Silvia R. Hernandez

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.

Enhanced application of square wave voltammetry with glassy carbon electrode coupled to multivariate calibration tools for the determination of B6 and B12 vitamins in pharmaceutical preparations

Vitamins B(6) (VB(6)) and B(12) (VB(12)) were simultaneously determined in pharmaceutical preparations by using square wave voltametry (SWV) together with artificial neural networks (ANNs). Supporting electrolyte solution, pH and voltametric technique were optimised. The calibration set was built with several artificial samples containing both active ingredients and excipients. Deviations from linearity were observed for both analytes. It is probably due to interactions among the electro active components and competition by the electrode surface, fact that supports the use of ANNs. Recoveries when analysing a nine sample validation set, of 100.2 and 96.4 were calculated for VB(6) and VB(12), respectively. Commercial samples were analysed with reasonably good results considering the complexity of the mixture studied.

Magneto immunosensor for gliadin detection in gluten-free foodstuff: Towards food safety for celiac patients

Gliadin is a constituent of the cereal protein gluten, responsible for the intolerance generated in celiac disease. Its detection is of high interest for food safety of celiac patients, since the only treatment known until now is a lifelong avoidance of this protein in the diet. Therefore, it is essential to have an easy and reliable method of analysis to control the contents in gluten-free foods. An electrochemical magneto immunosensor for the quantification of gliadin or small gliadin fragments in natural or pretreated food samples is described for the first time and compared to a novel magneto-ELISA system based on optical detection. The immunological reaction was performed on magnetic beads as solid support by the oriented covalent immobilization, of the protein gliadin on tosyl-activated beads. Direct, as well as indirect competitive immunoassays were optimized, achieving the best analytical performance with the direct competitive format. Excellent detection limits (in the order of μg L(-1)) were achieved, according to the legislation for gluten-free products. The matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs (skimmed milk and beer), obtaining excellent recovery values in the results.

Determination of anticholinesterase activity for pesticides monitoring using a thiocholine sensor.

Abstract An acetylthiocholine sensor based on a disposable screen-printed carbon electrode has been assembled for measuring organophosphorus and carbamate pesticides in river water samples through the degree of inhibition of the enzyme acetylcholinesterase (AChE). The carbon working electrode surface was modified by deposition of a mediator, tetracyanoquinodimethane (TCNQ), and Nafion. Acetylcholinesterase catalyses the cleavage of acetylthiocholine to thiocholine, which is measured by differential pulse voltammetry and directly related to the enzyme activity. The scan speed, the pulse amplitude of the differential pulse voltammetry and several parameters in the procedure were optimised. An inhibition calibration curve was obtained using carbofuran. The method was also applied to water samples, showing its suitability as a rapid screening assay (15 min per test) for anticholinesterase activity detection.

Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H₂O₂ as substrates by square wave voltammetry was correlated with the ATG2. Graphite-epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%.

Biotin determination in food supplements by an electrochemical magneto biosensor

An electrochemical magneto biosensor for the rapid determination of biotin in food samples is reported. The affinity reaction was performed on streptavidin-modified magnetic microbeads as a solid support in a direct competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP) competes with free biotin in the sample for the binding sites of streptavidin on the magnetic microbeads. The modified magnetic beads were then easily captured by a magneto graphite-epoxy composite electrode and the electrochemical signal was based on the enzymatic activity of the HRP enzyme under the addition of H(2)O(2) as the substrate and o-phenilendiamine as cosubstrate. The response was electrochemically detected by square wave voltammetry. The limit of detection was 8.4×10(-8) mol L(--1) of biotin (20 μg L(--1)) with a dynamic range from 0.94 to 2.4×10(-7) mol L(--1). Biotin-fortified commercial dietary supplement and infant formula samples were evaluated obtaining good performances in the results. Total time of analysis was 40 min per 20 assays.

Enzymatic electrochemical detection coupled to multivariate calibration for the determination of phenolic compounds in environmental samples.

An approach based on the electrochemical detection of the horseradish peroxidase enzymatic reaction by means of square wave voltammetry was developed for the determination of phenolic compounds in environmental samples. First, a systematic optimization procedure of three factors involved in the enzymatic reaction was carried out using response surface methodology through a central composite design. Second, the enzymatic electrochemical detection coupled with a multivariate calibration method based in the partial least-squares technique was optimized for the determination of a mixture of five phenolic compounds, i.e. phenol, p-aminophenol, p-chlorophenol, hydroquinone and pyrocatechol. The calibration and validation sets were built and assessed. In the calibration model, the LODs for phenolic compounds oscillated from 0.6 to 1.4 × 10(-6) mol L(-1). Recoveries for prediction samples were higher than 85%. These compounds were analyzed simultaneously in spiked samples and in water samples collected close to tanneries and landfills.

Magneto immunofluorescence assay for diagnosis of celiac disease.

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit.

Screening fluorescent method for the fluoroquinolone family in groundwater samples from intensive livestock production systems

ABSTRACT A fast and simple screening fluorescent method was developed and applied for detection of quinolones in groundwater samples. The experimental conditions for quinolone family detection in 96-well plates were acid media (pH = 4.85) from acetate buffer solution (0.1 mol L−1) and 5.8 × 10−3 mol L−1 of anionic surfactant sodium dodecyl sulfate (SDS) as micellar media at excitation and emission wavelengths of 280 and 450 nm, respectively. The developed method was validated to guarantee the quality of the results reported. Thus, the decision limit (CCα) of ciprofloxacin of 6.8 μg L−1, the most prescript quinolone in our country, was selected as the cut-off level to classify the water samples as ‘suspect’ or ‘negative’ referred to quinolone content. The method showed good recoveries ranging between 80 and 114% for 6.8 μg L−1 ciprofloxacin with relative standard deviation values lower than 13%. Moreover, other families of antibiotics such as aminoglycoside, penicillin, macrolide, sulfonamide and tetracycline did not present interference in the quinolone detection. Groundwater samples from Argentine regions with intensive livestock activities were analysed by this method, and the results had a good correlation with a reference method based on ultraperformance liquid chromatography coupled to mass spectrometry.