RESUMO
A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.
Assuntos
Técnicas Biossensoriais , Sondas de DNA , Técnicas de Amplificação de Ácido Nucleico , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , DNA Viral/análise , DNA Viral/genética , DNA/química , DNA/genética , Espectrometria de Fluorescência/métodos , Fluorescência , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , Limite de Detecção , HIV/genéticaRESUMO
Cancer detection is still a major challenge in public health. Identification of oncogene is the first step toward solving this problem. Studies have revealed that various cancers are associated with miRNA expression. Therefore, the sensitive detection of miRNA is substantially important to solve the cancer problem. In this study, let-7a, a representative substance of miRNA, was selected as the detection target. With the assistance of magnetic beads commonly used in biosensors and self-synthesized graphene oxide materials, specificity and sensitivity detection of the target gene let-7a were achieved via protease-free signal amplification. The limit of detection (LOD) was as low as 15.015pM. The fluorescence signal intensity showed a good linear relationship with the logarithm of let-7a concentration. The biosensor could also detect let-7a in complex human serum samples. Overall, this fluorescent biosensor is not only simple to operate, but also strongly specificity to detect let-7a. Therefore, it has substantial potential for application in the early diagnosis of clinical medicine and biological research.