmed by PCR assay based on aptamer-spore complex formation and filtration step. Obtaining these aptamers is the starting point for the future development of biosensors dedicated to the detection of Gbs spores.
Asunto(s)
Aptámeros de Nucleótidos , Geobacillus stearothermophilus , Geobacillus stearothermophilus/genética , Esporas Bacterianas/genética , Bacterias , Alimentos , Reacción en Cadena de la Polimerasa , Aptámeros de Nucleótidos/genética , Técnica SELEX de Producción de AptámerosRESUMEN
A sensitive and robust electrochemical cholinesterase-based sensor was developed to detect the quaternary ammonium (QAs) biocides most frequently found in agri-food industry wash waters: benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC). To reach the maximum residue limit of 28 nM imposed by the European Union (EU), two types of cholinesterases were tested, acetylcholinesterase (AChE, from Drosophila melanogaster) and butyrylcholinesterase (BChE, from horse serum). The sensors were designed by entrapping AChE or BChE on cobalt phthalocyanine-modified screen-printed carbon electrodes. The limits of detection (LOD) of the resulting biosensors were 38 nM for DDAC and 320 nM for BAC, using, respectively, AChE and BChE. A simple solid-phase extraction step was used to concentrate the samples before biosensor analysis, allowing for the accurate determination of DDAC and BAC in tap water with limits of quantification (LOQ) as low as 2.7 nM and 5.3 nM, respectively. Additional assays demonstrated that the use of a phosphotriesterase enzyme allows for the total removal of interferences due to the possible presence of organophosphate insecticides in the sample. The developed biosensors were shown to be stable during 3 months storage at 4 °C.
RESUMEN
This study focuses on the spectroelectrochemical quantification of four UV filters, butylmethoxy dibenzoylmethane (BM), benzophenone-3 (BP3), ethylhexyl methoxycinnamate (EM) and octocrylene (OC) used as sunscreens in cosmetics. Three of them exhibited electrochemical activity resulting in the modification of their absorption spectrum under the application of an oxidizing potential of +1.8 V vs. Ag. When working with a mixture containing both electroactive and nonelectroactive UV filters, UV-vis absorption spectra recorded before and after the application of the potential differed. The combination of spectral deconvolution of the spectra pair allowed a more accurate identification and quantification of UV filters than spectral deconvolution of the initial absorbance spectrum alone. This method was effective for the assessment of UV filters in model mixtures and commercial sun creams.
Asunto(s)
Cosméticos , Protectores Solares , Rayos UltravioletaRESUMEN
A novel dual electrochemical immunosensor was fabricated for the rapid and sensitive detection of enrofloxacin (EF) antibiotic in meat. Anti-quinolone antibody was immobilized onto screen-printed dual carbon electrodes via carbodiimide coupling. A new electrochemical probe was synthesized by conjugating difloxacin and aminoferrocene, whose oxidation was measured at + 0.2 V vs. Ag/AgCl by differential pulse voltammetry. The detection principle was based on the competitive binding of this conjugate and free EF on immobilized antibodies. The proposed immunosensor allowed detection of EF at concentrations ranging from 0.005 µg.mL-1 to 0.01 µg.mL-1 with a detection limit of 0.003 µg.mL-1. The immunosensor was stable for at least 1 month at 4 °C and displayed a good specificity for other fluoroquinolones. The new dual electrode design offered an improved accuracy as one electrode was used as negative control. The efficiency of the sensor and the adequacy of the extraction process were finally validated by detecting EF in different meat samples.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Electrodos , Enrofloxacina , Inmunoensayo , Límite de Detección , CarneRESUMEN
Chlortoluron (3-(3-chloro-p-tolyl)-1,1-dimethyl urea) is an herbicide widely used in substitution to isoproturon to control grass weed in wheat and barley crops. Chlortoluron has been detected in groundwaters for more than 20 years; and dramatic increases in concentrations are observed after intense rain outbreaks. In this context; we developed an immunosensor for the determination of chlortoluron based on competitive binding of specific monoclonal antibodies on chlortoluron and immobilized biotinylated chlortoluron; followed by electrochemical detection on screen-printed carbon electrodes. The optimized immunosensor exhibited a logarithmic response in the range 0.01-10 µg·L-1; with a calculated detection limit (LOD) of 22.4 ng·L-1; which is below the maximum levels allowed by the legislation (0.1 µg·L-1). The immunosensor was used for the determination of chlortoluron in natural groundwaters, showing the absence of matrix effects.
Asunto(s)
Técnicas Biosensibles , Monitoreo del Ambiente , Agua Subterránea , Herbicidas , Compuestos de Fenilurea/análisis , Contaminantes Químicos del Agua/análisis , Anticuerpos Monoclonales , Técnicas Electroquímicas , Electrodos , Herbicidas/análisis , Inmunoensayo , Límite de DetecciónRESUMEN
The bis-triazole ligand and its corresponding copper complexes were synthesized and characterized for the first time and proposed as new labels for the development of electrochemical aptasensors. The bis-triazole ligand was prepared from methyl 1,6-heptadiyne-4-carboxylate and 2-(azidomethyl)phenol using classical CuAAC in presence of different copper salts. The X-ray structure of bis-triazole showed a symmetry center (C1). UV-Vis and X-band EPR spectra showed that the coordination capacity of the bis-triazole ligand was improved in the presence of triethylamine due to deprotonation of the triazole and phenolate moieties. After complexation with copper, the obtained complex was successfully attached to an anti-estradiol aptamer through thiol-maleimide coupling, and the resulting labelled aptamer was immobilized on a carbon screen-printed electrode by carbodiimide coupling. The electrochemical response of the resulting sensor was shown to decrease in the presence of estradiol, demonstrating that the developed complexes can be applied for the development of aptasensors.
Asunto(s)
Técnicas Biosensibles , Cobre , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , TriazolesRESUMEN
This work presents the synthesis and characterization of salen/salan metal complexes for their future application as electrochemical labels in affinity sensors. Due to its stability and electrochemical properties, an oxovanadium salan complex was selected and coupled to an estradiol-specific aptamer. The response of the resulting aptasensor was shown to decrease with increasing estradiol concentration.
RESUMEN
Rapid detection of the foodborne pathogen Salmonella in food processing is of crucial importance to prevent food outbreaks and to ensure consumer safety. Detection and quantification of Salmonella species in food samples is routinely performed using conventional culture-based techniques, which are labor intensive, involve well-trained personnel, and are unsuitable for on-site and high-throughput analysis. To overcome these drawbacks, many research teams have developed alternative methods like biosensors, and more particularly aptasensors, were a nucleic acid is used as biorecognition element. The increasing interest in these devices is related to their high specificity, convenience, and relative rapid response. This review aims to present the advances made in these last years in the development of biosensors for the detection and the quantification of Salmonella, highlighting applications on meat from the chicken food chain.
RESUMEN
An ultrasensitive label-free amperometric immunosensor for the detection of chloramphenicol (CAP) residues in milk has been developed by using a screen-printed carbon electrode laminated with a layer of poly (vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane that is covalently immobilized with a CAP antibody (anti-CAP). The performance of the PVA-co-PE nanofiber membrane (PVA-co-PE NFM) on the electrode was compared with a PVA-co-PE casted membrane (PVA-co-PE CM), necessary fabrication steps and performance of the sensors were investigated by electrochemical impedance spectroscopy (EIS). The application of the PVA-co-PE NFM decreased the electron-transfer-resistance by about 4 times compared with a conventional PVA-co-PE casted membrane. Under the optimal conditions, the established immunosensor exhibited high sensitivity for determination of CAP in a range 0.01-10â¯ngâ¯mL-1, with a limit of detection of 0.0047â¯ngâ¯mL-1. In addition to the good selectivity, reusability and stability over time, the prepared immunosensor was successfully used in the detection of CAP in milk samples without any pretreatment.
Asunto(s)
Antibacterianos/análisis , Cloranfenicol/análisis , Residuos de Medicamentos/análisis , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Leche/química , Nanofibras/química , Polietileno/química , Alcohol Polivinílico/química , Animales , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica , Transporte de Electrón , Límite de Detección , Membranas Artificiales , Propiedades de SuperficieRESUMEN
Electrochemical molecularly imprinted polymers (e-MIPs) were for the first time introduced in screen-printed carbon electrodes (SPCE) as the sensing element for the detection of an organic pollutant. To play this sensing role, a redox tracer was incorporated inside the binding cavities of a cross-linked MIP, as a functional monomer during the synthesis step. Ferrocenylmethyl methacrylate was used for this purpose. It was associated with 4-vinylpyridine as a co-functional monomer and ethylene glycol dimethacrylate as cross-linker for the recognition of the endocrine disruptor, Bisphenol A (BPA), as a target. Microbeads of e-MIP and e-NIP (corresponding non-imprinted polymer) were obtained via precipitation polymerization in acetonitrile. The presence of ferrocene inside the polymers was assessed via FTIR and elemental analysis and the polymers microstructure was characterized by SEM and nitrogen adsorption/desorption experiments. Binding isotherms and batch selectivity experiments evidenced the presence of binding cavities inside the e-MIP and its high affinity for BPA compared to carbamazepine and ketoprofen. e-MIP (and e-NIP) microbeads were then incorporated in a graphite-hydroxyethylcellulose composite paste to prepare SPCE. Electrochemical properties of e-MIP-SPCE revealed a high sensitivity in the presence of BPA in aqueous medium compared to e-NIP-SPCE with a limit of detection (LOD) of 0.06â¯nM. Selectivity towards carbamazepine and ketoprofen was also observed with the e-MIP-SPCE.
Asunto(s)
Compuestos de Bencidrilo/aislamiento & purificación , Técnicas Biosensibles , Impresión Molecular , Fenoles/aislamiento & purificación , Piridinas/química , Adsorción , Compuestos de Bencidrilo/toxicidad , Carbono/química , Electrodos , Límite de Detección , Metacrilatos/química , Microesferas , Fenoles/toxicidad , Polímeros/química , Agua/químicaRESUMEN
One of the main pursuits, yet most difficult, in monitoring studies is to identify the sources of environmental pollution. In this study, we have identified health-care facilities from south European countries as an important source of pharmaceuticals in the environment. We have estimated that compounds consumed in by the elderly and released from effluents of senior residences can reach river waters at a concentration higher than 0.01 µg/L, which is the European Medicines Agency (EMA) threshold for risk evaluation of pharmaceuticals in surface waters. This study has been based on five health institutions in Portugal, Spain, and France, with 52 to 130 beds. We have compiled the pharmaceuticals dispensed on a daily base and calculated the consumption rates. From 54.9 to 1801 g of pharmaceuticals are consumed daily, with laxatives, analgesics, antiepileptics, antibiotics, and antidiabetic agents being the main drug families administered. According to excretion rates, dilution in the sewerage system, and elimination in wastewater treatment plants, macrogol, metformin, paracetamol, acetylcysteine, amoxicillin, and gabapentin, among others, are expected to reach river waters. Finally, we discuss the risk management actions related to the discharge of pharmaceuticals from senior residences to surface waters.
Asunto(s)
Monitoreo del Ambiente/métodos , Hogares para Ancianos , Viviendas para Ancianos , Preparaciones Farmacéuticas/análisis , Aguas Residuales/química , Contaminantes Químicos del Agua/análisis , Anciano , Francia , Humanos , Portugal , Medición de Riesgo , Ríos/química , EspañaRESUMEN
New chemiluminescence-based immunoassays for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies. Under optimized conditions, the chemiluminescence immunoassays showed a highly sensitive response to E2 and EE2, with respective detection limits of 0.5 and 1.2 ng L-1. The LOD achieved using biotinylated E2 was in the same order of magnitude as those obtained using commercially available E2-bovine serum albumin conjugate (E2-BSA). The developed devices were successfully applied to analysis wastewater treatment plants effluents (WWTP) with negligible matrix effect.
Asunto(s)
Técnicas de Química Analítica/métodos , Monitoreo del Ambiente/métodos , Estradiol/análisis , Etinilestradiol/análisis , Inmunoensayo , Mediciones Luminiscentes , Animales , Biotinilación , Bovinos , Límite de Detección , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/química , Contaminantes Químicos del Agua/análisisRESUMEN
Molecularly imprinted polymers (MIPs) for phosmet insecticide were synthesized by batch polymerization. The affinity of functional monomers to phosmet was tested using an original method involving an electrochemical biosensor based on acetylcholinesterase inhibition. It was demonstrated that association of phosmet with appropriate functional monomers resulted in a decrease of enzyme inhibition. Using this method, it was shown that N,N-methylenebis(acrylamide) displayed the highest interactions with phosmet using DMSO as solvent. These results were in good accordance with those obtained by conventional computational modeling. Molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs) were synthesized and adsorption isotherms were studied to describe their interaction with phosmet. Freundlich isotherm was able to fit phosmet adsorption on MIPs with good agreement (R2 = 0.9). The pre-exponential factor KF determined for MIPs was 1.439mg(1-N)g-1LN, more that 10 times higher than for NIPs (0.125mg(1-N)g-1. LN), indicating an increase of binding sites number and average affinity.
Asunto(s)
Técnicas Biosensibles/métodos , Inhibidores de la Colinesterasa/análisis , Insecticidas/análisis , Impresión Molecular , Fosmet/análisis , Polímeros/síntesis química , Adsorción , Inhibidores de la Colinesterasa/química , Electroquímica , Insecticidas/química , Cinética , Modelos Moleculares , Conformación Molecular , Fosmet/química , Polimerizacion , Polímeros/química , Extracción en Fase Sólida , Solventes/químicaRESUMEN
This paper describes for the first time the selection of aptamers selective to penicillin. Aptamers were selected using a specific process called Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique is based on the selection of DNA aptamers using penicillin G in solution while the ssDNA library is fixed on a support. One aptamer showing a good affinity to penicillin was finally selected and tested in electrochemical sensor configuration, using electrochemical impedance spectroscopy as detection technique. The developed aptasensor allowed the detection of penicillin in a wide concentration range, comprised between 0.4 and 1000µgL-1 Such performance was compatible with milk analysis, as the maximum residue limit tolerated in this matrix is 4µgL-1. The selectivity of the developed sensor was also studied, showing that the sensor was also able to bind other beta-lactam antibiotics, although with a weaker affinity. Finally the sensor was used for detection of penicillin G in milk. It was shown that a simple sample treatment with isopropanol followed by filtration was sufficient to eliminate matrix effects, allowing the determination of penicillin in milk at concentrations compatible with legislation requirements.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica/métodos , Penicilina G/química , Técnica SELEX de Producción de Aptámeros/métodos , Animales , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Unión Competitiva , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Leche/química , Penicilina G/análisis , Penicilina G/metabolismo , Reproducibilidad de los Resultados , beta-Lactamas/análisis , beta-Lactamas/química , beta-Lactamas/metabolismoRESUMEN
An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2-150 ng/L (LOD=1.15 ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2 µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000 ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50 ng/kg) and infants (25 ng/kg).
Asunto(s)
Aflatoxina M1/análisis , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Contaminación de Alimentos/análisis , Leche/química , Animales , Impedancia Eléctrica , Electroquímica , Glicoles de Etileno/químicaRESUMEN
An amperometric biosensor was designed for the determination of sulcotrione, a ß-triketone herbicide, based on inhibition of hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme allowing the oxidation of hydroxyphenylpyruvate (HPP) in homogentisic acid (HGA). HPPD was produced by cloning the hppd gene from Arabidopsis thaliana in E. coli, followed by overexpression and purification by nickel-histidine affinity. The electrochemical detection of HPPD activity was based on the electrochemical oxidation of HGA at +0.1 V vs. Ag/AgCl, using a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate-modified screen-printed electrode. Assays were performed at 25°C in 0.1 M phosphate buffer pH 8 containing 0.1M KCl. The purified HPPD was shown to display a maximum velocity of 0.51 µM(HGA) min(-1), and an apparent K(M) of 22.6 µM for HPP. HPPD inhibition assays in presence of sulcotrione confirmed a competitive inhibition of HPPD, the calculated inhibition constant K(I) was 1.11.10(-8) M. The dynamic range for sulcotrione extended from 5.10(-10) M to 5.10(-6) M and the limit of detection (LOD), estimated as the concentration inducing 20% of inhibition, was 1.4.10(-10) M.
Asunto(s)
4-Hidroxifenilpiruvato Dioxigenasa/antagonistas & inhibidores , Técnicas Biosensibles/métodos , Ciclohexanonas/análisis , Inhibidores Enzimáticos/análisis , Herbicidas/análisis , Mesilatos/análisis , Arabidopsis/enzimología , Calibración , Ciclohexanonas/farmacología , Electroquímica , Inhibidores Enzimáticos/farmacología , Herbicidas/farmacología , Límite de Detección , Mesilatos/farmacología , Poliestirenos/química , Tiofenos/químicaRESUMEN
This paper presents for the first time the integration of a biosensor for the on-line, real-time monitoring of a photocatalytic degradation process. Paracetamol was used as a model molecule due to its wide use and occurrence in environmental waters. The biosensor was developed based on tyrosinase immobilization in a polyvinylalcohol photocrosslinkable polymer. It was inserted in a computer-controlled flow system installed besides a photocatalytic reactor including titanium dioxide (TiO2) as photocatalyst. It was shown that the biosensor was able to accurately monitor the paracetamol degradation with time. Compared with conventional HPLC analysis, the described device provides a real-time information on the reaction advancement, allowing a better control of the photodegradation process.
Asunto(s)
Acetaminofén/análisis , Monitoreo del Ambiente/métodos , Monofenol Monooxigenasa/química , Fotólisis , Eliminación de Residuos Líquidos , Contaminantes Químicos del Agua/análisis , Técnicas Biosensibles , Cromatografía Líquida de Alta Presión , ElectrodosRESUMEN
We report here a novel method to detect methidathion organophosphorous insecticides. The sensing platform was architected by the combination of molecularly imprinted polymers and sol-gel technique on inexpensive, portable and disposable screen printed carbon electrodes. Electrochemical impedimetric detection technique was employed to perform the label free detection of the target analyte on the designed MIP/sol-gel integrated platform. The selection of the target specific monomer by electrochemical impedimetric methods was consistent with the results obtained by the computational modelling method. The prepared electrochemical MIP/sol-gel based sensor exhibited a high recognition capability toward methidathion, as well as a broad linear range and a low detection limit under the optimized conditions. Satisfactory results were also obtained for the methidathion determination in waste water samples.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas/métodos , Insecticidas/análisis , Impresión Molecular/métodos , Compuestos Organotiofosforados/análisis , Polímeros/química , Contaminantes Químicos del Agua/análisis , Impedancia Eléctrica , Electrodos , Geles , Insecticidas/química , Límite de Detección , Compuestos Organotiofosforados/química , Polimetil Metacrilato/químicaRESUMEN
A combination of molecular modelling and a screening of the library of non-imprinted polymers (NIPs) was used to identify acrylamide as a functional monomer with high affinity towards fenthion, organophosphate insecticide, which is frequently used in the treatment of olives. A good correlation was found between the screening tests and modelling of monomer-template interactions performed using a computational approach. Acrylamide-based molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) were thermally synthesised in dimethyl formamide (porogen) using ethylene glycol dimethacrylate as a cross-linker and 1,1-azo-bis (isobutyronitrile) as an initiator. The chemical and physical properties of the prepared polymers were characterised. The binding of fenthion by the polymers was studied using solvents with different polarities. The developed MIP showed a high selectivity towards fenthion, compared to other organophosphates (dimethoate, methidathion malalthion), and allowed extraction of fenthion from olive oil samples with a recovery rate of about 96%. The extraction of fenthion using MIPs was much more effective than traditional C18 reverse-phase solid phase extraction and allowed to achieve a low detection limit (LOD) (5 µg L(-1)).
Asunto(s)
Cromatografía Líquida de Alta Presión , Fentión/análisis , Insecticidas/análisis , Impresión Molecular/métodos , Polímeros/química , Acetonitrilos/química , Técnicas de Química Analítica , Contaminación de Alimentos/análisis , Límite de Detección , Modelos Moleculares , Aceite de Oliva , Aceites de Plantas/análisisRESUMEN
Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of ß-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 µM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.