RESUMEN
MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg2+-dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg2+, trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis.
Asunto(s)
Técnicas Biosensibles/métodos , ADN Catalítico/química , ADN/química , Fluorescencia , MicroARNs/análisis , División del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Límite de Detección , Magnesio/química , Neoplasias/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y EspecificidadRESUMEN
Cobalt (II) polyamidomine dendrimer was prepared by the reaction of cobalt chloride, glyoxal and polyamidomine dendrimer of 5.0 generation. The interaction of cobalt (II) polyamidomine dendrimer complex with herring sperm (hsDNA) was carried out using methylene blue (MB) as the probe molecule by absorption and fluorescence spectroscopy and synchronous fluorescence spectroscopy. The results showed that the intensity of absorption peaks and fluorescence peaks increased when the complex interacted with hsDNA. The effect of sodium chloride showed that sodium ion can significantly constrain the interaction of cobalt(II) polyamidomine dendrimer with hsDNA. The curves indicated the competitive inhibition of MB binding to hsDNA in the presence of cobalt (II) polyamidomine dendrimer complexes, also MB could insert into interior of cobalt (II) polyamidomine dendrimer complexes. The results suggested that the complex mainly interacted with negatively charged phosphate moieties on hsDNA through electrostatic attraction and stacked on the surface of double stranded hsDNA, which may reduce the binding affinity of MB to hsDNA in the surrounding site. It was indicated that sodium ion might neutralize the negatively charged phosphate backbone of hsDNA, and then weaken the electrostatic attraction between complexes and hsDNA.