Electrochemical nanoprobe-based immunosensor for deoxynivalenol mycotoxin residues analysis in wheat samples.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by several species of Fusarium fungi, which can be predominantly found in agricultural crops such as wheat. In livestock, deoxynivalenol-contaminated grain can produce vomiting, feed refusal, weight loss, and diarrhea. This paper reports an electrochemical immunosensor for the detection of residual DON mycotoxin in food samples. The device uses electrochemical nanoprobes (CdSNP-AbDON) and antigen biofunctionalized magnetic µ-particles (DON-BSAMP) to detect the mycotoxin. CdSNP-AbDON are prepared by labelling the DON-specific antibodies with CdS nanoparticles (CdSNPs). Nanoparticle size and CdSNP-AbDON conjugation ratio are characterized using TEM images. The metal ions released by the CdSNP are reduced at the working electrode and read by anodic stripping voltammetry. DON can be detected in PBST buffer with an IC50 of 6.74 ± 0.19 µg L-1. The high detectability of the immunosensor developed allows detection of DON residues in 50-fold diluted wheat extracts. The limit of detection (LOD, IC90) accomplished in wheat is of 342.4 µg kg-1, which is below the maximum residue limit (MRL, 1750 µg kg-1 for unprocessed durum wheat, 750 µg kg-1 for cereals intended for direct human consumption) established by the EU for this mycotoxin. The working range is in the interval between 610 and 6210 µg kg-1. The performance of the immunosensor was compared with the ELISA assay. DON naturally contaminated wheat samples were analyzed with the immunosensor, showing acceptable recoveries. Graphical abstract.

Rapid immunochemical analysis of the sulfonamide-sugar conjugated fraction of antibiotic contaminated honey samples.

A rapid high-throughput immunochemical screening (HtiS) procedure for the analysis of the sulfonamide (SA)-sugar conjugated fraction of antibiotic contaminated honey samples has been developed. Studies performed with this matrix have indicated that sulfonamide antibiotics are conjugated to sugars rapidly and quantitatively, providing samples with very low SA immunoreactivity. Therefore, sulfonamides must be first released before the analysis, and for this purpose, a simple and fast sample preparation procedure has been established consisting of hydrolyzing the sample for 5 min, adjusting the pH and buffering the sample prior to the immunochemical analysis. Under these conditions, honey samples could be directly analyzed without any additional sample treatment, other than dilution. Recovery values of the whole analytical procedure were greater than 85%. The analysis of the same samples without the hydrolysis provided recovery values below 5%. Selectivity studies performed in hydrolyzed honey samples revealed that nine relevant sulfonamide antibiotics can be detected with limit of detection (LOD) values below the action limits established by some EU countries (Belgium, 20 µg kg(-1), United Kingdom or Switzerland, 50 µg kg(-1)).

A portable electrochemical magnetoimmunosensor for detection of sulfonamide antimicrobials in honey.

A new electrochemical magnetoimmunosensor (EMIS) has been developed for the screening of residues of sulfonamide antimicrobials in honey samples. The immunosensor is able to detect up to ten different sulfonamide congeners at levels below the action points established in some European countries (25 µg kg(-1)) after a hydrolysis step in which the sulfonamides are released from the corresponding conjugates formed in samples of this type. In spite of the complexity of the sample after the hydrolysis procedure, the EMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. For example, sulfapyridine, used as a reference, can be detected in hydrolyzed honey with a limit of detection (IC90) of 0.1 ± 0.03 µg kg(-1). Considering that the use of antibiotics for bee treatment is prohibited in the European Union, the immunosensor presented here could be an excellent screening tool. Moreover, several samples can be processed in parallel, which facilitates the analysis, reducing the necessity to use more costly confirmatory methods for just screening. As a proof of concept, a set of blind honey samples (spiked and incurred) were analyzed and the results were compared with those obtained by high-performance liquid chromatography-tandem mass spectrometry, demonstrating the potential of the EMIS as a screening tool.

Development of a Coulombimetric immunosensor based on specific antibodies labeled with CdS nanoparticles for sulfonamide antibiotic residues analysis and its application to honey samples.

A new electrochemical immunosensor has been developed to detect sulfonamide antibiotic residues in food samples. The immunosensor presented uses immunoreagents specifically developed for the broad recognition of the sulfonamide antibiotic family, a graphite composite electrode (GEC), biofunctionalized magnetic µ-particles and electrochemical nanoprobes prepared by labeling the specific antibodies with CdS nanoparticles (CdSNP). After the immunochemical reaction, the CdSNP are dissolved and the metal ions released are reduced at the electrode and read as in the form of current or charge signal, by the well-known anodic stripping technique. Due to the amplification effect on the amperometric/coulombimetric signal produced by the CdSNP, a high detectability can be reached. Thus, sulfapyridine (SPY), one of the most widely used sulfonamide congeners, can be detected in buffer with an IC50current of 0.20±0.25µgL(-1). The immunosensor has been applied to the analysis of residues of this antibiotic in honey samples. Due to the reported formation of sulfonamide-sugar conjugates in this type of matrix, honey samples are first hydrolyzed in acidic media. The use of magnetic particles minimizes the matrix effect allowing to reach a detectability (LOD, limit of detection) of 0.11µgkg(-1) (current measurements), far below the limits established in some countries for these types of residues in honey samples. Due to the use of magnetic racks, multiple samples can be run simultaneously. The whole analysis process can be performed in around 22min.

Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine.

A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2 microg L(-1). The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods.

Utilidad de las técnicas de imagen y biomarcadores en la predicción del riesgo cardiovascular en pacientes con enfermedad renal crónica en España: Proyecto NEFRONA* / Usefulness of imaging techniques and novel biomarkers in the prediction of cardiovascular risk in patients with chronic kidney disease in Spain: The NEFRONA project

Justificación: La enfermedad cardiovascular (ECV) es la primera causa de mortalidad en pacientes con enfermedad renal crónica (ERC). La valoración del riesgo cardiovascular a partir de los factores tradicionales es poco útil en esta población debido al fenómeno de «reverse epidemiology» y a la existencia de factores específicos derivados de la uremia. En este trabajo presentamos el protocolo del proyecto NEFRONA, un estudio prospectivo con el objetivo de evaluar la utilidad de técnicas de imagen y biomarcadores en la predicción de la ECV en la ERC. Métodos: A partir de noviembre 2009 se reclutarán 2.661adultos asintomáticos con ERC (estadios 3-5D) procedentes de consultas ambulatorias de nefrología y centros de diálisis distribuidos a lo largo del territorio español. Asimismo, se incluirán843 participantes sin ERC (grupo control). Además, semestralmente se registrará la aparición de acontecimientos cardiovasculares y mortalidad. Un equipo itinerante realizará una ecografía carotíde a para valorar el grosor íntima-media y la presencia de placas, y determinará el índice tobillo-brazo para la clasificación de la enfermedad ateromatosa. Para el estudio de las calcificaciones vasculares se utilizará un score basado en la presencia de calcificaciones en las arterias carótidas, femorales y braquiales, y en las válvulas cardíacas, mediante ecografía. Finalmente, se recogerán muestras de sangre para la determinación de biomarcadores. Discusión: El proyecto NEFRONA nos permitirá evaluar la utilidad de las técnicas de imagen y biomarcadores en la valoración de la enfermedad ateromatosa y su valor predictivo en la población española con ERC (AU)

Background: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with chronic kidney disease(CKD). Cardiovascular risk assessment in this population is hampered by the failure of traditional risk factors to fully account for the elevated CVD risk, mainly due to the reverse epidemiology effect, and the presence of risk factors specifically related to uremia. Hereby, we present the protocol of a prospective study aimed to assess the predictive value of imaging techniques and biomarkers for CVD in patients with CKD. Methods: From November 2009, 2.661asymptomatic adult patients with stages 3-5D CKD will be recruited from nephrology services and dialysis units throughout Spain. Eighthundred forty-three participants without CKD (control group) will be also recruited. During the follow-up, CVD events and mortality will be recorded from all CKD patients. One trained itinerant team will carry out a carotid ultrasound to assess intima-media thickness and presence of plaques. A composite atherosclerosis score will be constructed based on carotid ultrasound data and ankle-brachialindex. Presence and type of calcifications will be assessed in carotid, femoral and brachial arteries, and in cardiac valves, by ultrasound. Finally, blood samples will be collected from all participants to study biomarkers. Discussion: The NEFRONA study will allow us to examine the usefulness of imaging techniques and biomarkers to assess atherosclerosis development and their predictive value in a Spanish population with CKD (AU)

Detection of pesticide residues using an immunodevice based on negative dielectrophoresis.

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 microm diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgG) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 microgL(-1) and in pre-treated wine samples of 6.8 microg L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 microg L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples.

Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors.

An immunoassay-based strategy for folic acid in vitamin-fortified milk with electrochemical detection using magneto sensors is described for the first time. Among direct and indirect competitive formats, best performance was achieved with an indirect competitive immunoassay. The immunological reaction for folic acid (FA) detection was performed, for the first time on the magnetic bead as solid support by the covalent immobilization of a protein conjugate BSA-FA on tosyl-activated magnetic bead. Further competition for the specific antibody between FA in the food sample and FA immobilized on the magnetic bead was achieved, followed by the reaction with a secondary antibody conjugated with HRP (AntiIgG-HRP). Then, the modified magnetic beads were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with electrochemical detection using magneto sensors was successfully evaluated using spiked-milk samples and compared with a novel magneto-ELISA based on optical detection. The detection limit was found to be of the order of microgl(-1) (13.1 nmoll(-1), 5.8 microgl(-1)) for skimmed milk. Commercial vitamin-fortified milk samples were also evaluated obtaining good accuracy in the results. This novel strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological and food samples.

Electrochemical magneto immunosensing of antibiotic residues in milk.

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.

Electrochemical magnetoimmunosensing strategy for the detection of pesticides residues.

A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.

Immunochemical determination of xenobiotics with endocrine disrupting effects.

This paper is a review with more than 100 references discussing the immunochemical methods reported in the literature for the most important man-made chemicals with suspected endocrine disrupting activity. Details regarding immunizing hapten design, antibody production, and the features (limit of detection, dynamic range, specificity) of the most important immunochemical methods developed (ELISA, FIIA, immunosorbents, immunosensors, etc.) are presented for important environmental pollutants such as bisphenol A, phthalates, alkylphenol polyethoxylates, alkylphenols, polychlorinated biphenyl compounds, and dioxins. Availability of commercial reagents and methods is reported.

Influencia del polimorfismo del gen receptor de la vitamina D y de la 25-hidroxivitamina D en la tensión arterial de individuos sanos / Vitamin D receptor genotype and 25 hydroxyvitamin D influence on blood pressure in healthy individuals

El efecto de la vitamina D sobre la tensión arterial (TA) no está bien establecido. No existen estudios que relacionen el polimorfismo del gen del VDR con la TA. Objetivo: Analizar la posible influencia del genotipo Bsm I y de la 25 hidroxivitamina D3 (25OHD3) en la TA en individuos sanos y normotensos. Métodos: Analizamos en 590 individuos sanos (260 varones y 330 mujeres) la posible asociación de la edad, sexo, IMC, creatinina, calcio, fósforo, PTHi, 25OHD3 y genotipo Bsm I con la tensión arterial sistólica (TAS) y diastólica (TAD) mediante un análisis de regresión lineal múltiple. Resultados: El sexo se asoció fuertemente a la TAS (β: –12,01, p: 0,000) y a la TAD (β: –4,78, p: 0,000), por lo que se realizó un análisis multivariante en función del mismo. En varones, la TAS se asoció a: 25OHD3 (β: 0,36, p: 0,000), genotipo (β: –3,90, p: 0,002) e IMC (β: 0,83, p: 0,001); y la TAD a: 25OHD3 (β: 0,16, p: 0,018) y edad (β: 0,28, p: 0,000). El análisis de la varianza mostró que los varones con genotipo bb presentaron una TAS superior al resto de genotipos (p: 0,007). En las mujeres, no encontramos asociación de la 25OHD3 ni del genotipo con la TA. Conclusiones: Los varones con mayores niveles de vitamina D presentan una mayor TAS y TAD. Los varones con genotipo bb tienen una mayor TAS. No existe dicha relación en el sexo femenino. Ello sugiere un posible nexo fisiopatológico entre las hormonas sexuales y la vitamina D en el control de la tensión arterial (AU)

The role of vitamin D in the regulation of blood pressure is unclear. There are no studies that relate Bsm I polymorphism with blood pressure. Objective: To analyze if Bsm I polymorphism and 25-hydroxyvitamin D (25OHD3) influence blood pressure in healthy individuals with normal blood pressure. Methods: Systolic (SBP) and diastolic (DBP) blood pressure, Body Mass Index (BMI), plasma creatinine, serum calcium, serum phosphorus, serum iPTH, serum 25OHD3 and Bsm I genotype were determined in 590 healthy individuals (260 men and 330 women). Data were analysed using a multiple linear regression model. SBP and DBP were defined as dependent variables and the rest of variables as independent. Results: Gender was strongly associated with both SBP (β: –12.01, p: 0.000) and DBP (β: –4.78, p: 0.000). Therefore, a separate analysis was performed according to gender. In males, SBP was associated with BMI (β: 0.83, p: 0.001), 25OHD3 (β: 0.36, p: 0.000) and genotype (β: –3.90, p: 0.002); and DBP with 25OHD3 (β: 0.16, p: 0.018) and age (β: 0.28, p: 0.000). Differences of blood pressure among the three genotypes were explored by analysis of variance. SBP was higher in men with bb genotype than in the other genotypes (p: 0.007). In females, 25OHD3 and genotype were not associated with blood pressure. Conclusions: Healthy men with higher levels of vitamin D have higher levels of SBP and DBP. Moreover, men with bb genotype have the highest levels of SBP. Blood pressure levels in women are not influenced by vitamin D nor by BsmI genotype. Our data suggest a possible pathophysiological interaction between vitamin D and sex hormones in blood pressure control (AU)

Efecto diferencial de los análogos de la vitamina D en la proliferación de células de músculo liso vascular / Vascular and tissue calcifications in haemodialysis patients

Existen datos experimentales contradictorios respecto al comportamiento de las células de músculo liso vascular (CMLV) expuestas al calcitriol. Determinar el efecto del calcitriol y de sus análogos a nivel vascular tiene una considerable trascendencia clínica ya que la proliferación de las CMLV está implicada en el mecanismo patogénico de la arteriosclerosis y de la resistencia tras angioplastia. En este trabajo demostramos mediante incorporación de BrdU que el calcitriol estimula la proliferación en las CMLV. La proliferación es menor al añadir al medio de cultivo Paracalcitol o EB1089 a dosis equimolar. En concordancia con estos hechos, también observamos que el calcitriol induce la expresión del mRNA VDR mientras que no existe este efecto con ninguno de los análogos estudiados. En conclusión, el calcitriol tiene un efecto directo estimulador de la proliferación de las CMLV que no se observa con el Paracalcitol y EB1089 a concentración equimolar (AU)

Atherosclerosis is the principal cause of myocardial infarction, stroke, and peripheral vascular disease, accounting for nearly half of all mortality in developed countries. The excessive growth of vascular smooth muscle cells is an important component in the development of atherosclerotic lesion. The direct effect of calcitriol and vitamin D analogs on the VSMCs proliferation is not clear. In this study we have analysed if calcitriol, Paracalcitol (19-nor-1,25-dihydroxyvitamin D2) and EB1089 (experimental analog used as anticancerous) modify proliferation and the expression of vitamin D receptor (VDR) gene that is regulated at the transcriptional level by itself in the VSMCs. VSMCs proliferation was analysed by BrdU incorporation and VDR gene expression using RT-PCR. VSMCs proliferation was stimulated when calcitriol was added to the culture. VSMCs proliferation was significantly lower with analogs at the same dose. With regard to the functional study, the expression of VDR gene was upregulated by calcitriol at a concentration of 100 nM. There were no changes in this expression with the analogs. In conclusion, calcitriol, do not modify VSMCs proliferation. Therefore, Paracalcitol could have a minor proliferating effect on the wall of vessels that vitamin D (AU)