Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen 518055, China.
Anal Chem. 2021 Jun 15;93(23):8128-8133. doi: 10.1021/acs.analchem.1c01594. Epub 2021 May 28.
The optical background such as autofluorescence and light scattering poses a big challenge to quantify nucleic acids with conventional fluorescence-based methods. We report here high-contrast nucleic acid detection with photoswitch-mediated fluorescence resonance energy transfer (FRET), which strongly occurs between the open forms of the photoswitch (a naphthopyran) and the signal fluorophores brought to the surface of the nanoprobes (≲15 nm). The fluorescence change (Δ) upon UV irradiation is highly sensitive and more robust to quantify the target DNAs than traditional intensity measurements. Therefore, the method works in samples with strong background fluorescence from the unbound fluorophores. The photoswitchable nanoprobes could be easily prepared and interrogated in capillaries for high-throughput measurements. The method was evaluated in both sandwich-like hybridization and DNA label-free detection with a nucleic stain SG. Without DNA amplification and sample pretreatment of blood serum, the photoswitchable nanoprobes provided a limit of detection of 0.5 nM, which is ∼6 to 20 times lower than conventional FRET.
传统的荧光方法受自发荧光和光散射等背景光的影响，难以对核酸进行定量分析。本研究报告了一种利用光开关介导的荧光共振能量转移（FRET）实现高对比度核酸检测的方法，该方法在光开关（萘并吡喃）的开环形式和被带到纳米探针表面的信号荧光团（≲15nm）之间强烈发生。与传统的强度测量相比，紫外光照射下的荧光变化（Δ）对定量目标 DNA 更敏感、更稳健。因此，该方法适用于结合荧光团的背景荧光较强的样品。这种可光开关的纳米探针可在毛细管中轻松制备和检测，用于高通量测量。该方法在夹心式杂交和核酸染色剂 SG 的无 DNA 标记检测中进行了评估。无需对血清样本进行 DNA 扩增和预处理，该可光开关的纳米探针的检测限为 0.5nM，比传统的 FRET 低约 6 至 20 倍。
