The first part is focused on the evaluation of various parameters on the properties and functionality of gold (AuNPs) and carbon nanoparticles (CNPs) conjugates with enzymes and/or antibodies. Covalent binding and adsorption strategies are compared by means of conjugation of AuNPs with the enzyme iron superoxide dismutase (FeSOD) through a linker. Covalent binding seems more favorable in terms of coverage and enzymatic activity, and also offers better stability under pH and ionic strength changes, although both methodologies lead to low reproducibility. Additionally, in a bienzyme complex of the sequential enzymes glucose oxidase (GOx) and peroxidase (HRP) on CNPs, the enzyme arrangement can be manipulated by changing the incubation order and protein:CNP proportions, which influences complex activity. GOx shows higher affinity for CNPs, although its activity is strongly affected by CNPs binding. On the contrary, HRP catalytic efficiency is enhanced by CNPs. On the other hand, different methodologies for AuNPs conjugation with antibodies and HRP, i.e. direct adsorption, covalent binding and directional, are compared in order to know their potential to improve ELISA sensitivity. Direct adsorption reveals higher enhancing capacity, leading to a six fold increase in the sensitivity of the detection of gliadin (allergen) by and indirect ELISA.
The second part presents the characterization of a novel optical biosensor based on periodic nanostructures of resonant nanopillars (RNP) formed by multilayers of SiO2/Si3N4. The development of the biofunctionalization process for these materials disposed in nanopillars is described. For it, several strategies for the activation, silanization and covalent binding of biomolecules on the surface have been applied, leading to a stable and reproducible biofunctionalization procedure. Additionally, a protocol for the detection of antigen-antibody interaction has been developed, applying the IgG-anti-IgG model and a direct format, showing the suitability of the RNP as real time and label-free biosensors. Moreover, this interaction is regenerable, allowing the use of the surface cyclically. This procedure has been applied for the development of measurement protocols for the detection of low molecular weight analytes (okadaic acid and polychlorinated biphenyl 169) in the range of ng/ml applying a competitive format.