基于DNA基质流体室温磷光原理的显色分析

Due to the longer lifetime over fluorescence, phosphorescence analysis attracted great attention in spectral analysis, since the short-lived background fluorescence can be eliminated through delay. Since the pioneering work reported by Paynter et al. using filter paper as substrate for inducing room-temperature phosphorescence (RTP), remarked progress has been made in RTP analysis. However, problems still exist for RTP analysis. First, in current RTP analysis, the phosphorescence is mainly from the analyte itself. For non-phosphorescent analytes, RTP analysis may be not applicable. Besides, the phosphorescence peak from the analyte may overlap with those from other interferents, which decrease the selectivity. Second, the phosphorescence intensity is much weaker than the fluorescence. Moreover, phosphorescence is much easier to be inactivated. Therefore, the sensitivity of RTP analysis is not satisfied. In this proposal, we proposed the employment of double strand DNA (dsDNA) as a new substrate for inducing fluid room-temperature phosphorescence. Due to the rigid structure of the dsDNA helix, the non-radiative pathways (rotation and vibration) of DNA-binding dyes may be blocked, leading to the activation of the triplet state of these dyes and thus phosphorescence (after de-oxygenation). Such activation can be selective due to the selective recognition of DNA. Meanwhile, the energy of the excited triplet state can be easily transferred to oxygen, leading to the generation of singlet oxygen. Singlet oxygen can directly oxidize chromogenic substrates to give color signal. Therefore, the weak phosphorescence signal can be amplified to color signal via the singlet oxygen bridge. In combination with DNA amplification techniques with dsDNA as products, the proposed RTP analysis can be derivatized into ultrasensitive DNA analytical methods for disease gene detection. In summary, this proposal devote to reform the classical RTP analysis.

由于磷光寿命较荧光更长，可通过设置延迟时间消除短寿命背景荧光的干扰，因而在光谱分析中备受关注。自1974年Paynter等人报道了利用滤纸作基质的诱导室温磷光以来，室温磷光分析技术得到了长足的发展。然而，室温磷光分析还存在以下问题：首先，现有的室温磷光信号大都源自于分析物本身，因而难以分析非磷光物质，且易受到谱峰宽度及其它潜在磷光发射物质的影响，其选择性受限；其次，相较于荧光，磷光信号本来就弱，且更容易失活，因而分析灵敏度受限。本项目拟将双链DNA作为一种新型流体室温磷光基质，以其刚性双螺旋结构诱导DNA结合染料的室温磷光，并通过DNA的特异性识别作用赋予其选择性；同时，根据磷光与单线态氧之间的关联，将弱的室温磷光信号通过单线态氧的桥梁作用转变为显色信号予以放大。结合DNA扩增技术，发展高灵敏显色分析用于疾病病原体基因的检测。在此基础上，实现传统室温磷光分析的革新。

由于磷光寿命较荧光更长，可通过设置延迟时间消除短寿命背景荧光的干扰，因而在光谱分析中备受关注。针对于传统磷光分析的选择性较低和分析灵敏度受限等问题，本项目提出将双链DNA作为一种新型流体室温磷光基质，以其刚性双螺旋结构诱导DNA结合染料的室温磷光，并通过DNA的识别作用赋予其选择性；同时，根据磷光与单线态氧之间的关联，将弱的室温磷光信号通过单线态氧的桥梁作用转变为显色信号予以放大。首先，研究了部分非标记核酸染料与DNA结合之后的激发三线态性质；通过EPR和光敏显色等方式考察了该过程中单线态氧的产生；并在此基础上，将所发展的光敏显色分析与核酸扩增结合，发展了系列新型基于室温磷光原理的高灵敏生物分析方法。其次，评价了部分显色底物（TMB、DAB等）在光敏显色中的性能，筛选了最适宜的显色底物。最后，发现了部分带正电荷的非标记核酸染料（SYBR系列染料和ThT）诱导金纳米（AuNPs）高效聚集的现象，并据此发展了若干高效比色分析方法。本项目共发表SCI论文17篇，包括CCS Chem 1篇、J Phys Chem Lett 1篇、Anal Chem 7篇等。