Development of a multi-electrode impedimetric biosensor: detection of pathogenic bacteria and mycotoxins

Abstract

[spa] La detección de bacterias patógenas y micotoxinas es la clave para la prevención y la identificación de los problemas relacionados con la salud pública y seguridad alimentaria. En esta tesis hemos desarrollado una nueva plataforma de múltiples electrodos (multi-electrodo) para detección impedimétrica de bacterias patógenas y micotoxinas. Nos hemos centrado en la detección de bacterias E. coli O157: H7, ya que son responsable por brotes de origen alimentario graves. Se han caracterizado y estudiado la influencia de la (bio)interfaz del sensor para el desarrollo de un biosensor altamente sensible. Por esta razón, se probaron diferentes estrategias y materiales (óxido de indio - estaño y oro). Todos los pasos fueron completamente caracterizados por medio de múltiples técnicas y los resultados obtenidos mostraron que los biosensores desarrollados tenían una excelente respuesta en términos de sensibilidad y selectividad. Además, se obtuvieron resultados prometedores usando multi-electrodos. Estos fueron fabricados en oro y consistían en múltiples electrodos iguales e independientes que permiten el alto rendimiento y experimentos en paralelo en las mismas condiciones experimentales. Los multi-electrodos fueron caracterizados por varias técnicas de análisis de la superficie y técnicas electroquímicas, confirmando la calidad del proceso de fabricación. Hemos demostrado las capacidades de biosensores del multi-electrodo para la detección de bacterias patógenas utilizando diferentes bio-receptores, incluyendo anticuerpos y péptidos antimicrobianos. También se aplicaron los multi-electrodos para el desarrollo de sensores basados en aptámeros para la detección de micotoxinas. Nos centramos en concreto en el caso de la ocratoxina A (OTA), una de las más abundantes que contaminan los alimentos. Se presentaron dos estrategias para la inmovilización de aptámeros, ambas basadas en la hibridación de la superficie del biosensor a través de oligonucleótidos parcialmente complementarios. Se utilizaron técnicas electroquímicas para caracterizar todas las etapas de funcionalización. El multi-sensor desarrollado es capaz de detectar concentraciones de OTA y los prometedores resultados obtenidos demuestran la aplicación para la detección de micotoxinas y las ventajas de utilizar multi-electrodos.

[eng] This thesis aims the development of a multi-electrode platform with applications on different biosensing fields: (i) detection of pathogenic bacteria E.coli O157:H7 and (ii) detection of the mycotoxin Ochratoxin A. For most label-free biosensors, including impedimetric biosensors, the principal limitation on multiplexing arises from the affinity step. Therefore, a great part of the research work described here deals with the characterization, optimization and evaluation of different functionalization strategies for biosensing. These surface functionalization strategies developed here are applied for the final development and fabrication of the multi­electrode biosensor. In Chapter 2, we take advantage of the outstanding properties of indium tin oxide (ITO) material for the development of an ITO-based immunosensor for detecting pathogenic E. coli O157:H7 bacteria. The sensor build-up consisted on a simple, efficient and direct covalent binding of anti-E. coli O157 antibodies onto the ITO substrates. The functionalization methodology was fully characterized by multiple techniques, showing the specific binding of E. coli O157:H7 to the antibody­functionalized surface. The detection capacity of the ITO-based immunosensor was finally tested by EIS and a novel highly sensitive and selective sensor was obtained. In Chapter 3, we develop a gold-based electrochemical immunosensor for the detection of pathogenic E. coli O157:H7 bacteria. Gold is bio-compatible, can be easy obtained and it is easy to pattern using photolithography. In order to enhance the sensor performance, the functionalization protocol was optimized and antibodies were immobilized onto gold electrodes following two different strategies. Both functionalization strategies were evaluated and characterized by several techniques and the strategy showing better antibody immobilization was selected for the development of a highly sensitive label-free immunosensor. The immunosensor showed a very low limit of detection and low interference with other pathogenic bacteria. In Chapter 4, we take advantage of the functionalization strategies developed in the previous chapter 3 to develop a miniaturized multi-electrodes array for the detection of pathogenic bacteria. The multi-electrodes were fabricated in gold and consisted of multiple equally independent electrodes. This allowed high-throughput and independent experiments, in parallel and under the same experimental conditions. Multi-electrodes were fabricated by standard photolithography techniques and characterized by several surface analysis and electrochemical techniques, confirming the quality of the fabrication process. We demonstrated the biosensing capabilities of the multi­electrode platform for the detection of pathogenic bacteria using different bioreceptors, including antibodies and antimicrobial peptides. In Chapter 5, we applied the multi-electrodes platform for the development of an aptamer­based sensor for the detection of mycotoxins. We focused on the specific case of ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins. Two strategies for aptamer immobilization were presented, both based on the hybridization onto the biosensor surface through partially complementary oligonucleotides. Electrochemical techniques were used to characterize all the functionalization steps. The developed multi-sensor was capable to detect OTA concentrations and the promising results obtained prove the successful application of the multi-electrodes strategy for the detection of mycotoxins and the advantages of using multi-electrode platform.