Percorrer por autor "BRAGA, DIOGO CUNHA"
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- Desenvolvimento de um teste rápido para detetar resistência antimicrobiana ambientalPublication . BRAGA, DIOGO CUNHA; Guerreiro, Joana Rafaela LaraAntimicrobial resistance (AMR) has become a global public health issue that threatens the effective treatment of infectious diseases and is one of the major issues of our times. Resistance genes can be acquired by bacteria, such as the qnr gene associated with quinolones resistance. The acquired resistance mechanisms enable the transfer of the genetic material to other non-resistant bacteria, both in the environment or hospital settings. The current methods for AMR detection are the microbiology and molecular biology methods such as PCR (Polymerase Chain Reaction), however, they are often labour-intensive, costly, and require specialized, high-tech equipment and trained technicians. Thus, there is a need to respond to the urgent demand for a rapid, cost-effective and accessible alternative for in-situ testing of environmental monitoring. This work, developed at LabRISE/ISEP (Porto Higher Institute of Engineering), aimed to design and validate a colorimetric biosensor based on gold nanoparticles (AuNPs) for the detection of qnr 3, a gene associated with quinolones in E. coli. The AuNPs were synthesized using a modified Turkevich-Frens method. Functionalization with thiolated DNA sequences was achieved using pH-assisted methods and incremental salt addition, the latter proving to be more robust, allowing denser sequence coverage and greater tolerance to the increase of ionic strength. Hybridization tests indicated that the co-immobilization of spacer molecules, such as mercaptohexanol (MCH), enhanced the biosensor’s sensitivity by achieving a 2-fold increase compared to longer incubation times or absence of such modifiers. Optimization studies further showed that the optimal MCH/DNA ratio was 3000:1. The effect of ionic strength on DNA-DNA hybridization was also tested and analyzed using naked eye detection, while quantitative analysis was performed with both spectral ratio and RGB-based approaches. The results demonstrated that spectral ratio sensitivity increased 1.7-fold from the lowest ionic strength to 0.45 M NaCl, while the RGB-based sensitivity increased approximately 2-fold when comparing the lowest ionic strength (0.25 M) with the optimal condition at 0.45 M NaCl, showing a similar trend for both methods. The comparative analysis between these two methods confirms their reliability to assess a colorimetric detection, showing the best sensing performance for the ionic strength of 0.45 M. The system was also evaluated using real environmental samples collected from the Douro River. Under the optimized conditions, the spiked samples exhibited a 3.3-fold increase in sensitivity compared to measurements in buffer, while the linear range decreased from 0–80 nM to 0–40 nM, representing a 2-fold reduction in the upper limit of the linear range. The limit of detection (LOD) was 25 nM in buffer and 17 nM in real samples. In conclusion, the developed AuNP-based colorimetric biosensor demonstrated robust analytical performance, with dual validation through spectral ratio and RGB-based analysis enabling accurate quantitative assessment beyond naked eye detection. The developed colorimetric biosensor was successfully applied to the detection of the target DNA in river waters samples, confirming its reliability and applicability under real environmental conditions. Overall, the biosensor proved to be an effective, portable, and a simple tool suitable for in-situ environmental antimicrobial resistance monitoring.