Development of biosensors for mycotoxins detection in food and beverages

Mycotoxins are secondary metabolites of mould, which are ubiquitous in a large variety of food and feed commodities. Thousands of mycotoxins exist, but only a few present significant damages and poisonous properties. Among them, the aflatoxins and ochratoxins are considered to be the most toxic and widely spread in the world and therefore, represent a real threat for human/animal life. Depending on a number of factors like the intake levels, duration of exposure, mechanisms of action, metabolism and defense mechanisms, mycotoxins elicit a wide spectrum of toxicological effects leading to both acute and chronic disease, liver and kidney damage, skin irritation, cancer, immune suppression, birth defects or even death. To address the adverse effects of mycotoxin contaminants in food and feed, health authorities in many countries all over the world have become active in establishing regulations to protect their citizens and livestock from the potential damages caused by those compounds. The European Commission, the US Food and Drug Administration (FDA), the World Health Organization and the Food and Agriculture Organization of the United Nations have set up regulations and maximum levels for major mycotoxins in foods and feeds. To fulfill expectations of these regulatory limits, there is an increasing need for the development and validation of new, simple, fast and precise methods for toxins detection. Therefore, this thesis reveals different strategies for rapid, cost-effective and ultrasensitive bioanalysis of two major mycotoxins: aflatoxin M1 and ochratoxin A. Inhibition competitive assays with surface plasmon resonance spectroscopy (SPR, optical technique), quartz crystal microbalance (QCM, acoustic device) and electrochemical based readout were developed and compared. Presented biosensors were challenged in a red wine and milk samples with no need for pre-treatment or pre-concentration of the sample extract. In order to prevent fouling on the sensor surface by the constituents present in milk samples, the gold surface of the sensor chip was modified and different surface architecture and compared (antifouling polymer brushes and self-assembled monolayer - SAM). Complete resistance to the non-specific interactions was observed for coating with p(HEMA) brushes resulting in two times lower LOD compared to that on thiol SAM. The SPR biosensor for AFM1 allowed for highly sensitive detection in milk with an excellent precision (the average calculated CV was below 4%), limit of detection of 18 pg mL−1 for p(HEMA) brushes and 38 pg mL-1 for thiol SAM and with the analysis time of 55 min. It is worth highlighting that it is the first time that an SPR chip modified with such polymer brushes was used for real time detection of a small target antigen opening a new avenue for highly precise analysis. In the case of wine samples tested for OTA detection, a simple but very effective pre-treatment procedure was successfully applied. It was proved that the addition of the 3% of the binding agent poly(vinylpyrrolidone) (PVP) to red wine completely reduces non-specific interactions by binding polyphenolic compounds (which may be responsible for inactivation of antibody and blocking the sensor surface) through hydrogen bonding, making their elimination easier. Moreover, in this study, the authors evaluated the influence of gold nanoparticles (AuNPs) on signal enhancement and thereby biosensor sensitivity. For this purpose two assays were performed: with and without implementation of NPs. Obtained results allowed for OTA detection at concentrations as low as 0.75 ng mL−1 however, its limit of detection was improved by more than one order of magnitude to 0.068 ng mL−1 by applying AuNPs as a signal enhancer. The combination of indirect competitive assay and AuNPs with QCM-D gave a straightforward tool, which can simultaneously measure frequency and dissipation changes resulting in information about the sensitivity but also about the mass attached to the sensor surface as well as viscoelastic properties and the hydration state of the film. A linear detection range of 0.2–40 ng mL-1 has been achieved with LOD of 0.16 ng mL-1. The same assay format was also tested in voltammetric detection of mycotoxins using modified gold screen printed electrodes (AuSPE). An excellent LOD of 15 ng mL-1 for OTA and 37 pg mL-1 for AFM1 were obtained. Additionally, AuSPE modified with SAMs based on different types of alkanethiols (long and short chains) were tested and compared in terms of electron transfer resistance. Proposed biosensors offer vast range of advantages such as high sensitivity (at pg or ng levels), short analysis time (55 min) in comparison to for example, ELISA which require multiple steps that translates to prolonged analysis time, possibility for online monitoring, characterization of binding kinetics, low consumption of primary antibody (cost reduction), excellent antifouling surface and simple pre-treatment procedure. Combining all most desirable aspects of a good biosensor such as high sensitivity, low costs, short analysis time and simple but effective cleaning-up technique make proposed approaches an important and very promising tools for widespread biosensing applications

Design and synthesis of biofunctional magnetic/fluorescent glyco-nanoparticles and quantum dots and their application as specific molecular imaging probes

206 p.[EN]Nanotechnology presents very promising characteristics for its application in the biomedicine area. By now the most advanced application of nanoparticles in medicine is the use of iron oxide nanoparticles embedded in biocompatible polymers as magnetic resonance imaging (MRI) contrast agents. Nowadays MRI application relies either on inherent tissue contrast differences or on the unspecific accumulation of contrast agents in certain tissues. This status can be extended to other imaging techniques. These limitations do not enables the application of imaging techniques to many diagnostic problems such as the imaging of small or diffuse populations of tumour cells, or require the administration of large amounts of the contrast agents to achieve a clear diagnostic. Considering this, there is a clear need for the development of new probes able to target specifically cells, organs or tissues for high resolution imaging. In this thesis, we have first prepared nanomaterials with suitable properties to be used as imaging probes; on one hand magnetic nanoparticles based on water soluble goldcoated glyco-nanoferrites to be used as MRI T2 contrast agents, and on the other, CdTebased quantum dots with fluorescent emissions in the visible to the near infrared spectra to be used in optical molecular imaging. The biofunctionalization of these nanoprobes with different biomolecules has been explored. Magnetic glyconanoparticles and quantum dots (QDs) coupled to protein G and IgG antibodies have been prepared to get specific targeted imaging probes (immuno-nanoparticles) for MRI and optical imaging. Functionalised magnetic glyconanoparticles with single chain DNA molecules have also been produced as potential specific probes for genetic material sensing. Both kinds of immuno-nanoparticles have been applied in vitro for the specific labelling of a cell population within a simple mixture, or of sub-cellular structures (cytoskeleton). The application of immuno-magnetic glyconanoparticles (immuno-MGNPs) has been taken further with specific ex vivo labelling experiments in whole human blood. Finally, the immuno-MGNPs have been applied in in vivo studies to label and track endogenous neural stem cells to answer a fundamental question in neurobiology: Do neural stem cells migrate towards damaged brain areas? In conclusion, we have been able to design, prepare, and apply, specific biofunctional nanoprobes as targeted contrast agents for in vivo imaging techniques, especially for MRI.[ES]La nanotecnología en general presenta características muy prometedoras para su aplicación en el área de biomedicina. Hasta el momento, la aplicación más avanzada en este área es el uso de nanopartículas de oxido de hierro encapsuladas en polímeros biocompatibles como agentes de contraste en resonancia magnética de imagen (MRI). Actualmente la aplicación de MRI confía, bien en diferencias inherentes de contraste entre tejidos, o bien en una acumulación inespecífica del agente de contraste en ciertos tejidos. Esta misma situación es compartida por otras técnicas de imagen. Estas limitaciones no permiten la aplicación de técnicas de imagen a muchos problemas diagnósticos, como por ejemplo la visualización de poblaciones pequeñas o difusas de células tumorales, o requiere de la administración de grandes cantidades del agente de contraste para la obtención de un diagnóstico claro. Considerando esto, hay una necesidad clara de desarrollo de nuevas sondas capaces de hacer un marcaje específico de células, tejidos u órganos para su uso en técnicas de imagen de alta resolución. En esta tesis, hemos preparado primero nanomateriales con propiedades adecuadas para su utilización como sondas de imagen; por una parte nanopartículas magnéticas basadas en glico-nanoferritas recubiertas con oro solubles en agua para ser usadas como agentes de contraste T2 en MRI, y por otra parte quantum dots derivados de CdTe con emisión de fluorescencia en la zona del visible al infrarrojo cercano para ser usados en imagen óptica molecular. También hemos explorado la biofuncionalización de estas nanosondas con diferentes biomoléculas. Gliconanopartículas magnéticas (MGNPs) y quantum dots (QDs) unidos a proteína G y anticuerpos IgG han sido preparadas para obtener sondas de imagen especificas (immuno-nanopartículas) para MRI e imagen óptica. MGNPs funcionalizadas con moléculas de DNA de una cadena también han sido preparadas como sondas para la potencial detección de material genético. Ambas clases de immuno-nanopartículas han sido aplicadas in vitro al marcaje específico de una población celular dentro de una mezcla simple de células, o al marcaje de estructuras sub-celulares (citoesqueleto). La aplicación de immunogliconanopartículas magnéticas (immuno-MGNPs) se ha llevado más lejos con experimentos de marcaje específico ex vivo en sangre humana. Finalmente, las immuno- MGNPs han sido aplicadas en estudios in vivo para marcar y hacer un seguimiento de células madre neuronales endógenas para contestar una pregunta fundamental en neurobiología: Migran las células madre neuronales hacia lugares en los que se produce un daño cerebral? En conclusión, hemos sido capaces de diseñar, preparar y aplicar nanosondas biofuncionales específicas como agentes de contraste dirigidos para su uso en técnicas de imagen in vivo, especialmente MRI.This thesis has been carried out in the Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit of the Centro de Investigación Cooperativa en Biomateriales (CIC biomaGUNE). The research was financially supported by a fellowship from CIC biomaGUNE, the Spanish Ministry of Science and Innovation (grant CTQ2008-04638), and the Department of Industry of the Basque Country (grant ETORTEK 2009 IE09-257)

Evaluation of Colloidal Stability and Ecotoxicity of Metal-based Nanoparticles in the Aquatic and Terrestrial Systems

Intrinsic to the many nano-enabled products are atomic-size multifunctional engineered nanomaterials, which upon release contaminate the environments, raising considerable health and safety concerns. This Ph.D. dissertation is designed to investigate (i) whether metals or oxide nanoparticles are more toxic than ions, and if MetPLATETM bioassay is applicable as a rapid nanotoxicity screening tool; (ii) how variable water chemistry (dissolved organic carbon (DOC), pH, and hardness) and organic compounds (cysteine, humic acid, and trolox) modulate colloidal stability, ion release, and aquatic toxicity of silver nanoparticles (AgNP); and (iii) the developmental responses of crop plants exposed to Ag- or ZnO- (zinc oxide) nanoparticles. Results suggest that the MetPLATEcan be considered a high-throughput screening tool for rapid nanotoxicity evaluation. Detectable changes in the colloidal diameter, surface charge, and plasmonic resonance revealed modulating effects of variable water chemistry and organic ligands on the particle stability, dissolution, and toxicity of AgNPs against Escherichia coli or Daphnia magna. Silver dissolution increased as a function of DOC concentrations but decreased with increasing hardness, pH, cysteine, or trolox levels. Notably, the dissociated Ag+ was inadequate to explain AgNP toxicity, and that the combined effect of AgNPs and dissolved Ag+ under each ligand treatment was lower than of AgNO3. Significant attenuation by trolox signifies an oxidative stress-mediated AgNP toxicity; its inability to attenuate AgNO3 toxicity, however, negates oxidative stress as Ag+ toxicity mechanism, and that cysteine could effectively quench free Ag+ to alleviate AgNO3 toxicity in D. magna. Surprisingly, DOC-AgNPs complex that apparently formed at higher DOC levels might have led daphnids filter-feed on aggregates, potentially elevating internal dose, and thus higher mortality. Maize root anatomy showed differential alterations upon exposure to AgNPs, ZnONPs, or their ions. Overall, various metal-based nanoparticles revealed lower toxicity than their ions against multiple organisms. This study showed that particle size, surface properties, and ion release kinetics of AgNPs modify following release into aquatic environment, suggesting potential implications to ecosystem health and functions, and that caution be applied when extending one species toxicity results to another because obvious differences in organism biology—supporting species sensitivity paradigm—can significantly alter nanoparticle or ionic toxicity

Evaluation of Colloidal Stability and Ecotoxicity of Metal-based Nanoparticles in the Aquatic and Terrestrial Systems

Intrinsic to the many nano-enabled products are atomic-size multifunctional engineered nanomaterials, which upon release contaminate the environments, raising considerable health and safety concerns. This Ph.D. dissertation is designed to investigate (i) whether metals or oxide nanoparticles are more toxic than ions, and if MetPLATETM bioassay is applicable as a rapid nanotoxicity screening tool; (ii) how variable water chemistry (dissolved organic carbon (DOC), pH, and hardness) and organic compounds (cysteine, humic acid, and trolox) modulate colloidal stability, ion release, and aquatic toxicity of silver nanoparticles (AgNP); and (iii) the developmental responses of crop plants exposed to Ag- or ZnO- (zinc oxide) nanoparticles. Results suggest that the MetPLATEcan be considered a high-throughput screening tool for rapid nanotoxicity evaluation. Detectable changes in the colloidal diameter, surface charge, and plasmonic resonance revealed modulating effects of variable water chemistry and organic ligands on the particle stability, dissolution, and toxicity of AgNPs against Escherichia coli or Daphnia magna. Silver dissolution increased as a function of DOC concentrations but decreased with increasing hardness, pH, cysteine, or trolox levels. Notably, the dissociated Ag+ was inadequate to explain AgNP toxicity, and that the combined effect of AgNPs and dissolved Ag+ under each ligand treatment was lower than of AgNO3. Significant attenuation by trolox signifies an oxidative stress-mediated AgNP toxicity; its inability to attenuate AgNO3 toxicity, however, negates oxidative stress as Ag+ toxicity mechanism, and that cysteine could effectively quench free Ag+ to alleviate AgNO3 toxicity in D. magna. Surprisingly, DOC-AgNPs complex that apparently formed at higher DOC levels might have led daphnids filter-feed on aggregates, potentially elevating internal dose, and thus higher mortality. Maize root anatomy showed differential alterations upon exposure to AgNPs, ZnONPs, or their ions. Overall, various metal-based nanoparticles revealed lower toxicity than their ions against multiple organisms. This study showed that particle size, surface properties, and ion release kinetics of AgNPs modify following release into aquatic environment, suggesting potential implications to ecosystem health and functions, and that caution be applied when extending one species toxicity results to another because obvious differences in organism biology—supporting species sensitivity paradigm—can significantly alter nanoparticle or ionic toxicity

Recent Developments in Atomic Force Microscopy and Raman Spectroscopy for Materials Characterization

This book contains chapters that describe advanced atomic force microscopy (AFM) modes and Raman spectroscopy. It also provides an in-depth understanding of advanced AFM modes and Raman spectroscopy for characterizing various materials. This volume is a useful resource for a wide range of readers, including scientists, engineers, graduate students, postdoctoral fellows, and scientific professionals working in specialized fields such as AFM, photovoltaics, 2D materials, carbon nanotubes, nanomaterials, and Raman spectroscopy

Biomarkers Used for the Diagnosis of Diseases

The detection and quantification of with high precision nucleic acid biomarkers and protein biomarkers in resource-limited settings is key to the early diagnosis of diseases and for monitoring the effects of treatments. As there is an enormous demand for high-quality biomarker detection platforms that are robust and highly applicable in resource-limited settings, this book is devoted to exploring methods for detection and quantification of biomarkers, focusing on the recent advances in this field