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Biosensors (Basel) ; 12(2)2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-35200358


The ability to detect double-stranded DNA (dsDNA) as a biomarker without denaturing it to single-stranded DNA (ss-DNA) continues to be a major challenge. In this work, we report a sandwich biosensor for the detection of the ds-methylated MGMT gene, a potential biomarker for brain tumors and breast cancer. The purpose of this biosensor is to achieve simultaneous recognition of the gene sequence, as well as the presence of methylation. The biosensor is based on reduced graphene oxide (rGO) electrodes decorated with gold nanoparticles (AuNPs) and uses Peptide Nucleic Acid (PNA) that binds to the ds-MGMT gene. 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 a higher C/O ratio, showed a smaller crystallite size of the sp2 lattice, and was more stable during measurement. It was also revealed that the electro-deposition of the AuNPs was more successful on the ErGO surface due to the higher At% of Au on the ErGO electrode. Therefore, the ErGO/AuNPs electrode was used to develop biosensors to detect the ds-MGMT gene. PNA, which acts as a bio-recognition element, was used to form a self-assembled monolayer (SAM) on the ErGO/AuNPs surface via the amine-AuNPs interaction, recognizing the ds-MGMT gene sequence by its invasion of the double-stranded DNA and the formation of a triple helix. The methylation was then detected using biotinylated-anti-5mC, which was then measured using the amperometric technique. The selectivity study showed that the proposed biosensor was able to distinguish between blank, non-methylated, non-complementary, and target dsDNA spiked in mouse plasma. The LOD was calculated to be 0.86 pM with a wide linear range of 1 pM to 50 µM. To the best of our knowledge, this is the first report on using PNA to detect ds-methylated DNA. This sandwich design can be modified to detect other methylated genes, making it a promising platform to detect ds-methylated biomarkers.

Técnicas Biossensoriais , Grafite , Nanopartículas Metálicas , Ácidos Nucleicos Peptídicos , Animais , Biomarcadores , Técnicas Biossensoriais/métodos , DNA/química , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro , Grafite/química , Nanopartículas Metálicas/química , Camundongos
Nanomaterials (Basel) ; 11(4)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921234


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.

Mikrochim Acta ; 187(6): 338, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32430539


The published version of this article, unfortunately, contains errors. Corrections in references were incorrectly carried out. Also, the reduction of graphene oxide was carried out between the potential of -1.5 and 0.5 V, instead of 0.5 and 1.5 V.

Mikrochim Acta ; 187(5): 288, 2020 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-32333119


A label-free biosensor is developed for the determination of plasma-based Aß1-42 biomarker in Alzheimer's disease (AD). The platform is based on highly conductive dual-layer of graphene and electrochemically reduced graphene oxide (rGO). The modification of dual-layer with 1-pyrenebutyric acid N-hydroxysuccinimide ester (Pyr-NHS) is achieved to facilitate immobilization of H31L21 antibody. The effect of these modifications were studied with morphological, spectral and electrochemical techniques. The response of the biosensor was evaluated using differential pulse voltammetry (DPV). The data was acquired at a working potential of ~ 180 mV and a scan rate of 50 mV s-1. A low limit of detection (LOD) of 2.398 pM is achieved over a wide linear range from 11 pM to 55 nM. The biosensor exhibits excellent specificity over Aß1-40 and ApoE ε4 interfering species. Thus, it provides a viable tool for electrochemical determination of Aß1-42. Spiked human and mice plasmas were used for the successful validation of the sensing platform in bio-fluidic samples. The results obtained from mice plasma analysis concurred with the immunohistochemistry (IHC) and magnetic resonance imaging (MRI) data obtained from brain analysis. Graphical abstract Schematic representation of the electrochemical system proposed for Aß1-42 determination: (a) modification of graphene screen-printed electrode (SPE) with monolayer graphene oxide (GO) followed by its electrochemical reduction generating graphene/reduced graphene oxide (rGO) dual-layer (b), modification of dual-layer with linker (c), Aß1-42 antibody (H31L21) (d), bovine serum albumin (BSA) (e) and Aß1-42 peptide (f).

Peptídeos beta-Amiloides/sangue , Técnicas Biossensoriais , Técnicas Eletroquímicas , Grafite/química , Fragmentos de Peptídeos/sangue , Animais , Biomarcadores/sangue , Humanos , Camundongos , Estrutura Molecular , Oxirredução
Biomed Mater Eng ; 28(5): 489-501, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28854491


BACKGROUND: Polytetrafluoroethylene (PTFE) is poorly biocompatible due to its low surface energy and hydrophobicity, which cause weak cell attachment and proliferation and complicate its use in implants. OBJECTIVE: NH3 plasma was used for surface modification and binding of amine groups on the PTFE surface. Collagen was immobilized on the plasma-treated PTFE in order to enable it to support enhanced cell adhesion and growth. METHODS: PTFE was exposed to NH3 plasma and collagen was immobilized on the NH3 plasma-treated surface. ATR-IR, SEM, EDXA and contact angle were conducted to determine the composition, microstructure and wettability of samples. The cytocompatibility of the samples was assessed via the growth HUVEC cells using MTT assay. RESULTS: Plasma treatment resulted in an incorporation of functional groups, containing N2 and O2 that caused the PTFE surface to become hydrophilic with contact angle 68°. Also, a reduction in F/C ratio was observed after collagen immobilization that indicates the presence of collagen. Cells proliferated in greater numbers on the collagen immobilized-PTFE as compared to the plasma-treated one. CONCLUSIONS: Plasma treatment incorporates functional polar moieties on the PTFE surface, causing enhanced wettability, collagen immobilization and cell viability. Collagen-immobilized PTFE may offer a valuable solution in biomedical applications such as vessel grafts.

Materiais Biocompatíveis/química , Adesão Celular , Células Imobilizadas/citologia , Colágeno/química , Células Endoteliais da Veia Umbilical Humana/citologia , Politetrafluoretileno/química , Células Cultivadas , Humanos , Gases em Plasma , Propriedades de Superfície
Glob J Health Sci ; 7(5): 91-5, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26156909


Geographic tongue is a benign lesion at the dorsum and margins of the tongue that sometimes causes pain and burning sensation. This lesion is characterized by an erythematous area with white or yellow folded edges. The predisposing factors of this lesion include heredity, allergies, psoriasis, stress, fissured tongue and consumption of some foods. The present study was conducted to investigate the prevalence of geographic tongue and its related factors among the 7-18 year-old students in Kermanshah, Iran. This descriptive cross-sectional study was carried out in three schools in Kermanshah using multi-stage random cluster sampling method. A total number of 3600 students were examined (1800 girls and 1800 boys). Demographic data and the results of examinations were recorded in a questionnaire. The factors affecting the incidence of geographic tongue were analyzed by the SPSS-20 software and the Chi-square test.The prevalence of geographic tongue was 7.86% (283 individuals). The incidence of this lesion was significantly higher in males than in females (p<0.01). There was no relationship between geographic tongue and psoriasis or fissured tongue.  Pain and discomfort during eating was more prevalent in those with geographic tounge compared to those without this condition (p<0.02). The prevalence of geographic tongue among the studied population was 7.86%, and the prevalence of geographic tongue in male students was higher than in female students.

Glossite Migratória Benigna/epidemiologia , Adolescente , Causalidade , Criança , Estudos Transversais , Feminino , Glossite Migratória Benigna/complicações , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Dor/etiologia , Prevalência , Psoríase/complicações , Distribuição por Sexo , Inquéritos e Questionários