Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads
by
, , , ,
Narshone Soda
1,2, 
Zennia Jean Gonzaga
3, 
Amandeep Singh Pannu
4,5,
Navid Kashaninejad
2
,
Richard Kline
6,
Carlos Salomon
7,8,9
,
Nam-Trung Nguyen
2
,
Prashant Sonar
4,5
,
Bernd H. A. Rehm
3,10,*,† and 
Muhammad J. A. Shiddiky
1,2,*,† 
1
School of Environment and Science (ESC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia
2
Queensland Micro- and Nanotechnology Centre (QMNC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia
3
Centre for Cell Factories and Biopolymers (CCFB), Griffith Institute for Drug Discovery (GRIDD), Griffith University, Nathan, QLD 4111, Australia
4
Centre for Material Science, School of Chemistry and Physics, Queensland University of Technology, Brisbane, QLD 4001, Australia
5
Centre for Biomedical Technologies, School of Chemistry and Physics, Queensland University of Technology (QUT), Brisbane, QLD 4001, Australia
6
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, LA 70121, USA
7
Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, LA 70121, USA
8
Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD 4029, Australia
9
Departamento de Investigación, Postgrado y Educación Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad Pedro de Valdivia, Santiago 8320000, Chile
10
Menzies Health Institute Queensland (MHIQ), Griffith University, Gold Coast Campus, Gold Coast, QLD 4222, Australia
*
Authors to whom correspondence should be addressed.
†
Equal Senior Authors.
Cancers 2021, 13(15), 3787; https://doi.org/10.3390/cancers13153787
Submission received: 31 May 2021
/
Revised: 19 July 2021
/
Accepted: 21 July 2021
/
Published: 27 July 2021
/
Corrected: 8 April 2024
Simple Summary
Genomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses.
Abstract
DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.
