RESUMEN
Lactate oxidase (LOx), recognized to selectively catalyze the lactate oxidation in complex matrices, has been highlighted as preferable biorecognition element for the development of lactate biosensors. In a previous work, we have demonstrated that LOx crosslinking on a modified screen-printed electrode results in a dual range lactate biosensor, with one of the analysis linear range (4 to 50â¯mM) compatible with lactate sweat levels. It was advanced that such behavior results from an atypical substrate inhibition process. To understand such inhibition phenomena, this work relies in the study of LOx structure when submitted to increased substrate concentrations. The results found by fluorescence spectroscopy and dynamic light scattering of LOx solutions, evidenced conformational changes of the enzyme, occurring in presence of inhibitory substrate concentrations. Therefore, the inhibition behavior found at the biosensor, is an outcome of LOx structural alterations as result of a pH-dependent mechanism promoted at high substrate concentrations.
Asunto(s)
Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Reactivos de Enlaces Cruzados/química , Enzimas Inmovilizadas/química , Oxigenasas de Función Mixta/química , Proteínas Bacterianas/metabolismo , Técnicas Biosensibles/instrumentación , Dispersión Dinámica de Luz , Electrodos , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ácido Láctico/química , Ácido Láctico/metabolismo , Oxigenasas de Función Mixta/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The World Health Organization considers iodide deficiency diseases (IDD) to be a public health problem. The main indicator to access IDD is urinary iodide, since approximately 90% of the ingested iodide uses this clearance path, with urine being a preferable target for the analysis. In this work, two screen-printed carbon electrode (SPCE) based sensors were developed to determine iodide by using only a single drop of sample. A first approach based on a SPCE proves to selectively determine iodide through the control of the cathodic stripping voltammetric (CSV) parameters. However, this strategy exhibits a gap in determining trace iodide concentrations, which is improved by modifying the working electrode surface with a chitosan coating. The performance of this new CS/SPCE-based sensor was compared with that of the previous SPCE-based sensor, showing improved iodide determination sensitivity. A limit of detection of 1.0â¯×â¯10-8 M and a linear analysis range of 0.15-500⯵M were achieved with this sensor. The application of both sensors to real-life samples found values close to those determined by the standard Sandell-Kolthoff spectrophotometric method, proving them to be powerful analytical tools for iodide determination in different kinds of samples, including biological matrices.
Asunto(s)
Técnicas Electroquímicas , Yoduros/análisis , Carbono/química , Electrodos , Modelos LinealesRESUMEN
The performance of several MF and UF ceramic membranes that filter the seawater surrounding mussel rafts is studied for preventive detection of toxic episodes. The modified fouling index applied to UF membranes (MFI-UF) is used to compare fouling rates and membrane fouling levels. The reduction of several quality parameters such as turbidity, alkalinity, chemical oxygen demand (COD), and chlorophyll content is explained by the higher quality of the UF rather than the MF permeates. Membrane rejection rates of Pb+2 and okadaic acid, the main toxin that provokes toxic episodes due to bloom-forming algae, are measured under different pH and pressures. Measurements are taken particularly at filtration times before and after the formation of stable caking on the membrane surface. The results indicated that trace concentrations of heavy ions were mainly rejected by the membrane charge, until the saturation point was reached, and that no rejections occurred when the pH was lower than the isoelectric point of the membranes. However, most of the okadaic acid was rejected due to the formation of cake on the membrane surface. The rejection of okadaic acid depended on the membrane pore size and transmembrane pressure, yielding negative rejections under specific filtration conditions.
Asunto(s)
Purificación del Agua , Cerámica , Membranas Artificiales , Ácido Ocadaico , Agua de Mar , UltrafiltraciónRESUMEN
Lactate concentration is studied as an indicator of physical performance in sports activities, and is also analyzed in health care applications, as well as in the food and cosmetic industries. This organic acid is routinely determined in different concentration ranges, depending on the type of samples for analysis. This paper describes the development of a screen-printed lactate oxidase (LOx) based biosensor to determine lactate in broad concentration range. The Cu-MOF (copper metallic framework) crosslinking of 0.25U LOx in a chitosan layer, allows to determine the enzymatic product generated on a platinum modified working electrode, at 0.15â¯V (vs SPE Ag/AgCl). The biosensor responds linearly in two different concentration ranges: a first catalysis range of 14.65⯵Aâ¯mM-1, from 0.75⯵M to 1â¯mM, followed by a saturation zone from 1 to 4â¯mM, after which a substrate enzymatic inhibition of 0.207⯵Aâ¯mM-1, is observed up to 50â¯mM. These two ranges of analysis would allow the biosensor to be used for the determination of lactate in different types of samples, with low and high content of lactate. The method reproducibility was kept below 7% and a limit of detection of 0.75⯵M was obtained. The device was successfully used in the determination of lactate in sweat and saliva, as a low cost noninvasive analysis, and also in wine samples.
RESUMEN
Human cytomegalovirus (HCMV) is a herpes virus that can cause severe infections. Still, the available methods for its diagnostic have the main disadvantage of requiring long time to be performed. In this work, a simple magnetic particle-based enzyme immunoassay (mpEIA) for the quantification of glycoprotein B of Human cytomegalovirus (gB-HCMV) in urine samples is proposed. The immunosensor scheme is based on the analyte protein gB-HCMV sandwiched between a primary monoclonal antibody, (MBs-PrG-mAb1), and a secondary anti-gB-HCMV antibody labelled with Horseradish peroxidase (Ab2-HRP) to allow spectrophotometric detection. The mpEIA analytical performance was tested in urine samples, showing a linear dependence between gB-HCMV concentration and the absorbance signal at 450â¯nm in a range of concentrations from 90 to 700â¯pgâ¯mL-1. The calculated detection limits for gB-HCMV were 90⯱â¯2â¯pgâ¯mL-1 and the RSD was about 6.7% in urine samples. The immunosensor showed good selectivity against other viruses from Herpesviridae family, namely varicella zoster and Epstein Barr viruses. The recoveries of spiked human urine samples at 0.30-0.50â¯ngâ¯mL-1 concentration levels of gB-HCMV ranged between 91 to 105%. The proposed mpEIA method was validated following the guidelines of the European Medicines Agency (EMEA-2014), and allows rapid, successful and easy quantification of gB-HCMV in urine samples.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/inmunología , Técnicas para Inmunoenzimas/métodos , Proteínas del Envoltorio Viral/orina , Anticuerpos Monoclonales/inmunología , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/orina , Infecciones por Citomegalovirus/virología , Voluntarios Sanos , Humanos , Imanes , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Espectrofotometría , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
This paper shows the ability of simple modified screen-printed devices for the electrochemical detection of ascorbic acid in different samples, including serum samples. Even if the presence of uric acid does not greatly affect the analysis of ascorbic acid using screen-printed carbon electrodes modified by gold nanoparticles, dopamine must be considered an interferent. Thus, the joint determination of the three compounds has been set out using methods of multivariate calibration. The validated models, built by partial least squares regressions, have been used to determine the concentration of ascorbic acid, dopamine and uric acid of spiked serum samples, with successful results.
Asunto(s)
Ácido Ascórbico/sangre , Análisis Químico de la Sangre/instrumentación , Carbono/química , Oro/química , Nanopartículas del Metal/química , Impresión , Dopamina/sangre , Electroquímica , Electrodos , Humanos , Ácido Úrico/sangreRESUMEN
This work describes the development of a new, simple and inexpensive method for the determination of chloride ions, by using voltammetric disposable sensors. The sensor includes three screen printed electrodes: a working, an auxiliary (both carbon based paste electrodes), and a pseudo-reference Ag/AgCl paste based electrode. Since the presence of chloride ions in the solution modifies the equilibrium potential of Ag/AgCl electrode, the concentration of this analyte has been determined through the systematic shift of the voltammetric peak potential of a control species such as potassium ferricyanide, potassium ferrocyanide or ferrocenemethanol. These control species can be used in solution or mixed into the carbon paste of the working screen printed electrode. In order to characterize the developed methods, reproducibility, repeatability and detection limit of the sensors were calculated in each case. Reproducibility values below 3% (n=5) were obtained. When ferrocenemethanol was used as control species, the lowest quantity of chloride ions detected was 10.0mM. A comprehensive study of interfering ions was also carried out. These sensors were successfully applied to determine the chloride content in sea water and in a commercial saline solution sample.
RESUMEN
Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h).
Asunto(s)
Cocaína/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Magnetismo , Saliva/química , Cocaína/sangre , Cocaína/orina , Límite de DetecciónRESUMEN
Considering the enzymatic activity of the cytochrome P450 2D6 on substrates such as codeine, the current paper includes the development of an enzymatic biosensor for detection of this drug. Home-made screen-printed electrodes were used as electrochemical transducers of the biosensor, in which the enzyme was covalently attached to the carbon surface of the working electrode, this type of modification being the most suitable for the immobilization of the biological element. Chronoamperometric measurements were carried out under optimum conditions of pH and working potential, pH 7 and +200 mV vs. screen-printed Ag/AgCl electrode, giving a reduction signal related to the concentration of codeine in solution. Consecutive additions of a solution of codeine were performed to obtain calibration curves in order to validate the electrochemical method in terms of precision and calculate its capability of detection. These biosensors were used for the determination of codeine in urine and commercial pharmaceutical samples.
Asunto(s)
Técnicas Biosensibles , Codeína/química , Citocromo P-450 CYP2D6/química , Electroquímica/métodos , Enzimas Inmovilizadas/química , Adsorción , Calibración , Carbono/química , Química Farmacéutica/métodos , Codeína/orina , Reactivos de Enlaces Cruzados/química , Relación Dosis-Respuesta a Droga , Electrodos , Reacciones Falso Positivas , Humanos , Concentración de Iones de Hidrógeno , Reproducibilidad de los Resultados , UrinálisisRESUMEN
This paper describes the voltammetric determination of cocaine in presence of three different interferences that could be found in street samples using disposable sensors. The electrochemical analysis of this alkaloid can be affected by the presence of codeine, paracetamol or caffeine, whose oxidation peaks may overlap and lead to false positives. This work describes two different solutions to this problem. On one hand, the modification of disposable carbon sensors with carbon nanotubes allows the voltammetric quantification of cocaine by using ordinary least squares regressions in the concentration range from 10 to 155 µmol L(-1), with a reproducibility of 5.6% (RSD, n = 7. On the other hand, partial least squares regressions are used for the resolution of the overlapped voltammetric signals when using screen-printed carbon electrodes without any modification. Both procedures have been successfully applied to the evaluation of the purity of cocaine street samples.
Asunto(s)
Anestésicos Locales/análisis , Cocaína/análisis , Técnicas Electroquímicas/instrumentación , Nanotubos de Carbono/química , Detección de Abuso de Sustancias/instrumentación , Acetaminofén/análisis , Cafeína/análisis , Codeína/análisis , Electrodos , Análisis de los Mínimos Cuadrados , Límite de Detección , Nanotubos de Carbono/ultraestructura , Oxidación-Reducción , Reproducibilidad de los ResultadosRESUMEN
Chronoamperometric assays based on tyrosinase and glucose oxidase (GOx) inactivation have been developed for the monitoring of Cr(III) and Cr(VI). Tyrosinase was immobilized by crosslinking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as electron transfer mediator. The tyrosinase/SPC(TTF)E response to pyrocatechol is inhibited by Cr(III). This process, that is not affected by Cr(VI), allows the determination of Cr(III) with a capability of detection of 2.0±0.2 µM and a reproducibility of 5.5%. GOx modified screen-printed carbon platinised electrodes (SPCPtEs) were developed for the selective determination of Cr(VI) using ferricyanide as redox mediator. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr(III) and to detect the toxic species Cr(VI). Chronoamperometric response of the biosensor towards glucose decreases with the presence of Cr(VI), with a capability of detection of 90.5±7.6 nM and a reproducibility of 6.2%. A bipotentiostatic chronoamperometric biosensor was finally developed using a tyrosinase/SPC(TTF)E and a GOx/SPC(Pt)E connected in array mode for the simultaneous determination of Cr(III) and Cr(VI) in spiked tap water and in waste water from a tannery factory samples.
Asunto(s)
Técnicas Biosensibles , Cromo/clasificación , Técnicas Electroquímicas , Electrodos , Cromo/análisisRESUMEN
Screen-printed carbon electrodes have been modified with tetrathiafulvalene and sulfite oxidase enzyme for the sensitive and selective detection of sulfite. Amperometric experimental conditions were optimized taking into account the importance of quantifying sulfite in wine samples and the inherent complexity of these samples, particularly red wine. The biosensor responds to sulfite giving a cathodic current (at +200 mV vs screen-printed Ag/AgCl electrode and pH 6) in a wide concentration range, with a capability of detection of 6 µM (α=ß=0.05) at 60°C. The method has been applied to the determination of sulfite in white and red samples, with averages recoveries of 101.5% to 101.8%, respectively.
Asunto(s)
Técnicas Biosensibles , Carbono , Electrodos , Sulfito-Oxidasa/metabolismo , Sulfitos/análisis , Vino/análisis , Humanos , Proteínas Recombinantes/metabolismoRESUMEN
This paper describes the development of screen-printed cytochrome P450 2B4 based biosensors, from the fabrication of the device using a single screen-printing technology to its characterization and application in the determination of cocaine street samples. Voltammetric measurements were first performed in order to study the electrochemical behavior of the enzyme trapped in the structure of the working electrode, as well as its interaction with one of its substrates, cocaine. Then, chronoamperometric measurements were carried out for the characterization of the biosensors. Calibration curves under optimum conditions of the experimental variables have been used for establishing the reproducibility (5.91%, n=5), the repeatability (10.45%, n=3) and the capability of detection (0.2mM, α=ß=0.05) of the described method in the calibration range from 0.2 to 1.2mM. Finally, the performance of the developed biosensors has been successfully probed by the analysis of the purity of cocaine street samples.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Técnicas Biosensibles , Cocaína/análisis , Técnicas Electroquímicas , Familia 2 del Citocromo P450RESUMEN
The current paper presents the chronoamperometric determination of codeine using screen-printed carbon electrodes that incorporate tetrathiafulvalene in the matrix of the working electrode, as mediator, and cross-linked acetylcholinesterase. Applying a potential of +250 mV, a 1mM solution of acetylthiocholine in electrolyte solution pH 7 gives an oxidation signal due to the dimerization of its metabolite after the reaction with the enzyme. This electrochemical signal is decreased by consecutive additions of a solution of codeine, which allows the performance of curves of calibration for the validation of this electrochemical method, giving a reproducibility of 3.31% (n=6) and a capability of detection of 20 µM. This type of inhibition has been studied by means of a Lineweaver-Burk plot. Additionally, the developed biosensor was used for the determination of the quantity of codeine in pharmaceutical commercial tablets and urine samples.
Asunto(s)
Acetilcolinesterasa/metabolismo , Técnicas Biosensibles/métodos , Codeína/análisis , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/instrumentación , Calibración , Codeína/orina , Técnicas Electroquímicas/instrumentación , Pruebas de Enzimas/métodos , Enzimas Inmovilizadas/metabolismo , Humanos , Cinética , Reproducibilidad de los Resultados , Comprimidos/químicaRESUMEN
An amperometric assay based on acetylcholinesterase (AChE) inactivation has been developed for the monitoring of permethrin using a screen-printed three-electrode system. The enzyme AChE catalyzes the hydrolysis of acetylthiocholine to thiocholine, which can be electrochemically oxidized. The presence of permethrin inhibits the AChE activity, resulting in a lower thiocholine production and thus, a decrease in the amperometric oxidation current. Immobilization of AChE was performed by cross-linking giving a capability of detection of 8.1±0.4 µM. Repeatability and reproducibility of the developed AChE biosensor were also calculated, yielding values of 9.6% (n=4) and 5.4% (n=5), respectively related to the slopes of the calibration curves performed in the range from 6.2 up to 41 µM. The method was successfully applied to the determination of permethrin content in a commercial lice gel.
Asunto(s)
Acetilcolinesterasa/química , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/análisis , Insecticidas/análisis , Permetrina/análisis , Técnicas Biosensibles , Inhibidores de la Colinesterasa/análisis , Inhibidores de la Colinesterasa/química , Monitoreo del Ambiente/instrumentación , Contaminantes Ambientales/química , Insecticidas/química , Permetrina/químicaRESUMEN
Screen-printed carbon electrodes (SPCEs) electrochemically platinised (Pt-SPCEs) and screen-printed platinised carbon electrodes (SPC(Pt)Es) have been chronoamperometrically characterized for the determination of formaldehyde (FA). The oxidation current registered at 600 mV in the FA concentration range from 0.99 to 9.09 mmol L(-1), led to higher precision values in terms of repeatability and reproducibility when using SPC(Pt)Es. SPC(Pt)Es have been also used for alcohol oxidase (AOX) cross-linked immobilization in the development of enzymatic biosensors for FA. The enzymatic reaction produces hydrogen peroxide, which has been chronoamperometrically monitored and related to FA concentration in different kinds of samples. Experimental design methodology has been performed to optimize the pH and the applied potential. This method has shown a repeatability and reproducibility of 3% (n=4) and 6% (n=4) respectively, related to the slopes of the calibration curves performed in the range from 60 to 460 µmol L(-1). The use of this kind of biosensor, which has a detection capability of 60 µmol L(-1) for a given probability of false positive and negative equal to 0.05, has been probed in the determination of FA in commercial samples for histology.
Asunto(s)
Técnicas Biosensibles/instrumentación , Equipos Desechables , Formaldehído/análisis , Electrodos de Iones Selectos , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodosRESUMEN
This work summarizes the manufacturing procedure of Horseradish peroxidase (HRP) based biosensors for the determination of the mycotoxin Ochratoxin A (OTA). The biosensors have been fabricated using the single technology of screen-printing. That is to say, an HRP containing ink has been directly screen-printed onto carbon electrodes, which offers a higher rapidity and simplicity in the manufacturing process of biosensors for OTA determination. The formal redox potential of the Fe(III/II) moiety of HRP has been used to demonstrate the effective loading of enzyme into the ink. The chronoamperometric oxidation current registered has been successfully related to the concentration of OTA in solution from different samples, including beer ones. Under the optimum conditions of the experimental variables, precision in terms of reproducibility and repeatability has been calculated in the concentration range from 23.85 to 203.28 nM. A relative standard deviation for the slopes of 10% (n = 4) was obtained for reproducibility. In the case of repeatability, the biosensor retained a 30% of the initial sensitivity after the third calibration. The average capability of detection for 0.05% probabilities of false positive and negative was 26.77 ± 3.61 nM (α = 0.05 and ß=0.05, n = 3).
Asunto(s)
Técnicas Biosensibles/métodos , Ocratoxinas/análisis , Carbono/química , Técnicas Electroquímicas , Electrodos , Peroxidasa de Rábano Silvestre/metabolismo , Hierro/química , Oxidación-ReducciónRESUMEN
A new electrochemical method has been described and characterized for the determination of cocaine using screen-printed biosensors. The enzyme cytochrome P450 was covalently attached to screen-printed carbon electrodes. Experimental design methodology has been performed to optimize the pH and the applied potential, both variables that have an influence on the chronoamperometric determination of the drug. This method showed a reproducibility of 3.56% (n=4) related to the slopes of the calibration curves performed in the range from 19 up to 166nM. It has been probed the used of this kind of biosensors in the determination of cocaine in street samples, with an average capability of detection of 23.05±3.53nM (n=3, α=ß=0.05).
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Técnicas Biosensibles/métodos , Cocaína Crack/análisis , Carbono/química , Cocaína Crack/metabolismo , Familia 2 del Citocromo P450 , Electroquímica/métodos , Enzimas Inmovilizadas/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The measurement of antiepileptic drugs (AEDs) in different samples has received considerable attention due to the directly correlation between clinical effects and plasma concentration. Numerous methods have been extensively applied to the analysis of AEDs since many years ago providing reliable and accurate results. This paper provides an overview of electrochemical techniques used for the analysis of different AEDs. More than sixty papers from refereed analytical chemistry journals on the analysis of AEDs in pharmaceutical formulations and biological samples are included. The present review shows the development of novel measurement electrochemical based on the use of different types of electrodes including mercury electrodes, screen-printed electrodes (SPEs) and electrodes modified with metal films and nanoparticles. Electrochemical biosensors and immunosensors developed for the analysis of AEDs have been also reviewed.
Asunto(s)
Anticonvulsivantes/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Electroquímica , Electrodos , InmunoensayoRESUMEN
This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 microM and from 0.4 to 2.4 microM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18+/-0.01 microM in the case of DAO/HRP and 0.40+/-0.04 microM (alpha=0.05 and beta=0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.