| dc.description.abstract | DNA methylation naturally happens at the fifth carbon position of cytosine base within
the CpG dinucleotides, plays a significant role in the numerous biological events, such as
gene expression, cellular proliferation, embryonic developments, and chromosome
instability. Aberrancies in DNA methylation pattern can lead to the genomic instability,
resulting in the development of various human diseases including cancer, considered as
one of the promising epigenetic (diagnostic and prognostic) biomarkers. Current research
shows that abnormality in DNA methylation pattern presents a signature for disease
diagnosis, therapeutic interventions, and prognosis of outcome. Therefore, current DNA
methylation research has a major focus and needs for the development of easy, reliable
and sensitive detection strategies.
Throughout the last few decades, extensive research has been reported towards
the quantification of DNA methylation in the mammalian genome. However, having their
respective advantages in analytical performance and reliability, most of these strategies
are confined to laboratory-based molecular biology techniques, such as real-time
quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarrays,
and sequence-based methods. These methods largely require either bisulfite treatment
(BT) or specific restriction enzyme digestion followed by a subsequence DNA
amplification or sequencing. BT converts unmethylated cytosine to uracils leaving
methylated cytosine unchanged, leveraging the methylated fragments distinguishable for
detection. Low conversion, non-specific responses, false reading, longer assay time,
amplification biased and complex chemistries significantly reducing the practice of BT
and PCR amplification in current DNA methylation analysis. Hence, development of new
analytical techniques without the BT steps and PCR amplification could be a valuable
and out-of-bench tool for sensitive, low-cost and rapid platform to accomplish the
emerging demand in genome-wide DNA methylation analysis in clinics. In this regard,
bisulfite-free electrochemical biosensors have gained much attention in recent years as
electrochemistry offers sensitive, cost-effective, portable, and simple biomolecules
recognition readout. Parallel with electrochemistry, biosensors with optical readout
enable direct real-time detection of biological molecules and are easily adaptable to
multiplexing. Incorporation of electrochemical and optical readouts to bisulfite-free DNA
methylation analysis is paving the way for translation of this important biomarker to
standard patient care even resource-poor settings.
This PhD thesis explores various electrochemistry along with colorimetric
approach based sensitive, specific, rapid and inexpensive biosensor platform for bisulfitefree
global DNA methylation analysis. Moreover, a series of commercial and in-house
synthesised novel superparamagnetic nanomaterials were integrated to enhance the
sensitivity and portability of the detection platform. A comprehensive literature review
entailing detailed mechanism in DNA methylation pattern, association of DNA
methylation with various human diseases, progress in DNA methylation biosensors
techniques with a special emphasis on electrochemical and optical detection platforms
have been reported. The challenges associated with current strategies have been outlined,
and a great deal of recommendations also addressed to overcome the existing techniques.
In the following chapters, four novel DNA methylation analysis strategies have reported
based on colorimetry, electrochemistry and engineered nanostructures based inorganic
enzymes (nanozymes) for the sensitive, rapid, and inexpensive analysis of global DNA
methylation. First, based on the nucleic-acid affinity with the gold surface and
methylation site-specific 5-mC antibody conjugated with horseradish peroxidase (HRP,
natural enzyme), an amplification-free electrochemical and colorimetric assay was
developed for the detection of global DNA methylation. In this strategy, the methylationsites
to 5-mC antibody recognition were carried out on a screen-printed electrode surface
and HRP catalysed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation were employed to
read out the recognition event. The widely used colorimetry and ultra-sensitive
chronoamperometry (i-t) were used to readout the DNA methylation pattern. Subsequent
to the development of this proof-of-concept sensor, we replaced the commercial HRP
enzyme with an in-house specially designed mesoporous iron-oxide for reading the
methylation site recognition events in genomic DNA obtained from the oesophageal
cancer cell lines. In this DNA biosensor, both the BT and PCR amplification were avoided
to overcome the challenges associated with them and to achieve relatively simple and
rapid DNA methylation detection. Prior to integration of mesoporous iron-oxides into the
sensor platform, the peroxidase-like activity was explored and a colorimetric glucose
sensor was developed. This intrinsic property was then combined with DNA methylation
site-5-mC antibody immunocomplex and hence a sensitive colorimetric and
electrochemical readout were reported for global DNA methylations. In the following
chapter, a more sensitive assay was stated by utilising the coupling of enzymatic and
electrocatalytic strengths of another natural (glucose oxidase) enzyme. In this assay,
chronocoulometric (CC) charge measurement from redox mediator [Ru(NH3)6]3+ in the
presence of glucose subtract via glucose oxidase enzyme was used for DNA methylation analysis. In our final readout strategies, we extended our approach towards an inexpensive
and portable assay platform which enabled naked-eye, colorimetric and electrochemical
interrogation of in-house synthesised porous graphene oxide-loaded iron oxide (GOFe2O3)
via (TMB)/Nanozyme)-based colorimetric assay.
All the readout platforms reported herein have shown excellent analytical
performance with high sensitivity (limit of detection-LOD for global DNA methylation
5%) and specificity. The applicability of the assays was also demonstrated in different
cancer cell line samples with high reproducibility. We envisage that this research efforts
will contribute to the on-going development of simple, inexpensive and sensitive
technology for global DNA methylation analysis towards commercial aspect of both the
laboratory settings and resource-poor clinical settings. | |
