Electrochemical and optical nanoparticlebased biosensors for point-of-care applications

Resumen

This Thesis describes the study and development of different strategies based on novel properties of nanoparticles and other micromaterials for the improvement of the performance of two biosensing platforms with interest for analytical applications. The first one consists in screen printed carbon electrodes (SPCEs) used in combination with iridium oxide nanoparticles (IrO2 NPs). These nanoparticles are employed both as novel electrocatalytic labels for biomarkers detection, and also as electrode surface modifiers for impedimetric detection of small molecules such as toxins. The second platform consists in lateral flow assay (LFA) strips used in combination with gold nanoparticle (AuNP) labels, where different amplification strategies are developed for the improvement of the assay sensitivity, applied for protein and DNA detection. In Chapter 1, a general overview about the use of nanoparticles in electrochemical and paper-based platforms is presented. The first part gives a general overview on electrochemical biosensors and the different related applications of nanoparticles either as labels or modifiers of the surface of the working electrode of screen-printed carbon electrodes (SPCE) used as electrotransducer. The second part is focused on the use of nanoparticles in paper-based sensors, including basic concepts and the state-of-the-art of their application in this type of sensors. In Chapter 2 the objectives of this Thesis are presented. The third chapter (Chapter 3) presents the evaluation of the electrocatalytic activity of citrate-capped IrO2 NPs toward the water oxidation reaction and their use as novel electrochemical labels in protein diagnostics. Magnetic beads modified with antibodies are used as platform for the immunoassay which is applied for the detection of Apolipoprotein E (ApoE), a biomarker of Alzheimer disease. The main feature of the developed device is the signal generation in the same medium where the immunoassay takes place avoiding the use of additional reagents, which is expected to allow a simpler and user-friendlier methodology for protein detection. In Chapter 4, a novel aptasensor based on SPCEs modified with conductive films of thionine and adsorption of citrate-capped IrO2 NPs is presented. This system is explored for label-free impedimetric detection of ochratoxin A (OTA), a highly toxic compound which represents a public health problem due to its presence in foods above the limit established by worldwide organizations. Each fabrication step has been characterized by electrochemical impedance spectroscopy (EIS) in the presence of a redox probe. The developed device is able to detect low concentrations of OTA showing also good specificity and reproducibility. A new strategy for improving the sensitivity of AuNPs-based LFAs by using printed barriers deposited onto the nitrocellulose membrane by wax printing technique is presented in Chapter 5. Pillars of different designs and distributions were printed, in order to create hydrophobic barriers that can cause flow delay. The controlled delays in microfluidics increase the binding time between the immunocomplex and the detection antibody, in addition to the generation of pseudo turbulences in the pillars zone that improves mixing between the analyte and the labeled antibody. This microfluidics delay in certain zones (incubation areas) combined with the generation of the pseudo turbulences directly affects the analytical performance of the LFA being transduced to a better sensitivity and detection limit. In Chapter 6, a novel LFA design with enhanced sensitivity able to detect very low quantities of isothermal amplified Leishmania DNA sequences with interest for animals diagnostics is presented. The enhanced methodology takes advantage of the use of AuNPs tags connected with polyclonal secondary antibodies which recognize primary ones. The polyclonal nature of the secondary antibodies allowed their multiple connections with primary ones, giving rise to the enhancement of the AuNP signal. Furthermore, an endogenous control was introduced so as to avoid false negatives and the analysis accordingly performed. Finally, general conclusions and the future perspectives are discussed in Chapter 7. Additionally, Annex 1 describes preliminary studies related to the size- and shape-effect on the electrochemical response of AuNPs (including nanospheres and concave nanocubes) using differential pulse voltammetry (DPV) on SPCEs. The Annex 2 lists the publications, book chapter and manuscript submitted that resulted from this thesis.