Author: Karen Bruce
Bruce, Karen, 2017 Development of Oligonucleotide Modified Substrates for the Selective Detection of Harmful Algal Bloom Causative Microalgae, Flinders University, School of Chemical and Physical Sciences
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Harmful algal bloom (HAB) events have become more prevalent in the last few decades, with each event leading to the subsequent release of biotoxins. Bloom monitoring is essential to prevent detrimental damage to the environment and human health. Traditional methods using light microscopy can encounter difficulties in discriminating between toxic and non-toxic species of the same genus due to similarities in cellular morphology. Alternatively, specific, and highly discriminative detection can be achieved using oligonucleotide probes that are able to target variable regions found within the rRNA of HAB species. Detection of HABs using these specifically designed oligonucleotides provides a highly sensitive and selective route for monitoring microalgae bloom formations.
This thesis reports the covalent attachment of oligonucleotides to solid supports for model hybridisation with fluorescently labelled target oligonucleotide sequences specific to HAB species Alexandrium catenella in the aim to develop substrates for the sensitive, selective detection of HAB microalgae. Initial investigation into substrate compared PDMS and glass microscope slides for their performance in hybridisation experiments with the target sequence. Glass exhibited superior properties when compared to PDMS, with a higher sensitivity and improved stability of attachment during storage. PDMS modified surfaces were subject to hydrophobic recovery resulting in a re-arrangement of the surface, minimising oligonucleotides available on the surface over time for hybridisation.
Evaluation of the effect of surface chemistry used in attachment was also completed. Here, the amine functionalisation was compared to a thiolene attachment using a vinyl functionalised glass slide. The vinyl modification was able to effectively eliminate non-specific binding. Furthermore, the vinyl functionalised surface performed better with sensitivity and also demonstrated the potential for quantitative analysis required for future bloom monitoring when applied to natural seawater samples. Use of the vinyl functionalised glass slide allowed for the selective discrimination when hybridised with a non-complimentary target sequence. Vinyl functionalised surfaces were also able to be regenerated after one cycle, with only a loss of 0.7 % reactivity upon re-hybridisation.
In order to further improve sensitivities of the oligonucleotide modified surfaces, vinyl terminated nanoparticles were trialled as attachment substrates. Initial experiments using 1-dodecanethiol as a model reagent demonstrated the successful attachment using a thiolene photoinitiated reaction. Attachment densities were also calculated using ATR-FTIR with a 1000 molar excess addition of 1-dodecanethiol generating an attachment density of 10.6 ± 5.6 attachments.nm-2. Issues with the reproducibility resulted in high variation between analysed samples. Alternatively, TGA was trialled to calculate theoretical attachment densities resulting in 2.4 attachments.nm-2. Attachment of thiol modified oligonucleotides was also tested as a proof of concept, but was unsuccessful as shifting the reaction to an aqueous solution resulted in aggregation of the hydrophobic vinyl terminated nanoparticles. Investigation into alternative ethanol solutions resulted in false positive signals from blank nanoparticle aggregations formed upon drying.
In summary, the use of thiolene attached oligonucleotides to glass surfaces provides a highly sensitive, selective surface for the detection of A. catenella target sequences in model hybridisation experiments. Future application of these surfaces on natural seawater samples would be required to determine if it is effective in the presence of other nucleic acids. Future use of vinyl terminated nanoparticles for oligonucleotide attachment requires more optimisation to prevent aggregation in solution which inhibits the reaction.
Keywords: Harmful algal bloom, DNA, oligonucleotide, DNA biosensor, Alexandrium catenella
Subject: Chemistry thesis
Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Chemical and Physical Sciences
Supervisor: Claire Lenehan