A disposable electrochemical magnetic immunosensor for the rapid and sensitive detection of 5-formylcytosine and 5-carboxylcytosine in DNA.
Biosens Bioelectron
; 262: 116547, 2024 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-38968775
ABSTRACT
5-formylcytosine (5 fC) and 5-carboxylcytosine (5caC) serve as key intermediates in DNA demethylation process with significant implications for gene regulation and disease progression. In this study, we introduce a novel electrochemical sensing platform specifically designed for the sensitive and selective detection of 5 fC and 5caC in DNA. Protein A-modified magnetic beads (ProtA-MBs) coupled with specific antibodies facilitate the immunorecognition and enrichment of these modified bases. Signal amplification is achieved through several chemical reactions involving the interaction between N3-kethonaxl and guanine, copper-free click chemistry for the attachment of dibenzocyclooctyne (DBCO)-Biotin, and the subsequent recognition by streptavidin-conjugated horseradish peroxidase (SA-HRP). The assay's readout is performed on a disposable laser-induced graphene (LIG) electrode, modified with the bead-antibody-DNA complex in a magnetic field, and analyzed using differential pulse voltammetry in a system employing hydroquinone (HQ) as the redox mediator and H2O2 as the substrate. This immunosensor displayed excellent sensitivity, with detection limits of 14.8 fM for 5 fC across a 0.1-1000 pM linear range and 87.4 fM for 5caC across a 0.5-5000 pM linear range, and maintained high selectivity even in the presence of interferences from other DNA modifications. Successful application in quantifying 5 fC and 5caC in genomic DNA from cell extracts, with recovery rates between 97.7% to 102.9%, underscores its potential for clinical diagnostics. N3-kethoxal was used for the first time in an electrochemical sensor. This work not only broadens the toolkit for detecting DNA modifications but also provides a fresh impetus for the development of point-of-care testing (POCT) technologies.
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
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Técnicas Biossensoriais
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Citosina
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Técnicas Eletroquímicas
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Limite de Detecção
Limite:
Humans
Idioma:
En
Revista:
Biosens Bioelectron
Ano de publicação:
2024
Tipo de documento:
Article