Martín A. Fernández-Baldo

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.

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

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.

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.

Microfluidic Immunosensor with Micromagnetic Beads Coupled to Carbon-Based Screen-Printed Electrodes (SPCEs) for Determination of Botrytis cinerea in Tissue of Fruits

A wide range of plant species, including economically important crops such as vegetables, ornamentals, bulbs, and fundamentally fruits, can be affected by gray mold caused by the fungal pathogen Botrytis cinerea . This paper describes the development of a microfluidic immunosensor with micromagnetic beads (MMBs) coupled to carbon-based screen-printed electrodes (SPCEs) for the rapid and sensitive quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (Williams) tissues. The detection of B. cinerea was carried out using a competitive immunoassay method based on the use of purified B. cinerea antigens immobilized on 3-aminopropyl-modified MMBs. The total assay time was 40 min, and the calculated detection limit was 0.008 μg mL(-1). Moreover, the intra- and interassay coefficients of variation were below 7%. The developed method allowed detects B. cinerea even in asymptomatic fruits and promises to be particularly useful for application in the agricultural industry.

Development of nitrocellulose membrane filters impregnated with different biosynthesized silver nanoparticles applied to water purification

Bactericidal water filters were developed. For this purpose, nitrocellulose membrane filters were impregnated with different biosynthesized silver nanoparticles. Silver nanoparticles (AgNPs) from Aspergillus niger (AgNPs-Asp), Cryptococcus laurentii (AgNPs-Cry) and Rhodotorula glutinis (AgNPs-Rho) were used for impregnating nitrocellulose filters. The bactericidal properties of these nanoparticles against Escherichia coli, Enterococcus faecalis and Pseudomona aeruginosa were successfully demonstrated. The higher antimicrobial effect was observed for AgNPs-Rho. This fact would be related not only to the smallest particles, but also to polysaccharides groups that surrounding these particles. Moreover, in this study, complete inhibition of bacterial growth was observed on nitrocellulose membrane filters impregnated with 1 mg L(-1) of biosynthesized AgNPs. This concentration was able to reduce the bacteria colony count by over 5 orders of magnitude, doing suitable for a water purification device.

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

BackgroundBotrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (Williams) tissues.ResultsThe method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation.ConclusionsThe developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

Integrated bio-affinity nano-platform into a microfluidic immunosensor based on monoclonal bispecific trifunctional antibodies for the electrochemical determination of epithelial cancer biomarker

BACKGROUND The epithelial cell adhesion molecule (EpCAM) is a biomarker that is highly overexpressed on the surface of epithelial carcinoma cells. In this study, silver nanoparticles covered with polyvinyl alcohol (AgNPs-PVA) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-EpCAM recombinant antibodies as a trapping agent. METHODS The concentration of trapped EpCAM is then electrochemically quantified by HRP-conjugated anti-EpCAM-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude (at a working voltage as low as -0.10V) is directly proportional to the concentration of EpCAM. RESULTS Under optimized conditions, the detection limits for the microfluidic immunosensor and a commercial ELISA were 0.8 and 13.9pg/L, respectively. The within-assay and between-assay coefficients of variation are below 6.5% for the proposed method. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained. CONCLUSIONS The good analytical performance is attributed to the efficient immobilization of the anti-EpCAM recombinant antibodies on the AgNPs-PVA, and its high specificity for EpCAM. This microfluidic immunosensor is intended for use in diagnosis and prognosis of epithelial cancer, to monitor the disease, and to assess therapeutic efficacy.

Development of a nanostructured immunosensor for early and in situ detection of Xanthomonas arboricola in agricultural food production

We report a microfluidic electrochemical immunosensor for Xanthomonas arboricola (XA) determination, based on the covalently immobilization of monoclonal anti-XA antibody (anti-XA) on a previously amino functionalized SBA-15 in situ synthesized in the central channel of a glass-poly(dimethylsiloxane) microfluidic immunosensor. The synthetized amino-SBA-15 was characterized by N2 adsorption-desorption isotherm, scanning electron microscopy and infrared spectroscopy. XA was detected by a direct sandwich immunoassay through an alkaline phosphatase (AP) enzyme-labeled anti-XA conjugate. Later, the substrate p-aminophenyl phosphate was converted to p-aminophenol by AP. The enzymatic product was detected at +100mV on a sputtered gold electrode. The measured current was directly proportional to the level of XA in walnut trees samples. The linear range was from 5 × 102 to 1 × 104CFUmL-1. The detection limit was 1.5 × 102CFUmL-1, and the within- and between-assay coefficients of variation were below 5%. Microfluidic immunosensor is a very promising tool for the early and in situ diagnosis of XA in walnuts avoiding serious economic losses.

Serological diagnosis of Toxoplasmosis disease using a fluorescent immunosensor with chitosan-ZnO-nanoparticles

This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T. gondii antigens. Bound antibodies were quantified by the addition of anti-IgG antibodies labeled whit alkaline phosphatase (ALP). ALP enzymatically converts the non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to soluble fluorescent methylumbelliferone that was measured using excitation at 355 nm and emission at 440 nm. The relative fluorescent response of methylumbelliferone is proportional to the concentration of anti-T. gondii IgG antibodies. The coefficients of variation are less than 4.73% for within-day assays and less than 6.34% for between-day assays. Results acquired by LIF immunosensor agree with those obtained by enzyme-linked immunosorbent assay method, suggesting that the designed sensor represents a promising tool for the quantitative determination of anti-T. gondii IgG antibodies of clinical samples.