Development of a detection method for butyric spores based on Real-Time PCR and biomagnetic separation in milk

La hinchazón tardía (HT) se produce en quesos de pasta dura y semi-dura como consecuencia de la fermentación producida por bacterias butíricas, dando lugar a cambios en el sabor y aroma, y a la aparición de agujeros o roturas. Los quesos afectados por HT no pueden ser comercializados lo que produce perdidas económicas en el sector de la industria láctea. Normalmente, estos quesos se destinan a queso rallado. Las bacterias acido butíricas son Gram-positivas y esporuladas. Sus esporos, la forma de resistencia de estos microorganismos, son muy difíciles de eliminar completamente de la leche cruda porque pueden sobrevivir a una gran variedad de tratamientos.Actualmente, no existe un método rápido para detectar las bacterias butíricas en leche cruda, que permita decidir el destino final de la leche contaminada para elaborar diferentes productos lácteos, excepto para quesos de pasta dura y semi-dura en los que tiene lugar la HT. El método de rutina para detectar las bacterias butíricas es el Numero Mas Probable (NMP), en el cual los resultados se obtienen entre dos y siete días. Por esta razón, métodos mas rápidos y específicos alternativos al NMP, son necesarios para prevenir la HT y reducir las perdidas económicas en la industria láctea.Aunque diferentes bacterias butíricas pueden producir la HT, C. tyrobutyricum es considerado como el principal agente causante. El principal objetivo de esta tesis doctoral ha consistido en elaborar un método de detección para esporos de C. tyrobutyricum en leche cruda basado en PCR a tiempo real (qPCR) y bioseparación magnética.La primera parte de esta tesis doctoral ha consistido en el cribado de diferentes métodos de ruptura de esporos de C. tyrobutyricum para obtener ADN genómico puro. Disponer de un método efectivo resulta clave para obtener suficiente cantidad de ADN para su análisis mediante qPCR, considerando la resistencia de los esporos en comparación con las células vegetativas. Teniendo en cuenta los resultados obtenidos, se seleccionaron los tratamientos por microondas (MW) y bead beating (BB), seguidos de una purificación del ADN mediante columna de sílica. Para recuperar los esporos, laleche se trato con subtilisina y después de una centrifugación, se aplicaron los tratamientos de MW y BB al precipitado de esporos para su posterior análisis mediante qPCR. Finalmente, el tratamiento de MW fue seleccionado como el mas apto en leche UHT como paso previo a la detección de los esporos de C. tyrobutyricum mediante qPCR.Aunque el tratamiento de MW y la posterior purificación del ADN mediante columna, se selecciono como el mejor método a aplicar en tampón acuoso (PBS) y leche UHT; la aplicación de este método en leches crudas procedentes de laboratorios de control de calidad de la leche españoles, dio una baja precisión en el ensayo de qPCR.Por esta razón, se evaluó un tercer método basado en un kit comercial y que precisa de un sistema automatizado denominado King Fisher (KF) Duo Prime System.La curva de calibración realizada con el método KF permitió conseguir valores de Ct y un limite de detección (LD) mas bajo en el ensayo de qPCR que el tratamiento de MW, como paso previo a la purificación del ADN con columna. Después de esta verificación, 202 muestras de vaca, oveja y cabra, procedentes de tres zonas geográficas españolas diferentes, fueron analizadas para determinar la concentración de esporos de C. tyrobutyricum. Los valores de concentración de esporos se encontraron en el rango de 102-103 esporos/mL, aunque un importante numero de las muestras analizadas seencontraron por debajo del LD y no pudieron ser cuantificadas. Por esta razón, aunque la precisión de la qPCR mejoro claramente al aplicar el método de KF, este método se ha considerado como cualitativo.Para verificar la presencia de C. tyrobutyricum en las muestras de leche cruda, estas fueron cultivadas en un medio selectivo para bacterias butíricas que contiene Dcicloserina y rojo neutro. Las colonias fueron analizadas mediante PCR multiplex y secuenciación del 16S rADN. Los resultados mostraron la presencia de otros microorganismos, como Lactobacillus y Paenibacillus, que también podían crecer en este medio. C. tyrobutyricum solo fue aislado en leche de vaca, y otras especies como C. sporogenes y C. perfringens, fueron identificadas, sugiriendo que también podrían contribuir a la HT. Ademas, las especies de Clostridium identificadas, fueron diferentes dependiendo del tipo de leche analizada (vaca, oveja o cabra).La segunda parte de esta tesis se ha centrado en el desarrollo de un sistema de captura de esporos basado en partículas magnéticas funcionalizadas con ligandos afines.Los resultados del análisis mediante qPCR de las leches crudas, había revelado que muchas de las muestras se encontraban por debajo del LD. Por esta razón, un paso de captura de los esporos directamente en la leche, podría ser una buena estrategia para mejorar la detección. Con este objetivo, se evaluó el péptido pCZS1 obtenido mediante la técnica de Phage Display y afín por los esporos de C. tyrobutyricum. En primer lugar, la afinidad del péptido pCZS1 por los esporos butíricos, se analizo mediante Calorimetría de Titulación Isotérmica (CTI) y citometría de flujo. Después, se evaluó la capacidad de captura de esporos de C. tyrobutyricum en PBS y leche UHT por partículas magnéticas funcionalizadas con pCZS1. Los resultados de la captura fueron positivos en PBS y en leche UHT tratada con subtilisina. Sin embargo, el principal objetivo, consistía en aplicar la captura de los esporos directamente en leche, por lo que se evaluaron otros ligandos con el fin de conseguir este objetivo.La proteina G3P transmembrana del fago M13, se expreso unida al péptido pCZS1 en el extremo N-terminal de la proteína para incrementar la exposición del péptido a los esporos. Primero, se expreso solo el dominio soluble de la proteína G3P junto con pCZS1, denominándola como tG3P. Después, se diseño una segunda proteína formada por la G3P completa y el péptido pCZS1, denominándola cG3P. Ambas proteínas fueron expresadas en Escherichia coli y purificadas mediante cromatografía de afinidad aplicando un método para solubilizar los cuerpos de inclusión. La proteína tG3P tuvo mejor rendimiento que la cG3P, probablemente porque la parte transmembrana pudiera afectar al proceso de purificación. La proteína tG3P fueevaluada mediante CTI y citometría de flujo confirmando su afinidad por los esporos butíricos. La evaluación mediante CTI de la proteína cG3P, obtuvo un valor de Kd muy similar al obtenido para la proteína tG3P. Sin embargo, debido a los problemas de estabilidad de la proteína cG3P, las partículas magnéticas solo se funcionalizaron con la proteina tG3P. Los esporos de C. tyrobutyricum fueron capturados con las partículas magnéticas funcionalizadas con tG3P, pero se obtuvieron valores muy bajos de captura tanto en PBS como en leche UHT. Por esta razón, se concluyo que la proteína G3P no aportaba ninguna ventaja frente al péptido pCZS1.La ultima parte de esta tesis se ha centrado en la purificación de anticuerpos policlonales procedentes del suero de conejos inmunizados con esporos de C. tyrobutyricum. Después de la purificación, los anticuerpos se unieron a partículasmagnéticas con proteína A y G. La eficiencia de captura de los esporos con las partículas funcionalizadas con los anticuerpos se evaluó en PBS y leche UHT, obteniendo valores de recuperación cercanos al 90%. Como aplicación final, la inmunocaptura se realizo en leche cruda y después se realizo el ensayo de qPCR para detectar los esporos. La extracción del ADN de los esporos se realizo mediante tratamiento de MW y purificación en columna. Las partículas con proteína A, proporcionaron valores de Ct y porcentajes de amplificación mejores que los ensayos realizados con las partículas con proteína G.Estos resultados confirman que la immunocaptura de esporos de C. tyrobutyricum directamente en leche y el posterior análisis mediante qPCR son posibles.Los resultados obtenidos en esta tesis sientan las bases para el desarrollo de un método de detección de esporos butíricos en leche cruda mediante qPCR revelando los puntos críticos. Ademas, se propone un sistema basado en la inmunocaptura de esporos con partículas magnéticas funcionalizadas con anticuerpos para recuperar los esporos de C. tyrobutyricum de la leche cruda y para su posterior detección mediante qPCR.Late blowing defect (LBD) is produced in hard and semi-hard cheeses due to the fermentation produced by butyric bacteria, leading into changes on flavor, smell and also to the appearance of cracks. The cheeses with LBD cannot be commercialized, which produces important economic loses for dairy industry. Normally, these cheeses are destinated to melted cheese. Butyric bacteria are Gram positive and sporulated, and spores, the resistance form of these microorganisms, are very difficult to remove completely from raw milk because they can survive to several treatments. Nowadays, there is not a fast method to detect butyric bacteria in raw milk, which would allow to use contaminated raw milk in different dairy products except for hard or semi-hard cheeses in which LBD can take place. The routine microbiological method used to detect butyric bacteria in raw milk is the Most Probable Number (MPN), which provides results between two and seven days. For this reason, faster and more specific methods alternative to MPN method are needed to prevent LBD and reduce economic losses in dairy industry. Although different species of butyric bacteria can produce LBD, C. tyrobutyricum is considered the main causative agent. The main objective of this thesis has been to develop a detection method for C. tyrobutyricum spores in raw milk based on Real-Time PCR and biomagnetic separation. The first part of this thesis has consisted on the screening of different disruption methods to obtain pure genomic DNA from C. tyrobutyricum spores. An effective disruption is a key point to have enough DNA for qPCR analysis taking into account the resistance of spores in comparison with vegetative cells. From the results obtained, microwaves (MW) treatment and bead beating (BB) followed by column purification were selected. To facilitate the recovery of spores, milk was treated with subtilisin and after centrifugation, MW and BB treatment were applied to the precipitate of spores for further analysis by qPCR. Finally, MW treatment was found successful in UHT milk as a previous step to the qPCR analysis to detect C. tyrobutyricum spores. Although MW treatment followed by column purification of DNA was found as the best choice in an aqueous buffer and UHT milk, the application of this method in cow raw milk samples coming from Spanish laboratories of milk quality control, gave results with low precision by qPCR. For this reason, a third method was evaluated based on a commercial kit and automated system named King Fisher (KF) Duo Prime System. This method disrupts the spores based on BB and the released DNA is recovered with magnetic particles. The calibration curve made with the KF method allowed to achieve better LOD and lower Ct by qPCR analysis than MW and column purification. After this verification step, 202 samples of cow, ewe and goat from three different geographical regions of Spain were analyzed to determine C. tyrobutyricum spore concentration. The spore levels found were in the range 10^2-10^3 spores/mL, although an important number of samples were below the LOD and could not be quantified. For this reason, this method was considered as qualitative, though qPCR precision was clearly improved by applying the KF method. To verify C. tyrobutyricum presence in raw milk samples, they were cultured in a selective media for butyric bacteria composed of RCM with D-cycloserine and neutral red. The grown colonies were analyzed by multiplex PCR and 16S rDNA sequencing. The results obtained showed that other microorganisms, such as Lactobacillus and Paenibacillus can grow in the selective medium. C. tyrobutyricum was only isolated in cow milk and other species, such as C. sporogenes and C. perfringens, were identified suggesting that they could contribute to LBD in cheese. Moreover, the identified species of Clostridium were different depending on the milk type analyzed (cow, ewe or goat). The second part of this thesis was focused on the development of a capture system based on magnetic particles functionalized with affine ligands. The results from qPCR analysis revealed that most of the samples analyzed were below the LOD, for this reason a step of spore capture directly from milk could be a good strategy to improve their detection. With this aim, the pCZS1 peptide obtained by Phage Display technique as affine for C. tyrobutyricum spores was evaluated. First, the affinity of pCZS1 for butyric spores was checked by isothermal titration calorimetry (ITC) and flow cytometry. After that, particles were functionalized with pCZS1 and C. tyrobutyricum spore capture was done in PBS and UHT milk. Positive results were achieved only in PBS and UHT milk treated with subtilisin. However, as the main objective was to apply the spore capture directly in milk, other ligands were assayed to achieve this goal. The transmembrane G3P protein from M13 phage was expressed with pCZS1 attached to the N-terminal end to increase the exposition of the peptide to the spores. First, only the soluble domain of G3P, named as tG3P, together with pCZS1 were expressed. A second ligand was designed based on the complete structure of tG3P together with pCZS1, named as cG3P, and pCZS1. Both proteins were expressed in Escherichia coli cells and purified by affinity chromatography by applying a protocol to solubilize inclusion bodies. tG3P protein provided better yields than cG3P protein probably because the transmembrane domain affects the purification process. tG3P was evaluated by ITC and flow cytometry confirming its affinity for butyric spores. cG3P was only evaluated by ITC with a similar Kd value in comparison with tG3P but due to the stability problems of tG3P, only tG3P was bound to magnetic particles. C. tyrobutyricum spores were captured with particles functionalized with tG3P reporting low capture values in PBS and milk with no successful results. For this reason it was concluded that G3P protein did not provide any advantage over pCZS1. The last part of this thesis has focused on the purification of polyclonal antibodies from the serum of rabbits immunized with C. tyrobutyricum spores. After the purification, Protein A and Protein G magnetic particles were functionalized with the specific antibodies. The capture efficiency for the spores was evaluated in PBS and UHT milk with both particles providing rates close to 90%. As a final application, the immunocapture was done in raw milk followed by qPCR detection. The DNA extraction from spores was made by MW treatment followed by column purification. Protein A particles reported better values in terms of amplification rates and Ct values than Protein G particles, confirming that the immunocapture of C. tyrobutyricum spores directly from milk, followed by qPCR detection is possible. The results obtained in this thesis set up the basis to develop a detection method for C. tyrobutyricum spores in raw milk by qPCR revealing the critical points. Furthermore, an immunocapture system based on magnetic particles coated with antibodies is proposed to recover C. tyrobutyricum spores from raw milk for further qPCR detection. <br /

Obtención de antígenos y anticuerpos para el desarrollo de un test para la detección de crustáceos en alimentos

Los crustáceos se encuentran entre los mariscos más comúnmente consumidos, pero también representan un grupo importante de alimentos que causa alergia alimentaria, siendo la tercera causa más importante de anafilaxia inducida por alimentos. La actual normativa de etiquetado obliga incluir los crustáceos si se utilizan como ingredientes en la elaboración de un alimento. Sin embargo, pueden también estar presentes como alérgenos ocultos debido a una contaminación cruzada durante el procesado, por lo que es necesario disponer de técnicas analíticas específicas y sensibles para su detección. La presente memoria forma parte de un proyecto cuyo objetivo es desarrollar técnicas inmunoquímicas rápidas para la detección de crustáceos en alimentos procesados. Para realizar este trabajo ha sido necesario poner a punto un método de purificación de la tropomiosina, la proteína diana seleccionada por ser abundante y la de mayor potencial alergénico de los crustáceos, además de presentar una elevada termoestabilidad. Estas características son esenciales para que las técnicas que se desarrollen puedan alcanzar una buena sensibilidad y se puedan aplicar en alimentos que hayan sido sometidos a tratamientos térmicos durante su procesado. Se han ensayado diferentes condiciones de preparación de las muestras de langostino (eliminando o no el exoesqueleto, aplicando o no un tratamiento térmico, entre otras), así como diferentes condiciones de extracción (pH y fuerza iónica del tampón, presencia de agentes desnaturalizantes, temperatura y tiempo de incubación, entre otros). Asimismo, se han ensayado dos procesos de purificación, uno mediante técnicas de precipitación isoeléctrica y salina y otro mediante técnicas de cromatografía de filtración en gel). El protocolo de purificación de la tropomiosina con el que se han obtenido los mejores resultados en cuanto a pureza y rendimiento ha incluido la extracción de las proteínas del tejido muscular del langostino con PBS tras un tratamiento en ebullición durante 5 min y la separación de las proteínas mediante una cromatografía de filtración en Sephadex G-50 en presencia de un agente quelante. La tropomiosina purificada se inoculó en conejos para obtener antisueros, que se han caracterizado para conocer su título y especificidad. Los antisueros, analizados mediante una técnica de ELISA indirecto, tienen un título alto y muestran reacción cruzada con extractos de otras especies de crustáceos, tanto crudos como tratados térmicamente, pero no con extractos de moluscos, pescado, carne de ternera o de pollo. Estos resultados indican que los antisueros obtenidos podrían ser usados para desarrollar técnicas inmunoquímicas para la detección de especies de crustáceos en alimentos procesados

A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs

IntroductionCoronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available.MethodsHerein, the first comparative study of six “in house” and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT–qPCR results) as a reference. Four “in house” tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera).Results and discussionELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (&amp;gt;90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs

RICORS2040 : The need for collaborative research in chronic kidney disease

Chronic kidney disease (CKD) is a silent and poorly known killer. The current concept of CKD is relatively young and uptake by the public, physicians and health authorities is not widespread. Physicians still confuse CKD with chronic kidney insufficiency or failure. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%. Thus health authorities may consider CKD a non-issue: very few persons eventually need KRT and, for those in whom kidneys fail, the problem is 'solved' by dialysis or kidney transplantation. However, KRT is the tip of the iceberg in the burden of CKD. The main burden of CKD is accelerated ageing and premature death. The cut-off points for kidney function and kidney damage indexes that define CKD also mark an increased risk for all-cause premature death. CKD is the most prevalent risk factor for lethal coronavirus disease 2019 (COVID-19) and the factor that most increases the risk of death in COVID-19, after old age. Men and women undergoing KRT still have an annual mortality that is 10- to 100-fold higher than similar-age peers, and life expectancy is shortened by ~40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth greatest global cause of death by 2040 and the second greatest cause of death in Spain before the end of the century, a time when one in four Spaniards will have CKD. However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded Centres for Biomedical Research (CIBER) network structure in Spain. Realizing the underestimation of the CKD burden of disease by health authorities, the Decade of the Kidney initiative for 2020-2030 was launched by the American Association of Kidney Patients and the European Kidney Health Alliance. Leading Spanish kidney researchers grouped in the kidney collaborative research network Red de Investigación Renal have now applied for the Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) call for collaborative research in Spain with the support of the Spanish Society of Nephrology, Federación Nacional de Asociaciones para la Lucha Contra las Enfermedades del Riñón and ONT: RICORS2040 aims to prevent the dire predictions for the global 2040 burden of CKD from becoming true

Análisis de proteínas de huevo en alimentos mediante un sistema rápido de lectura imparcial

Las alergias alimentarias constituyen un grave problema de salud pública debido a su alta prevalencia y al deterioro que suponen en la calidad de vida de las personas sensibilizadas. La única forma de prevenirlas es evitando el consumo del alimento alergénico. El control del cumplimiento de la legislación de etiquetado de alérgenos por los organismos competentes y la implementación de un plan de gestión de alérgenos en la industria alimentaria requieren métodos de detección sensibles, rápidos y sencillos.El huevo, muy utilizado por su calidad nutricional y propiedades tecnológicas, es uno de los alimentos más alergénicos. Para detectarlo, la empresa Zeulab ha desarrollado un test inmunocromatográfico, denominado Proteon Egg Express, basado en la detección de ovoalbúmina, la proteína más abundante y alergénica del huevo. El presente trabajo ha tenido como objetivo validar dicho test, siguiendo los protocolos establecidos en las guías internacionales, utilizando un material de referencia, alimentos modelo y comerciales. También se ha probado un lector óptico diseñado para obtener una lectura objetiva de los resultados del test. El test Proteon Egg Express es robusto y tiene una alta sensibilidad, pues permite detectar 1 ppm de huevo en polvo. No ha mostrado interferencias con una amplia gama de ingredientes básicos, por lo que es adecuado para analizar una gran variedad de matrices alimentarias. Su sensibilidad es menor en productos procesados, debido a la desnaturalización térmica de la ovoalbúmina. Se ha determinado la aplicabilidad del test Proteon Egg Express a superficies de trabajo, donde puede detectar 0,5 y 0,25 µg de huevo en polvo en acero inoxidable y melamina, respectivamente. En aguas de limpieza, es capaz de detectar 1 ppm de huevo en polvo en presencia de concentraciones de 0,5 N y 0,1 N de hidróxido sódico y de ácido clorhídrico, respectivamente. <br /

Disposable amperometric immunosensor for the detection of adulteration in milk through single or multiplexed determination of bovine, ovine, or caprine immunoglobulins G

This paper reports the first immunoplatforms for the detection of adulteration in milk with milk or colostrum from other animals. The developed electrochemical bioplatforms allow the reliable determination of immunoglobulins G (IgGs) from cows, sheeps, or goats. They rely on sandwiching each animal species-specific IgGs with selective antibody pairs [unconjugated and conjugated with horseradish peroxidase (HRP)] onto magnetic microbeads (MBs) used as solid supports and amperometric transduction with the H2O2/hydroquinone (HQ) system at disposable electrodes. The immunoplatforms allow achieving limits of detection (LODs) of 0.74, 0.82, and 0.66 ng mL–1 for bovine, ovine, and caprine IgGs, respectively, which are lower than those obtained with conventional enzyme-linked immunosorbent assay (ELISA) methodologies and in 2–5 times shorter time. The bioplatforms were successfully applied to the determination of the individual content of the target IgGs in milk samples of different animals (cow, sheep, and goat) and type (colostrum, raw, and pasteurized), without matrix effect and after just a sample dilution. They were also applied to the detection of adulteration with milks from other animals at levels below than those required by the European legislation (1.0%, v/v). The possibility to detect milk adulteration with colostrum using a strategy based on the measurement of the total content of the three target IgGs in raw milks is also demonstrated. Multiplexing platforms were constructed to be used in routine surveillance of milk. They are able to provide in a single run and in just 30 min relevant information regarding the milk sample including its animal origin, the undergone heat treatment, and whether it was adulterated with milk or colostrum from other species.The financial support of the Spanish Ministerio de Economía y Competitividad, CTQ2015-64402-C2-1-R Research Project, and the TRANSNANOAVANSENS Program from the Comunidad de Madrid (P2018/NMT-4349), and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.-V.M.) are gratefully acknowledged. S.B. acknowledges the financial support from MINECO through the “Juan de la Cierva” program.Peer reviewe

Fast and sensitive electrochemical immunoplatforms for detecting traces of soy in food

Resumen del póster presentado a la XXXVIII Reunión Bienal de la Real Sociedad Española de Química, celebrada en el Palacio de Congresos de Granada, del 27 de junio al 30 de junio de 2022.The prevalence of food allergies has been steadily increasing in developed countries in recent times and is now considered a serious public health problem affecting 2-4% of the population. Soybean is among the most frequent human food allergens, also known as the "big eight", accounting for 90% of all food allergies. Soy allergy has a high prevalence among children (0.5%), probably because soy is widely used as an ingredient in the preparation of many foods, and current EU and US legislations includes soy as one of the major allergens that must appear on the label of food products. Therefore, to protect allergic consumers, regulatory agencies are demanding highly sensitive and selective detection methods. Glycinin and β-conglycinin, accounting for about 70-80% of the total globulin fraction of the seed, are the main allergenic soy proteins. In this communication, the first electrochemical bioplatform for the determination of soy traces in food will be presented. The immunoplatform involves sandwich-type immunoassays using specific antibodies for β-conglycinin and glycinin and carboxylic acid-modified magnetic microbeads (MBs). The determination of both proteins can be carried out in only 1.5 h. It is important to note that, counting since the prepared capture antibody (cAb)-modified MBs, the determination can be performed in a short time as 30 min by direct assaying the food extract. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. It is also important to highlight that the results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005 ppm of a food allergen in model cookie extracts. This level of detection is significantly lower than that achieved with ELISA methodologies using the same immunoreagents (50-500 ppm).Peer reviewe

Ultrasensitive detection of soy traces by immunosensing of glycinin and β-conglycinin at disposable electrochemical platforms

This work reports the first electrochemical bioplatform for the determination of soy traces in food. The bioplatform involves sandwich-type immunoassays using specific antibodies for β-conglycinin and glycinin, which are the main allergenic soy proteins, and carboxylic acid-modified magnetic microbeads. Amperometric detection at −0.20 V (vs. an Ag pseudo-reference electrode) was performed using single or dual screen-printed carbon electrodes and the H2O2/hydroquinone (HQ) system. The measured variation in the cathodic current was directly proportional to the concentration of target allergenic proteins. The developed bioplatforms exhibit a good selectivity and sensitivity providing limits of detection (LOD) values of 0.03 and 0.02 ng mL−1 for β-conglycinin and glycinin, respectively. The determination of both proteins can be carried out in only 1.5 h. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. The results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005 ppm of a food allergen in model cookie extracts.The financial support of PID2019-103899RB-I00 to S.C., (Spanish Ministerio de Ciencia e Innovación) Research Project, and TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to S.C. are gratefully acknowledged. S.B. acknowledges a Juan de la Cierva Incorporación contract (Ref.: IJCI-2017-31345) from the Spanish Ministerio de Economía, Industria y Competitividad.Peer reviewe