Abstract
Every year, millions of cases of food-borne diseases occur worldwide, due to food contamination by different hazards (e.g., foodborne viruses, mycotoxins, residues of pesticides, and other chemical toxicants). Detection and quantification of these hazards in food is a matter of public health protection, necessary to guarantee food safety to the whole world population. This is why there is a high demand for sensitive and selective real-time analytical methods for hazard detection. In this context, biosensors - a new class of analytical devices - able to selectively detect these hazards are especially interesting due to their simplicity, high sensitivity, short analysis time, label-free detection, low fabrication cost, minimal sample preparation, and field applicability. This Ph.D. thesis focuses on the development and evaluation of biosensing platforms for the detection of widely known foodborne toxicants - biogenic amines such as histamine and spermidine in food samples, covering every aspect from fabrication to electrical characterization, as well as functionalization with specific bio-recognition elements (such as antibodies and aptamers) in order to achieve the desired biosensor selectivity to the analyte of interest. Due to the advantages offered by nanomaterials especially in terms of increasing overall biosensor sensitivity by amplification of their conductivity and catalytic activity, a significant part of this Ph.D. work focuses on carbon nanotubes (CNTs), in terms of ink preparation and integration into biosensing platforms. The first part of this research work focuses on the development of three-electrode system-based electrochemical biosensors for histamine detection. The biosensors were fabricated using screen-printing of silver/silver chloride conductive electrodes. To enhance the analytical performance of the biosensors, the working electrodes are modified with spray-deposited conductive CNTs. Different CNTs inks were prepared, in order to achieve the desired ink properties, by varying sodium dodecyl sulfate (used as surfactant) concentration, and sonication/centrifugation time of the prepared inks. The prepared inks were characterized by UV-Vis spectroscopy, atomic force microscopy (AFM), and sheet resistance measurements. To achieve specific detection of histamine, the sensors were functionalized with anti-histamine antibodies based on the direct ELISA (enzyme linked immunosorbent assay) principle. The realized biosensor showed a wide linear detection range from 0.005 to 50 ng/ml for histamine samples, with a coefficient of deVII VIII Abstract termination as high as 98.05% The biosensor performance was evaluated by detecting histamine in real fish samples matrix, with average recoveries in fish samples from 96.00% to 104.7%. The second part of this research focuses on the evaluation of a different sensing platform, to explore the possibility to further improve the detection of food toxicants. Specifically, the focus was on micro-fabrication, bio-functionalization, and characterization of biosensors employing an electrolyte-gated field-effect transistor (EG-FET) as a transduction element, using spray-deposited CNTs as semiconducting channels. As a proof of concept, the fabricated devices were functionalized with anti-spermidine antibodies and were used to detect spermidine prepared in 0.1x PBS (phosphate buffer saline), biosensor showed a wide linear detection range from 0.001 to 100 nM. A key component for optimal EG-CNTFET operation is the fabrication of the semiconducting channel, therefore, in a more thorough study, specific focus has been paid to the optimization of the spray-deposited CNT channel. Our findings clearly show that a CNT source-drain resistance of circa ≤ 10 kΩ should be reached to obtain an optimal EG-CNTFET behavior. Furthermore, in this study, we have demonstrated that the sensitivity of the resulting EG-CNTFETs strongly depends on the density of semiconducting CNT i.e., increased sensitivity in the function of the increasing number of layers (-1.03 up to -2.45 µA/decade). Moreover, the extensive literature research has led to the publication of an invited review paper to Applied Physics Review, which covers: working principle, fabrication, bio-functionalization, present issues, and challenges faced for the EG-CNTFET-based biosensors. Finally, to further enhance EG-CNTFET-based biosensor performance, in terms of overcoming the Debye length (λD) limitation (i.e., the device is sensitive only within/near the λD which in physiological solution is characterized by dimensions less than 1 nm), evaluation of a new functionalization strategy using aptamers as bio-recognition was employed. Aptamers present a promising approach to overcome the Debye length limitation thanks to the conformational changes upon target recognition which enabled signal transduction and amplification under physiological solution conditions within λD. The developed functionalization strategy was characterized by means of several analytical techniques including optical waveguide lightmode spectroscopy (OWLS), X-ray photoelectron spectroscopy (XPS), and fluorescence microscopy. The EG-CNTFETs functionalized with histamine aptamers were used for the detection of histamine, the preliminary results show a linear detection range from 1 µM to 100 µM for histamine samples prepared in 1x PBS (representing real physiological condition).