A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.
Mikrochim Acta
; 191(7): 405, 2024 06 19.
Article
em En
| MEDLINE
| ID: mdl-38896292
ABSTRACT
CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cobre
/
MicroRNAs
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Nanopartículas Metálicas
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Limite de Detecção
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Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
Mikrochim Acta
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China