Nuevas estrategias para el desarrollo de biosensores ópticos aplicados al análisis de micotoxinas y hongos toxigénicos en alimentos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Química Analítica, leída el 15-07-2019Mycotoxins are a diverse group of low molecular weight compounds produced as secondary metabolites by numerous species of filamentous fungi. This assemblage is chemically and toxigenically rather heterogeneous, but generally these toxins are known to cause disease and death in human and other vertebrates even at low concentrations. Mycotoxigenic fungi grow on a wide range of conditions and they can produce mycotoxins into the matrices on which they grow, often food intended for human consumption or animal feed. As a result of the ubiquitous nature of mycotoxigenic fungi, particularly in temperate and tropical regions of the world, mycotoxin contamination is often inevitable, and some calculations have estimated that approximately 25–50% of world crops are contaminated with these toxins. Although the awareness related to the hazards of mycotoxins as food and feed contaminants is growing, there are no absolute measures available for eliminating mycotoxins from agricultural products. While mycotoxin occurrence in the field can be decreased by good agronomic practices and planting resistant varieties, in the end, analytical methods capable of detecting mycotoxins and toxigenic fungi even at low concentration are of key importance for ensuring food safety...Las micotoxinas son metabolitos secundarios tóxicos producidos por algunas cepas de hongos que contaminan alimentos, especialmente cereales y hortalizas. Estos compuestos de bajo peso molecular son químicamente y toxigénicamente heterogéneos; sin embargo, muchas de estas toxinas pueden originar enfermedades, y en ocasiones la muerte, tanto en humanos como en otros vertebrados. Los hongos toxigénicos crecen en muchas condiciones muy diversas, lo que puede dar lugar a la aparición de micotoxinas en los alimentos destinados tanto al consumo humano como al animal. Los hongos toxigénicos están ampliamente distribuidos por todo el mundo, particularmente en las regiones templadas y tropicales, por lo que la contaminación natural por micotoxinas es casi inevitable. De hecho, se estima que aproximadamente el 25–50% de los cultivos mundiales están contaminados por estas toxinas, y la preocupación sobre los peligros asociados a su presencia en alimentos es cada día mayor. Actualmente, no existen alternativas viables para su eliminación en los productos agrícolas; aunque el empleo de buenas prácticas agrícolas o la plantación de variedades resistentes a los hongos, pueden ayudar a mejorar este problema. En cualquier caso, se requieren métodos analíticos sensibles y selectivos para la detección de micotoxinas y hongos toxigénicos, a bajas concentraciones, afin de garantizar la seguridad alimentaria...Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEunpu

Single-step noncompetitive immunocomplex immunoassay for rapid aflatoxin detection

Owing to the high carcinogenicity of aflatoxins, these toxic secondary metabolites pose a severe risk to human and animal health and can have major economic implications. Herein, we report the development of a noncompetitive immunoassay for aflatoxins based on a monoclonal capture antibody and a unique anti-immunocomplex (anti-IC) antibody fragment (scFv) isolated from a synthetic antibody repertoire. The anti-IC scFv recognizes the immunocomplex and enables the development of noncompetitive sandwich-type assays despite the small size of the analyte. The single-step assay developed in this work, with a detection limit of 70 pg mL−1, could detect aflatoxins within 15 min. The assay was applied to the analysis of spiked food samples, and the results showed that the method could provide a rapid and simple tool for aflatoxin detection. Moreover, the work demonstrates the potential of anti-IC antibodies and non-competitive immunoassays for the analysis of small molecule contaminants. </p

Optical Biosensors for Label-Free Detection of Small Molecules

Label-free optical biosensors are an intriguing option for the analyses of many analytes, as they offer several advantages such as high sensitivity, direct and real-time measurement in addition to multiplexing capabilities. However, development of label-free optical biosensors for small molecules can be challenging as most of them are not naturally chromogenic or fluorescent, and in some cases, the sensor response is related to the size of the analyte. To overcome some of the limitations associated with the analysis of biologically, pharmacologically, or environmentally relevant compounds of low molecular weight, recent advances in the field have improved the detection of these analytes using outstanding methodology, instrumentation, recognition elements, or immobilization strategies. In this review, we aim to introduce some of the latest developments in the field of label-free optical biosensors with the focus on applications with novel innovations to overcome the challenges related to small molecule detection. Optical label-free methods with different transduction schemes, including evanescent wave and optical fiber sensors, surface plasmon resonance, surface-enhanced Raman spectroscopy, and interferometry, using various biorecognition elements, such as antibodies, aptamers, enzymes, and bioinspired molecularly imprinted polymers, are reviewed

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 μg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.This work has been funded by the Ministry of Science, Innovation and Universities (MSIU) (RTI2018-096410-B-C21, PID2021-127457OB-C21 and PID2019-105237 GB-I00). FP acknowledges the MSIU for an FPU contract.S

Effect of Particle Size and Surface Chemistry of Photon Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL−1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Kohti hiilineutraalia kuntaa: ilmastoverkoston vaikutus kunnan ilmastotyöhön ja päästöihin

Kunnilla on suuri rooli ilmastonmuutoksen hillitsemisessä. Yhä useampi kunta on asettanut itselleen merkittäviä päästövähennystavoitteita. Tavoitteilla ja suunnitelmilla nähdään olevan tärkeä rooli kansallisen ilmastopolitiikan toteuttamisessa. Kuntien strategioiden yhteyttä päästövähennyksiin on ollut kuitenkin vaikea osoittaa. Edes lisääntyneen ilmastotyön yhteyttä päästövähennyksiin ei ole pystytty suoraan todentamaan. Tässä työssä selvitettiin Kohti hiilineutraalia kuntaa (Hinku) -verkoston vaikutusta kuntien kasvihuonekaasupäästöihin, aurinkosähkön käyttöönottoon ja kuntien ilmastotyöhön. Verkostoon liittyneet kunnat pyrkivät saavuttamaan 80 prosentin kasvihuonekaasupäästövähennyksen vuoteen 2030 mennessä verrattuna vuoden 2007 päästöihin. Työssä tarkasteltiin Manner-Suomen kuntien päästökehitystä tilastollisella analyysillä, jossa useita muun muassa kuntien rakenteeseen, energiajärjestelmään ja maantieteeseen liittyviä tekijöitä kontrolloitiin niin, että Hinku-verkoston vaikutukseen päästiin käsiksi. Tämän lisäksi haastateltiin 40 ennen vuotta 2019 verkostoon liittyneen Hinku-kunnan edustajat. Haastatteluissa kysyttiin Hinku-verkoston roolista kunnan ilmastotyössä. Lisäksi kysyttiin kuntien kohtaamista ilmastotyön haasteista ja onnistumisista, sekä eri toimijoiden rooleista kunnan ilmastotyössä. Työn tulokset osoittavat, että Hinku-verkoston jäsenyydellä ja kunnan kasvihuonekaasupäästöjen välillä on tilastollisesti merkitsevä yhteys. Analyysin mukaan Hinku-verkostoon liittyminen pienentää kunnan päästöjä, niiden ollessa noin 3,1 prosenttia alemmalla tasolla kuin mitä ne olisivat ilman Hinku-verkostoa, kun muut päästöihin vaikuttavat tekijät on huomioitu. Hinkuun liitytään kunnanvaltuuston päätöksellä, usein aktiivisen kunnan avainhenkilön tai verkostoa koordinoivan Suomen ympäristökeskuksen edustajan kannustuksesta. Haastattelujen mukaan johdon sitoutuminen on ollut tärkeää. Usein verkostoon liittyminen on ollut luonnollinen jatkumo kunnassa jo tehdylle ilmastotyölle, mutta liittyminen on voitu nähdä myös uutena avauksena tai imagoa nostattavana tekijänä. Haastattelujen mukaan verkostolta on saatu motivaatiota, inspiraatiota ja vertaistukea. Lähes puolessa haastatelluista kunnista verkoston kautta saatu asiantuntijatuki koettiin arvokkaaksi. Verkostolta oltiin saatu myös viestintätukea ja medianäkyvyyttä. Muutamassa kunnassa Hinkun myötä kunnan strategioihin oltiin lisätty ilmastotyön kirjauksia, joskin monissa kunnissa konkreettiset toimenpiteet olivat lähteneet liikkeelle hitaasti. Ilmastotyön haasteina oli koettu muun muassa organisaatiomuutosten ja henkilövaihdosten vaikutukset Hinku-työn eteenpäin viemiseen, sekä niukat taloudelliset resurssit. Konkreettisia ilmastotoimia oli toisaalta edistänyt se, että niitä oli voitu perustella päättäjille Hinku-jäsenyydellä. Kuntien edustajista 60 prosenttia koki, että Hinku-verkostoon liittyminen on edistänyt päästövähennyksiä konkreettisten toimenpiteiden kautta. Haastattelujen mukaan tärkeimpien konkreettisten toimien joukossa on ollut muun muassa energiantuotantoon, rakennusten energiatehokkuuteen, liikenteeseen ja aurinkosähköön liittyviä toimia. Hinku-kunnat eroavat keskenään suuresti ilmastotyön suunnitelmallisuudessa ja toimeenpanossa. Tässä työssä tunnistetut, ilmastotyössä onnistuneiden kuntien käyttöön ottamat toimenpiteet liittyvät käytännössä kunnan johdon sitoutumiseen, henkilöstön aktiivisuuteen ja työn riittävään resurssointiin. Onnistumisia on koettu, kun ilmastotyö on jalkautettu läpi toimialojen, kokemuksia on vaihdettu, ja ilmastotyöhön on osallistettu useita eri toimijoita. Työn tulosten perusteella Hinku-verkoston keskitetylle tuelle ja kuntien yhteistoiminnalle voidaan suunnitella uusia toimintamalleja

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL(-1) and an IC50 of 2.9 +/- 0.5 ng mL(-1). The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL(-1) in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Comparative Study of the Performance of Two Different Luciferases for the Analysis of Fumonisin B1 in Wheat Samples

The development of two different immunoassays for the determination of fumonisin B1 in wheat samples is reported. A previously described mimopeptide for fumonisin B1 (FB1) was used to produce fusion proteins in combination with two different luciferases: Gaussia luciferase (GLuc) and NanoLuc luciferase (NLuc). The production, expression and the development of two immunoassays based on these fusion proteins (A2- GLuc and A2-NLuc) is detailed. The assay showing the best performance, A2-NLuc, with a limit of detection of 0.61 ngmL 1 and a dynamic range from 1.9 to 95 ngmL 1 , was employed for the analysis of spiked wheat samples, a reference matrix material, as well as naturally contaminated wheat samples. The recoveries obtained in the spiked samples were acceptable, between 81.5 and 109%, with relative standard deviations lower than 14%. The analysis of naturally contaminated wheat was validated by a liquid chromatography coupled to tandem mass detection method