A Label-Free DNA-Immunosensor Based on Aminated rGO Electrode for the Quantification of DNA Methylation

Abstract

In this work, we developed a sandwich DNA-immunosensor for quantification of the methylated tumour suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT), which is a potential biomarker for brain tumours and breast cancer. The biosensor is based on aminated reduced graphene oxide electrode, which is achieved by ammonium hydroxide chemisorption and anti-5-methylcytosine (anti-5mC) as a methylation bioreceptor. 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. Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Scanning electron microscopy (SEM) techniques were used to characterise the electrode surface. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used for electrochemical measurements. Under optimised conditions, the proposed biosensor is able to quantify a linear range of concentrations of the MGMT gene from 50 fM to 100 pM with a limit of detection (LOD) of 12 fM. The sandwich design facilitates the simultaneous recognition and quantification of DNA methylation, and the amination significantly improves the sensitivity of the biosensor. This biosensor is label-, bisulfite- and PCR-free and has a simple design for cost-efficient production. It can also be tailor-made to detect other methylated genes, which makes it a promising detection platform for DNA methylation-related disease diagnosis and prognosis.

An innovative fluorometric bioanalysis strategy towards recognition of DNA methylation using opto‐active polymer: A new platform for DNA damage studies by genosensor technology

“…50 Also, a label-free and immune-based electrochemical biosensor was applied to detect methylation with a detection limit of 12 fM. 51 According to the results of the above-mentioned works, the low limit of quantification (LLOQ) genosensor in the current study shows the high sensitivity and efficiency designed genosensor (Table 1).…”

Molecularly Imprinted Electrochemical Sensor Based on the Self-Assembly of Cage-like Gold Nanoparticles and Amino-Functionalized rGO for the Detection of Diuron in Cotton Defoliant

“…The peaks at 289.3, 286.6, 285.1, and 284.6 eV were attributed to O–CO, C–O, C–N, and C–C, respectively (Figure Ga), manifesting many oxygen-rich groups on the NH 2 -rGO@AuNCs surface . It can be seen that the N 1s spectra (Figure Gb) are divided into two peaks of N–H at 401.3 eV and C–N–C at 399.8 eV, indicating the successful introduction of amino groups . In addition, two characteristic peaks of Au 4f were located at 87.7 and 84.1 eV, which were assigned to Au 4f 5/2 and Au 4f 7/2 , respectively (Figure Gc).…”

“…31 It can be seen that the N 1s spectra (Figure 1Gb) are divided into two peaks of N−H at 401.3 eV and C−N−C at 399.8 eV, indicating the successful introduction of amino groups. 32 In addition, two characteristic peaks of Au 4f were located at 87.7 and 84.1 eV, which were assigned to Au 4f 5/2 and Au 4f 7/2 , respectively (Figure 1Gc). The XRD patterns of rGO, NH 2 -rGO, AuNCs, and NH 2 -rGO@AuNCs were investigated.…”

Detection of a Double-Stranded MGMT Gene Using Electrochemically Reduced Graphene Oxide (ErGO) Electrodes Decorated with AuNPs and Peptide Nucleic Acids (PNA)

“…The peaks at around 1570 cm −1 are G bands which are attributed to in-plane vibrations of the sp 2 -bonded graphitic carbon atoms. The peaks at 1350 cm −1 are D bands and represent the out-of-plane vibration of the disordered structures [ 36 ]. I D /I G ratio, or the intensity ratio, is normally used to evaluate disorder level, or the ratio of structural defects in the GO or rGO layers.…”