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

Nanomaterials ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 985
Author(s):  
Mina Safarzadeh ◽  
Ahmed Suhail ◽  
Jagriti Sethi ◽  
Anas Sattar ◽  
David Jenkins ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Photoelectron Spectroscopy ◽  
Electrode Surface ◽  
Dna Methyltransferase ◽  
Limit Of Detection ◽  
Disease Diagnosis ◽  
Tumour Suppressor Gene ◽  
Efficient Production ◽  
Label Free ◽  
Potential Biomarker

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.

scholarly journals DNA Methylation in Circulating Tumour DNA as a Biomarker for Cancer

2007 ◽  
Vol 2 ◽  
pp. 117727190700200 ◽  
Author(s):  
Ruth E Board ◽  
Lucy Knight ◽  
Alastair Greystoke ◽  
Fiona H Blackhall ◽  
Andrew Hughes ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Primary Tumour ◽  
Predictive Biomarkers ◽  
Tumour Suppressor Gene ◽  
Tumour Suppressor ◽  
Tumour Suppressor Genes ◽  
Potential Biomarker ◽  
Frequent Event ◽  
Promotor Region ◽  
Circulating Tumour Dna

Free circulating DNA, which is thought to be derived from the primary tumour, can be detected in the blood of patients with cancer. Detection of genetic and epigenetic alteration in this tumour DNA offers a potential source of development of prognostic and predictive biomarkers for cancer. One such change is DNA methylation of the promotor region of tumour suppressor genes. This causes down regulation of tumour suppressor gene expression, a frequent event in carcinogenesis. Hypermethylation of the promotor region of a number of genes has been detected in many tumour types and more recently these changes have been detected in circulating tumour DNA. This review will summarise the literature detailing DNA methylation in circulating tumour DNA and discuss some of the current controversies and technical challenges facing its use as a potential biomarker for cancer.

scholarly journals Label-Free DNA Biosensor Using Modified Reduced Graphene Oxide Platform as a DNA Methylation Assay

Materials ◽  
2020 ◽  
Vol 13 (21) ◽  
pp. 4936 ◽  
Author(s):  
Eliska Sedlackova ◽  
Zuzana Bytesnikova ◽  
Eliska Birgusova ◽  
Pavel Svec ◽  
Amir M. Ashrafi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Graphene Oxide ◽  
Reduced Graphene Oxide ◽  
Limit Of Detection ◽  
Differential Pulse ◽  
Clinical Diagnostics ◽  
Label Free ◽  
Free Dna ◽  
Methylation Assay ◽  
Reduced Graphene

This work reports the use of modified reduced graphene oxide (rGO) as a platform for a label-free DNA-based electrochemical biosensor as a possible diagnostic tool for a DNA methylation assay. The biosensor sensitivity was enhanced by variously modified rGO. The rGO decorated with three nanoparticles (NPs)—gold (AuNPs), silver (AgNPs), and copper (CuNPs)—was implemented to increase the electrode surface area. Subsequently, the thiolated DNA probe (single-stranded DNA, ssDNA−1) was hybridized with the target DNA sequence (ssDNA-2). After the hybridization, the double-stranded DNA (dsDNA) was methylated by M.SssI methyltransferase (MTase) and then digested via a HpaII endonuclease specific site sequence of CpG (5′-CCGG-3′) islands. For monitoring the MTase activity, differential pulse voltammetry (DPV) was used, whereas the best results were obtained by rGO-AuNPs. This assay is rapid, cost-effective, sensitive, selective, highly specific, and displays a low limit of detection (LOD) of 0.06 U·mL−1. Lastly, this study was enriched with the real serum sample, where a 0.19 U·mL−1 LOD was achieved. Moreover, the developed biosensor offers excellent potential in future applications in clinical diagnostics, as this approach can be used in the design of other biosensors.

scholarly journals Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry

Biosensors ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 117
Author(s):  
Ivan Piovarci ◽  
Sopio Melikishvili ◽  
Marek Tatarko ◽  
Tibor Hianik ◽  
Michael Thompson
Keyword(s):  
Gold Nanoparticles ◽  
Protease Activity ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Enzymatic Reaction ◽  
Disease Diagnosis ◽  
Shear Mode ◽  
Label Free ◽  
Thickness Shear Mode ◽  
Thickness Shear

The determination of protease activity is very important for disease diagnosis, drug development, and quality and safety assurance for dairy products. Therefore, the development of low-cost and sensitive methods for assessing protease activity is crucial. We report two approaches for monitoring protease activity: in a volume and at surface, via colorimetric and acoustic wave-based biosensors operated in the thickness-shear mode (TSM), respectively. The TSM sensor was based on a β-casein substrate immobilized on a piezoelectric quartz crystal transducer. After an enzymatic reaction with trypsin, it cleaved the surface-bound β-casein, which increased the resonant frequency of the crystal. The limit of detection (LOD) was 0.48 ± 0.08 nM. A label-free colorimetric assay for trypsin detection has also been performed using β-casein and 6-mercaptohexanol (MCH) functionalized gold nanoparticles (AuNPs/MCH-β-casein). Due to the trypsin cleavage of β-casein, the gold nanoparticles lost shelter, and MCH increased the attractive force between the modified AuNPs. Consequently, AuNPs aggregated, and the red shift of the absorption spectra was observed. Spectrophotometric assay enabled an LOD of 0.42 ± 0.03 nM. The Michaelis–Menten constant, KM, for reverse enzyme reaction has also been estimated by both methods. This value for the colorimetric assay (0.56 ± 0.10 nM) is lower in comparison with those for the TSM sensor (0.92 ± 0.44 nM). This is likely due to the better access of the trypsin to the β-casein substrate at the surface of AuNPs in comparison with those at the TSM transducer.

scholarly journals Validation of DNA Methylation Biomarkers for Diagnosis of Acute Lymphoblastic Leukemia

2014 ◽  
Vol 60 (7) ◽  
pp. 995-1003 ◽  
Author(s):  
Zac Chatterton ◽  
Daniel Burke ◽  
Kerry R Emslie ◽  
Jeffery M Craig ◽  
Jane Ng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Limit Of Detection ◽  
Lymphoblastic Leukemia ◽  
False Negative ◽  
Disease Diagnosis ◽  
Patient Specific ◽  
Bone Marrow Biopsies ◽  
Maldi Tof

Abstract BACKGROUND DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. METHODS We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. RESULTS Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000–5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. CONCLUSIONS Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.

scholarly journals Fabrication of label-free electrochemical food biosensor for the sensitive detection of ovalbumin on nanocomposite-modified graphene electrode

2019 ◽  
Vol 9 (6) ◽  
pp. 4655-4662 ◽  
Keyword(s):  
Electrode Surface ◽  
Limit Of Detection ◽  
Pd Nanoparticles ◽  
High Electron ◽  
Label Free ◽  
Graphene Electrode ◽  
Electronic Current ◽  
Electrocatalytic Effect ◽  
Modified Graphene ◽  
Food Biosensor

In this work a highly sensitive label-free electrochemical food biosensor for the detection of egg allergen ovalbumin (Ova) was developed using nanocomposite consisting of iron oxide (Fe3O4) and palladium (Pd) nanoparticles with conducting polymer chitosan (CS). Pd nanoparticles were synthesized and seeded onto Fe3O4 nanoparticles, and dispersed in CS to form the nanocomposite. The nanocomposite (Fe3O4-Pd/CS) was drop-casted onto a screen printed graphene electrode (SPGE). Subsequently, 4-ABA was electrografted onto the modified SPGE/Fe3O4-Pd/CS. Carbodiimide chemistry was performed for the covalent conjugation of Ova-antibody (Ova-Ab) onto the electrode surface. The bimetallic nanocomposite portrayed excellent electrocatalytic effect due to the synergistic conductive effect of the Fe3O4-Pd nanomaterials and CS polymers and facilitated the electron movement between electrode surface and redox buffer. Additionally, ABA also enhanced electronic current on the surface of the electrode. Featuring high electron conductivity, the developed food biosensor demonstrated a large linear range to detect the Ova between 0.01 pg/mL – 1 µg/mL with a low limit of detection of 0.01 pg/mL. Furthermore, the proposed food biosensor portrayed very good selectivity with a high potential to detect Ova in real food sample.

scholarly journals Photonic Crystal Nanobeam Cavities for Nanoscale Optical Sensing: A Review

Micromachines ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 72 ◽  
Author(s):  
Da-Quan Yang ◽  
Bing Duan ◽  
Xiao Liu ◽  
Ai-Qiang Wang ◽  
Xiao-Gang Li ◽  
...  
Keyword(s):  
Photonic Crystal ◽  
Optical Waveguides ◽  
Optical Sensing ◽  
Early Stage ◽  
Disease Diagnosis ◽  
Label Free ◽  
Quality Factors ◽  
Integrated Sensor ◽  
Early Stage Disease ◽  
Wide Range

The ability to detect nanoscale objects is particular crucial for a wide range of applications, such as environmental protection, early-stage disease diagnosis and drug discovery. Photonic crystal nanobeam cavity (PCNC) sensors have attracted great attention due to high-quality factors and small-mode volumes (Q/V) and good on-chip integrability with optical waveguides/circuits. In this review, we focus on nanoscale optical sensing based on PCNC sensors, including ultrahigh figure of merit (FOM) sensing, single nanoparticle trapping, label-free molecule detection and an integrated sensor array for multiplexed sensing. We believe that the PCNC sensors featuring ultracompact footprint, high monolithic integration capability, fast response and ultrahigh sensitivity sensing ability, etc., will provide a promising platform for further developing lab-on-a-chip devices for biosensing and other functionalities.

scholarly journals Neuroblastoma and the epigenome

2021 ◽  
Author(s):  
Irfete S. Fetahu ◽  
Sabine Taschner-Mandl
Keyword(s):  
Dna Methylation ◽  
Histone Modifications ◽  
Disease Diagnosis ◽  
Dna Methyltransferases ◽  
Epigenetic Alterations ◽  
Aberrant Expression ◽  
Tet Proteins ◽  
Relative Paucity ◽  
Underlying Causes ◽  
Aberrant Dna Methylation

AbstractNeuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system and one of the most common solid tumors in infancy. Amplification of MYCN, copy number alterations, numerical and segmental chromosomal aberrations, mutations, and rearrangements on a handful of genes, such as ALK, ATRX, TP53, RAS/MAPK pathway genes, and TERT, are attributed as underlying causes that give rise to NB. However, the heterogeneous nature of the disease—along with the relative paucity of recurrent somatic mutations—reinforces the need to understand the interplay of genetic factors and epigenetic alterations in the context of NB. Epigenetic mechanisms tightly control gene expression, embryogenesis, imprinting, chromosomal stability, and tumorigenesis, thereby playing a pivotal role in physio- and pathological settings. The main epigenetic alterations include aberrant DNA methylation, disrupted patterns of posttranslational histone modifications, alterations in chromatin composition and/or architecture, and aberrant expression of non-coding RNAs. DNA methylation and demethylation are mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, respectively, while histone modifications are coordinated by histone acetyltransferases and deacetylases (HATs, HDACs), and histone methyltransferases and demethylases (HMTs, HDMs). This article focuses predominately on the crosstalk between the epigenome and NB, and the implications it has on disease diagnosis and treatment.

DNA methyltransferase inhibitors modulate histone methylation: epigenetic crosstalk between H3K4me3 and DNA methylation during sperm differentiation

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Liliana Burlibaşa ◽  
Alina-Teodora Nicu ◽  
Carmen Domnariu
Keyword(s):  
Dna Methylation ◽  
Histone Methylation ◽  
Dna Methyltransferase ◽  
Integrated Approach ◽  
Regulatory Mechanisms ◽  
Temporal Expression ◽  
Dna Methyltransferase Inhibitors ◽  
Expression Of Genes ◽  
Species Survival ◽  
Advanced Stages

Summary The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate ‘tuning’ of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial–temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

scholarly journals Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Donggee Rho ◽  
Seunghyun Kim
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Optical Cavity ◽  
Sample Volume ◽  
Small Sample ◽  
Label Free ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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