De Luca, Maria (2024) Plasmonic Nanostructures for Efficient Optical Biosensing. [Tesi di dottorato]

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Abstract

Nanotechnology has emerged as a pivotal area of research, profoundly impacting various scientific and industrial sectors. Advances in material science, combined with innovations in fabrication and characterization techniques, have enabled the precise manipulation of materials at atomic and molecular levels, paving the way for groundbreaking applications. Among these, plasmonic nanostructures, particularly those composed of noble metals, stand out for their remarkable optical properties, which arise from strong and resonant light-matter interactions. These properties, such as enhanced light absorption, scattering, and localized electromagnetic fields near nanoparticle surfaces, have made plasmonic materials indispensable for biosensing applications, enabling the detection of biological analytes at ultra-low concentrations. Furthermore, the integration of plasmonic nanostructures with fluidic systems enhances their biosensing potential. By controlling sample flow across sensor surfaces, fluidic systems improve analyte-antibody binding efficiency, reduce response times, and increase detection accuracy. Automated fluidic platforms minimize human error, enhance reliability, and support high-throughput, multiplexed analyses. The resulting synergy between plasmonic sensitivity and fluidic precision creates integrated, robust, and versatile diagnostic platforms that meet modern demands for rapid and efficient diagnostic tests. In this framework, we developed a versatile biosensing platform by integrating plasmonic nanostructures, composed of gold nanoparticles (AuNPs), with a custom-designed 3D-printed fluidic circuit. This integrated approach facilitated the realization of two distinct optical biosensors, showcasing its adaptability and potential for a wide range of applications. The first biosensor exploits the plasmon-enhanced fluorescence (PEF) phenomenon, which occurs when fluorescent molecules are in close proximity to plasmonic nanostructures under controlled spatial and spectral alignment conditions between the nanostructure and the fluorophore. The PEF immunosensor employed a sandwich scheme, capturing the target analyte with a lower layer of covalently immobilized antibodies on the AuNPs array, while detection occurred via an upper layer of antibodies labeled with the fluorophore Cy3. As a result, this biosensor successfully detected prostate-specific antigen (PSA) in real samples of human serum, achieving a limit of detection (LOD) of 150 pg/mL, significantly below the clinical threshold of 4 ng/mL, with a detection time of 45 minutes. The second biosensor employed Core-Satellite Magnetic Nanoparticles (CSMPs), comprising 250 nm superparamagnetic nanoparticles coated with 20 nm AuNPs, to enhance the optical response of the AuNPs array. The CSMPs combine both the optical properties of AuNPs and the magnetic properties of iron oxide composites in response to an external magnetic field. The CSMPs were employed in a sandwich scheme, binding from above to the target analyte, which had been previously captured by antibodies immobilized on the plasmonic platform. Furthermore, the CSMP binding induced a significant change in the extinction spectrum of the AuNPs array in the presence of a magnetic field, accelerating and enhancing analyte capture. The biosensor setup was tested against the inflammatory protein Pentraxin 3 (PTX3) in phosphate-buffered saline (PBS) solution at clinically relevant concentrations, achieving a LOD of 300 pg/mL with a detection time of 35 minutes. The innovative CSMPs demonstrated significant potential for exceptional sensing applications, effectively overcoming the limitations of passive diffusion.

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