Mina Safarzadeh

Abstract

DNA methylation is associated with various diseases including cancer, cardiovascular diseases and neurodegenerative disorders, making it a potential biomarker to diagnose and prognose these diseases. Hence, a tool to detect and quantify DNA methylation can be valuable to improve clinical tests. In this thesis, we report two different biosensors for the detection and quantification of methylated tumour suppressor gene, O-6-methylguanine-DNA methyltransferase (MGMT), which is a potential biomarker for brain tumours and breast cancer. Both of the reported biosensors in this thesis are based on screen-printed reduced graphene oxide electrodes (rGO SPEs) and are electrochemical biosensors. In the Ab/ssDNA biosensor the rGO SPEs were first aminated by ammonium hydroxide chemisorption prior to their incubation in the anti-5-methylcytosine monoclonal antibody (anti-5mC) which acted as a methylation bioreceptor. After that, the target single-strand (ss) MGMT oligonucleotide is first recognised by its hybridisation with complementary DNA to form double-stranded (ds) MGMT, which is then captured by anti-5mC on the electrode surface due to the presence of methylation. The amination of the rGO electrodes were confirmed using XPS and Raman spectroscopy techniques. The assay development was tested via voltammetry techniques, namely CV and DPV. Furthermore, some of the preparation steps were optimized to achieve a better performance of the biosensor. Our validation studies showed that the peak current generated by the biosensor is proportional to the concentration of MGMT (both single stranded and previously hybridized double stranded) in the range of 50 fM to 500 pM, demonstrating the efficacy of the sensor. A selectivity experiment was also carried out in order to confirm that the biosensor is uniquely selective towards the methylated gene. Although, this biosensor is selective to the target biomarker when applied to previously hybridized dsDNA, it is not selective when detecting ssDNA because it responds to the methyl group regardless of the ssDNA sequence, so a hybridization step is needed to select the target ssDNA sequence from a mixture of strands. This drawback was the driving force behind the design and development of the second biosensor. To the best of our knowledge, this biosensor is the first report on the detection of MGMT genes, using rGO electrodes. To achieve the ability to directly detect dsDNA as a biomarker without denaturating it to ssDNA, the PNA/Ab biosensor was designed. This biosensor is a sandwich assay based on gold nanoparticles (AuNPs) decorated rGO electrodes to achieve high conductivity and allow self-assembly of nucleic acids. Peptide nucleic acid (PNA) was used to form a self-assembled monolayer (SAM) on the surface via amine-AuNPs interaction. PNA acts as a bio-recognition layer for the target ds-MGMT gene sequence, by invasion of the double strand and the formation of triple helix. The methylation was subsequently captured by biotinylated-anti-5mC which was then detected via amperometry. This results in a biosensor concept that is uniquely sensitive to both the target MGMT gene as well as its methylation. PNA has a high affinity to its complementary DNA relative to other natural nucleic acids and allows ds-DNA detection directly. Direct detection of double stranded DNA biomarkers removes the need for sample preparation (such as denaturation or hybridization) and makes the biosensor faster and easier to use. It may also result in a higher reliability of the biosensor due to the elimination of the risk of mistakes in the sample preparation step. To achieve this, the reduction of GO was performed in two ways: electrochemically (ErGO) and thermally (TrGO). XPS and Raman spectroscopy as well as voltammetry techniques showed that the ErGO was more efficiently reduced, had higher C/O ratio as well as smaller crystallite size of the sp$^2$ lattice and also was more stable during voltammetric measurements. It was also shown that the electro-deposition of AuNPs was more successful on the ErGO surface due to the higher At\% of AuNPs. SEM images and EDS spectra also confirmed the presence of AuNPs on the surface. Therefore, the ErGO/AuNPs electrode was used to fabricate biosensors and to detect the ds-MGMT gene. To the best of our knowledge, this is the first report on using PNA to detect methylated DNA and the first report on the detection of double stranded methylated DNA. Both of the above biosensor designs can be modified and tailor-made to detect other methylated genes, making them promising platforms to detect a variety of methylated biomarkers.

Keywords

Graphene based biosensors, DNA methylation detection, MGMT gene, Reduced Graphene Oxide

Document Type

Thesis

Publication Date

2023

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