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Title: Development of functional nanoparticles for biosensing and antibacterial applications
Author: Lapitan, Lorico Delos Santos
ISNI:       0000 0004 7428 1490
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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PART 1 The ability of ultrasensitive detection of specific genes and discrimination of disease related single nucleotide polymorphisms (SNPs) is important for biomedical research and clinical disease diagnosis. Herein, we report an ultrasensitive approach for label-free detection and discrimination of full-match target-DNA from its cancer related SNPs by combining magnetic nanoparticle (MNP) capture and poly-enzyme nanobead signal amplification. It uses a MNP linked capture-DNA and a biotinylated signal-DNA to sandwich the target followed by ligation to offer high SNP discrimination: only the perfect-match target-DNA yields a MNP covalently linked biotinylated signal-DNA for subsequent binding to a neutravidin-horseradish peroxidase conjugate (NAV-HRP) and signal amplification. The use of polymer nanobeads each tagged with thousands of copies of HRPs greatly improves the signal amplification power, allowing for detection of 10 aM target-DNA with a large dynamic range of 5 orders of magnitude (0.01-1000 fM). Moreover, this sensor also offers excellent signal discrimination between the perfect-match gene and its cancer-related SNPs and can positively detect 1 fM perfect-match target-DNA in the presence of 100 fold excess of co-existing SNPs. Furthermore, it works robustly in clinical relevant media (e.g. 10% human serum) and gives almost identical SNP discrimination as that in clean buffers. This ultrasensitive SNP sensor appears to have excellent potential for rapid detection and diagnosis of genetic diseases. This study also reports the design of a MNP-DNAss-HRP nanoprobe for the label-free detection of DNA and ECOR-I. The Fe3O4 MNP were prepared by thermal decomposition and coated with silica by the reverse micelle method yielding core-shell nanoparticles. These silica coated MNP were modified with amino groups for further conjugation with DNA. In Design-1, the capture DNA contain DBCO and biotin group at the 5’ and 3’ end respectively. The capture DNA was linked to the amino modified MNP through copper free click chemistry approach. The NAV-HRP was linked to the DNA strands through biotin-strepatividin interaction. The MNP-DNA conjugates were further incubated with NHS-PEG~17-OCH3 to cap unreacted amines. In Design-2, the capture DNA also contain DBCO group at 5’ end but a thiol (-SH) group at 3’ end. The capture DNA strand was linked to MNP and the unreacted surface amines were blocked following the same strategy for Design-1. Incubation with maleimide-HRP led to the covalent linking of the enzyme to the DNA strands. The MNP-DNAss-HRP nanoprobes and target DNA are hybridized and the resulting double strand DNA contains specific sequence that is recognized and cleaved by ECOR-I. This releases thousands of HRP enzyme to the solution which in turn can catalyse a colourimetric reaction. Using Design-2, the optimal incubation temperature was 30 °C and 1 hour incubation time with ECOR-I. This approach can detect 10 U·mL-1 of ECOR-I and 1 nm of target DNA. PART 2 Silver nanomaterials have been widely utilized for antimicrobial applications. Silver nanoparticles (Ag NPs) have been used in many biomedical and environmental applications for many years. Interestingly, silver nanoclusters (Ag NCs) have emerged as a new class of silver nanomaterials and currently being investigated for its antibacterial properties. In this study, we report the antibacterial properties of Ag NPs and NCs that were synthesized using the same protocol and capped with the same dihydrolipoic acid (DHLA) based ligands against S. aureus and E. coli. These ligands were DHLA-EGn-NH2 (n=3,12), DHLA-PEGn-OCH3 (n ~17, 23) and DHLA-zwitterion. The Ag NC-DHLA and Ag NC-PEG~23-OCH3 inhibited S. aureus and E. coli with MIC results of 128, 64 and 64, 128 μg·mL-1 respectively. The Ag NPs were observed to be more effective antimicrobial agents as revealed by MIC results. The 5.7 nm Ag NP-zwitterion is the most potent antibacterial agent among all the Ag NPs tested with an MIC of 8 μg·mL-1 for both bacterial strains. This study demonstrated, for the first time, that the antibacterial properties of silver nanomaterials differ significantly when coated with different ligands. Moreover, surface coating and charge are most likely the key factors that control the antimicrobial efficacy of Ag NCs and NPs.
Supervisor: Zhou, Dejian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available