Author(s): Sande, Maria Georgina
Date: 2023
Persistent ID: https://hdl.handle.net/1822/82576
Origin: RepositóriUM - Universidade do Minho
Subject(s): Cell-SELEX; Electrochemical biosensor; Magnetic nanoparticles; UspA2; YadA; Biossensores eletroquímicos; Nanopartículas magnéticas
Diagnosis of bacterial infections is primarily accomplished by culture and laboratory analysis, immunological methods and nucleic-acid amplification techniques. However, these methods have limitations related to affordability, accessibility and time to inform a result. Moreover, the emergence of antibiotic resistant bacteria comprises a global threat to health, strengthening the relevance of early detection of pathogens to provide adequate and timely treatments. To address these concerns, point-ofcare (POC) diagnostic platforms arose as a solution to deliver affordable, equipment-free and rapid accurate testing especially in resource-limited settings. The combination of cell-SELEX with highthroughput sequencing and bioinformatic analysis led to the identification of a novel ssDNA aptamer sequence (Apt1YadA) with high affinity and selectivity towards the adhesin YadA from Yersinia enterocolitica. An electrochemical aptasensor POC platform was assembled using Apt1YadA on a gold screen printed electrode which successfully detected recombinant YadA. This was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and square wave voltammetry techniques, attaining a limit of detection of 7.0 × 104 CFU mL−1. The selectivity of Apt1YadA was validated in the presence of non-target bacteria. A novel aptamer Apt1_RCUspA2 specifically targeting UspA2 adhesin from Moraxella catarrhalis was also herein identified using a similar workflow. Apt1_RCUspA2 was characterized, demonstrating high affinity and selectivity towards recombinant UspA2. Likewise, an electrochemical aptasensor assembled using Apt1_RCUspA2 successfully detected UspA2 with a limit of detection of 4.0 × 104 CFU mL−1. To further enhance the sensitivity of biosensors, a previous enrichment step of samples, is envisaged. Since both YadA and UspA2 have an affinity for collagen, magnetic nanoparticles (MNPs) functionalized with hydrolyzed collagen were synthesized by a quick, green co-precipitation method and were characterized by ATR-FTIR. The collagen MNPs specifically interacted with YadA and UspA2, with capture efficacies of 94.7 ± 5.2% and 87.4 ± 4.7%, respectively, providing a viable and efficient tool for bacterial capture. The results gathered in this thesis contributed to the goal of developing novel POC platforms and diagnostic workflows for pathogenic biomarkers (YadA and UspA2). Moreover, the innovative workflows developed, can serve as a prototype for developing similar POC platforms for other bacterial pathogens.
