Julio Raba

Monitoring melatonin and its isomer in Vitis vinifera cv. Malbec by UHPLC-MS/MS from grape to bottle

Abstract:  Several studies have shown the presence of melatonin and related compounds in grapes and wines. The latter provides evidence of the possibility to enhance the nutraceutical properties of premium wines. However, there are many external factors that can influence the levels of this indolamine in grape and wines. In this study, the monitoring of melatonin and its tentatively identified isomer was carried out during the entire winemaking process in Vitis vinifera cv. Malbec by ultra high‐performance liquid chromatography‐tandem mass spectrometry. Laboratory and pilot studies were carried out to elucidate the role of grape, yeasts, and tryptophan in the evolution of the indolamines during the fermentation process. Melatonin was detected in grape extract within the range 120–160 ng/g while its isomer was found in musts and finished wines. Our results demonstrate that Saccaromyces cervisiae plays a decisive role in contributing to the content of melatonin and its isomer in wine.

Modified paramagnetic beads in a microfluidic system for the determination of ethinylestradiol (EE2) in river water samples.

In this work, we have developed and characterized a novel microfluidic immunoassay methodology for rapid and sensitive quantification of ethinylestradiol (EE2) in river water samples. The detection of EE2 was carried out using a competitive direct immunoassay method based on the use of anti-EE2 polyclonal antibodies immobilized on magnetic microspheres 3-aminopropyl-modified manipulated for an external removable magnet. The EE2 present in the water sample was allowed to compete with EE2-horseradish peroxidase (HPR) conjugated for the immobilized anti-EE2 antibody. The HPR, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of catechol (Q) whose back electrochemical reduction was detected on gold electrode at 0.0 V. The response current obtained from the product of enzymatic reaction is inversely proportional to the amount of EE2 in the water sample. The electrochemical detection can be done within 1 min and total assay time was 30 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.09 and 0.32 ng L(-1) respectively and the intra- and inter-assay coefficients of variation were below 5.8%. Our electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows the potential for assessment of EE2 in river water samples.

Citrinin (CIT) determination in rice samples using a micro fluidic electrochemical immunosensor.

The development of an electrochemical immunosensor incorporated in a micro fluidic cell for quantification of citrinin (CIT) mycotoxin in rice samples is described for the first time. Both CIT present in rice samples and immobilized on a gold surface electrodeposited on a glassy carbon (GC) electrode modified with a cysteamine self-assembled monolayer were allowed to compete for the monoclonal mouse anti-CIT IgG antibody (mAb-CIT) present in solution. Then, an excess of rabbit anti mouse IgG (H+L) labelled with the horseradish peroxidase (secAb-HRP) was added, which reacts with the mAb-CIT which is in the immuno-complex formed with the immobilized CIT on the electrode surface. The HPR, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of catechol (H(2)Q) whose back electrochemical reduction was detected on a GC electrode at -0.15 V vs Ag/AgCl by amperometric measurements. The current measured is proportional to the enzymatic activity and inversely proportional to the amount of CIT present in the rice samples. This immunosensor for CIT showed a range of work between 0.5 and 50 ng mL(-1). The detection (LOD) and the quantification (LOQ) limits were 0.1 and 0.5 ng mL(-1), respectively. The coefficients of variation intra- and inter-assays were less than 6%. The electrochemical detection could be done within 2 min and the assay total time was 45 min. The immunosensor was provided to undertake at least 80 determinations for different samples with a minimum previous pre-treatment. Our electrochemical immunosensor showed a higher sensitivity and reduced analysis time compared to other analytical methods such as chromatographic methods. This methodology is fast, selective and very sensitive. Thus, the immunosensor showed to be a very useful tool to determine CIT in samples of cereals, mainly rice samples.

Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 µg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.

Electrochemical detection of a powerful estrogenic endocrine disruptor: Ethinylestradiol in water samples through bioseparation procedure

The synthetic estrogen ethinylestradiol (EE2) is an active component of oral contraceptives (OCs), considered as an endocrine disrupting compound (EDC). It is excreted from humans and released via sewage treatment plant effluents into aquatic environments. EDCs are any environmental pollutant chemical that, once incorporated into an organism, affects the hormonal balance of various species including humans. Its presence in the environment is becoming of great importance in water quality. This paper describes the development of an accurate, sensitive and selective method for capture, preconcentration and determination of EE2 present in water samples using: magnetic particles (MPs) as bioaffinity support for the capture and preconcentration of EE2 and a glassy carbon electrode modified with multi-walled carbon nanotubes (MWCNTs/GCE) as detection system. The capture procedure was based on the principle of immunoaffinity, the EE2 being extracted from the sample using the anti-EE2 antibodies (anti-EE2 Ab) which were previously immobilized on MPs. Subsequently the analyte desorption was done employing a sulfuric acid solution and the determination of the EE2 in the pre-concentrated solution was carried out by square wave voltammetry (SWV). This method can be used to determine EE2 in the range of 0.035-70 ng L(-1) with a detection limit (LOD) of 0.01 ng L(-1) and R.S.D.<4.20%. The proposed method has been successfully applied to the determination of EE2 in water samples and it has promising analytical applications for the direct determination of EE2 at trace levels.

Determination of progesterone (P4) from bovine serum samples using a microfluidic immunosensor system

Progesterone (P4) is a steroidal hormone with a vital role in the maintenance of human and animal health. This paper describes the development of an immunosensor coupled to glassy carbon (GC) electrode and integrated to a microfluidic system to quantify P4 from bovine serum samples in a fast and sensitive way. The serum samples spiked with a given P4 concentration and a given P4 concentration bound to horseradish peroxide (HPR) were simultaneously added and, therefore, they competed immunologically with sheep monoclonal anti-P4 antibodies that were immobilized at a rotating disk. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the chatecol (H(2)Q) oxidation to benzoquinone (Q). Its reverse electrochemical reduction to H(2)Q can be detected at a GC electrode surface at -0.15 V by chronoamperometric measurements. These current responses are proportional to the enzyme activity and inversely proportional to the P4 amount present in bovine serum samples. This P4 immunosensor showed a linear working range from 0.5 to 12.5 ng mL(-1). The detection (DL) and quantification (QL) limits were 0.2 and 0.5 ng mL(-1), respectively. The electrochemical immunosensor had a higher sensitivity than the ELISA method using conventional spectrophotometric detections. However, both methods allowed us to obtain similar detection limits. The immunosensor allowed us to make up to 100 determinations on different samples without any previous pre-treatment. This behavior proved to be suitable to detect P4 in routine veterinary, clinical, biological, physiological, and analytical assays.

Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori.

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1min and total assay time was 25min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1UmL(-1), respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.

Screen-printed immunosensor modified with carbon nanotubes in a continuous-flow system for the Botrytis cinerea determination in apple tissues

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. This paper describes the development of an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method. Both the infected plant tissue sample and the B. cinerea-specific monoclonal antibody are allowed to react immunologically with the B. cinerea purified antigens immobilized on a rotating disk. Then, the bound antibodies are quantified by a horseradish peroxidise (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. The HRP, in the presence of hydrogen peroxide, catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone. The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at -0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. The time consumed per assay was 30 min and the calculated detection limits for electrochemical method and the ELISA procedure are 0.02 and 10 microg mL(-1), respectively. Moreover the intra- and inter-assay coefficients of variation were below 7%. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy plant prior to the development of the symptoms.

A novel solid phase extraction Ultra high performance liquid chromatography tandem mass spectrometry method for the quantification of ochratoxin A in red wines

A novel and advanced technology on solid phase extraction column prior to liquid chromatography coupled to tandem mass spectrometry has been used for the determination of ochratoxin A in red wine samples. Due to the need of a reliable and rugged method according to current regulations and with the aim of minimize heuristic efforts associated with analytical method development, the statistical design of experiment was employed. On other hand, the method validation according to European Commission 2002/657/EC was achieved. The values obtained for decision limit (CCα), detection capability (CCβ), limits of detection and quantification were 0.07 μg L(-1), 0.14 μg L(-1), 0.13 μg L(-1) and 0.41 μg L(-1), respectively. The recoveries values were ranged from 95.7% to 107.2%. These values were compatible with the 2.0 μg L(-1) maximum allowable concentration limit established by different international regulations.

Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues.

This work described the development and characterization of an electrochemical method using square wave voltammetry (SWV) combined with the use of modified magnetic nanoparticles (MNPs), which had shown a rapid and sensitive determination of ochratoxin A (OTA) in wine grapes (Cabernet Sauvignon, Malbec and Syrah) post-harvest tissues. The wine grapes were inoculated with Aspergillus ochraceus to obtain OTA in artificially infected samples. The OTA was directly determined using square-wave voltammetry. The current obtained is directly proportional to the concentration of OTA present in the samples. This method has been used for OTA determination in wine grapes and it was validated against a commercial ELISA test kit. The limits of detection calculated for electrochemical detection and the ELISA were 0.02 and 1.9 μg kg(-1), respectively and the coefficients of variation for accuracy and precision dates were below 5.5%. This method promises to be suitable for the detection and quantification of OTA in apparently healthy fruits post-harvest for assuring safety and quality of food as well as consumers health.