NANOTECHNOLOGICAL CANTILEVER-BASED ANALYSIS OF INFECTIOUS DISEASE BIOMARKERS

Abstract

Our history is pin-pointed with several events when a viral, parasite or bacterial pathogen disrupted its boundaries and influenced human culture and behaviour, shaping human past and present times. The continuous appearance of new threats enhances the demand for an improvement and optimisation of prompt detection methods of the relative condition. Indeed, each pathogen or disorder can be correlated with a wide number of specific and unique biomarkers. Therefore, reliable analytical devices able to perform quick and accurate analysis are urgently needed, with biosensors representing promising diagnostic tools. In order to accomplish this aim, a clinical diagnostic biosensor device was developed and optimised to function as a quantitative, label-free platform to investigate in parallel the presence of multiple epitope-specific interactions in complex biological matrices as serum samples. The scope of this thesis is to evidence and highlight the clear advancement obtained with this upgraded cantilever-based biosensor assay in the field of pathogen-related biomarker detection, specifically in correlation with malaria vaccines and COVID-19. First of all, we focused on analysing immunization clinical trial samples in serum gained from naïve and malaria pre-exposed volunteers injected with specific malaria candidate vaccines. Subsequent to the preliminary studies on pure solutions of antibodies or specific vaccine candidates, human serum samples have been analysed in order to evaluate and confirm the patients seroconversion against each candidate vaccine. Consequently, the project moved to the quantitative analysis of COVID-19 variant-specific biomarkers. First of all, a surface topographic analysis was carried out with focus on open or close spike protein configurations. A direct immune recognition comparison among pandemic hCoVs was successfully performed. Finally, variant-specific single amino acids mutations in the virus binding domain were used to carry out quantitative evaluations on immune evasion and infectiousness for three SARS-CoV-2 variants of concern. In conclusion, the clinical diagnostic platform with embedded nanomechanical sensor arrays performed as a robust method for quick and precise evaluation of a broad multitude of biomarkers present in human serum samples, exploiting its function as complementary tool to the commonly used standard procedures (PCR, ELISA and LFA). In detail, we achieve a limit of detection of few pg/ml (sub-pM concentration) in serum, full quantitative real-time kinetics and the inherent differential read-out with in situ controls reduces false positive results. The selectivity of the biomarker recognition as phase- or variant-specific assay with regards to a vaccine immunisation was indeed deeply evaluated with success.