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目的:建立一种基于纳米粒子的时间分辨(TR)荧光共振能量转移(FRET)生物传感器用于mi RNAs的无扩增检测。方法:以寡核苷酸单链probe I偶联Gd F3:Tb3+纳米粒子作为供体,以寡核苷酸单链probe II偶联金(Au)纳米粒子作为受体,利用供体与受体之间的FRET检测目标mi RNA hsa-mi R-122-5p浓度,并通过设置多功能酶标仪的检测延迟去除自发荧光背景以提高灵敏度。结果:该传感器能够高灵敏度、高特异性地检测目标分子,并且对于光照有良好的耐受性,而且能够避免自发荧光干扰。对hsa-mi R-122-5p的检测线性范围为0.1 fmol/L~100 pmol/L,与同类型的RNA荧光传感器的检测限具有可比性。结论:基于纳米粒子的TR-FRET生物传感器用于mi RNAs的无扩增检测具有良好的灵敏度和特异性。
OBJECTIVE: To establish a nanoparticle-based time-resolved (TR) fluorescence resonance energy transfer (FRET) biosensor for the detection of mi RNAs without amplification. Methods: Gd F3: Tb3 + nanoparticles were coupled with oligonucleotide single-stranded probe I as donor and oligonucleotide single-stranded probe II conjugated gold (Au) nanoparticles as acceptor. FRET was used to detect the mi RNA hsa-mi R-122-5p concentration and the autofluorescence background was removed by detection of a multifunctional microplate reader to increase sensitivity. Results: The sensor can detect target molecules with high sensitivity and high specificity, and is well tolerated by light and can avoid autofluorescence interference. The linear range of detection of hsa-mi R-122-5p was 0.1 fmol / L ~ 100 pmol / L, which was comparable to the detection limit of the same type of RNA fluorescence sensor. Conclusion: The nanoparticle-based TR-FRET biosensor has good sensitivity and specificity for non-amplification detection of mi RNAs.