功能性DNA双亲性嵌段聚合物的合成与应用

Synthesis and Application of Functional DNA Amphiphilic Block Polymer

李盼

11649103

硕士

材料物理与化学

田雷蕾

2018-06-01

2018-07-04

哈尔滨工业大学

深圳

聚合诱导发光（AIE）效应是一种全新的光物理现象，引起了材料学领域巨大的研究兴趣。这类荧光材料在单分散状态下不显示荧光发射，而在聚集态的情况下显示强荧光，基于这种 AIE 特性的荧光探针在生物传感和成像应用中表现出低信噪比和高灵敏度的特点。一般来说，AIE 效应的产生在于有效地限制 AIE 分子的分子内运动，这可以通过产生强分子间相互作用或聚集来实现。DNA 是实现 AIE调控的最佳选择之一：（1）AIE 可由 DNA 杂交控制。AIE 分子可插层在碱基对中，其分子运动受到强分子间相互作用的限制。在此机制的基础上，已经发展了若干的 DNA 探针来检测 DNA 杂交以及识别核苷酸突变。（2）除上述效应外，如果将疏水的 AIE 分子和 DNA 进行共价接枝，亲水的 DNA 部分将决定 AIE-DNA 结合物的水溶性。众所周知，DNA 可以响应各种外界刺激完成精确的构象变化，包括序列折叠、变性和酶切，进而调节 AIE-DNA 双亲分子的溶解度，最终实现可控的AIE 效应。这一策略将大大拓宽 AIE 探针设计的思路和应用范围。 本研究中，我们首先设计出具有 i-motif 结构的 DNA 片段，通过 DNA 合成仪合成与修饰，得到 5'端炔基修饰的 DNA-alkynyl 片段，用固相合成的方法将具有AIE 效应的叠氮基四苯基乙烯分子（Tetraphenylethylene 简称 TPE）分子通过 Click反应与 DNA-alkynyl 成功接枝，得到功能性 DNA 嵌段分子 TPE-DNA，可作为对pH 响应灵敏的新型荧光探针。我们还深入研究了该荧光探针在溶液中的 pH 响应范围，及其在活细胞层面上的传感应用，从而得出如下结论：（1）DNA 嵌段在不同 pH 值情况下构型的改变，可驱动作为疏水片段的 TPE分子的聚集和解聚集，因此可通过 AIE 效应产生的荧光强度变化指示 pH 的变化。通过 TPE-DNA 分子在不同 pH 值下荧光强弱的对比，得出 TPE-DNA 的 pH 有效响应范围为 4.0-7.0； （2）在不同 pH 值下 TPE-DNA 分子的聚集和分散过程完全可逆，且响应迅速，适于活细胞内生理活动的实时监测； （3）TPE-DNA 分子表现出了良好的生物相容性，较强的抗干扰能力和较高的 pH 检测灵敏性，可以用于活细胞内 pH 值的检测。

Representing a new and exciting photophysical phenomenon, aggregation-induced emission (AIE) effect has attracted enormous research interests. Especially, on account of its unique features, such as non-emission in the isolated state and light-up/turn-on emission in the aggregated state, AIE-based probe exhibited low signal-to-noise ratio and high sensitivity in the application of bio-sensing and imaging, showing clear superiority over many other fluorescent probes. During a sensing process, the recognition moiety of an AIE-based probe should respond to the presence of targets, and subsequently trigger the AIE effect and lead to turn-on fluorescence. Generally, the generation of an AIE effect lies in the efficient restriction of the intramolecular motion of AIE molecules, which can be achieved by either the formation of strong intermolecular interactions or the aggregation as a result of solubility change. For this purpose, DNA is one of the best choice for the construction of AIE-based probe by acting as a recognition moiety. (1) AIE can be controlled by DNA hybridization. AIE molecules stack onto the base-pairs, as a result, whose molecular motions are restricted by forming strong intermolecular interactions. On the basis of this mechanism, a couple of studies have developed AIE-based DNA probe to sense DNA hybridization as well as to recognize nucleotide mutation. (2) Beyond this effect, if a conjugation between a hydrophobic AIE moiety and DNA is synthesized, the hydrophilic DNA part will determine the water-solubility of the AIE-DNA conjugate. It is well-known that DNA can exactly and precisely undergo a conformation change in response to various external stimuli. Many conformational changes, including sequence folding, denature, and enzymatically cutting, probably can regulate the solubility of the AIE-DNA conjugate, and finally control the AIE effect. This strategy will greatly broaden the application field to the detection of pH, temperature, small molecules, and bio-macromolecules. In this thesis, a DNA fragment with an i-motif forming sequencewas conjugated to tetraphenylethylene (TPE) through a Click reaction carried on a DNA synthesizer. The final product, TPE-DNA showed sensitive response to pH variations and turn-on fluorescence in an acid environment. (1) In principle, the hydrophilic DNA fragment determines the solubility of TPE-DNA; at basic condition, the TPE-DNA is well dispersed in water as its DNA fragment shows an extended conformation; on the other hand, at acid condition, the DNA part will folded to form a i-motif structure, which reduces the solubility of TPE-DNA and drives the aggregation of the TPE part. Therefore we observed that TPE-DNA showed moreand more significant AIE effect when the pH value decreased, which exhibited aneffective pH response range of 4.0-7.0.(2) TPE-DNA showed turn-on fluorescence in an acidic condition and more sensitive to pH variations, which is different from DNA molecular beacon. More importantly, TPE-DNA showed reversibly respond to pH switches from 7.0 to 5.0, which demonstrated its good reliability.(3) According to MTT experiment, TPE-DNA showed low cytotocycity; and its response to pH change will not be interfered by the presence of metal ions. Finally, TPE-DNA has been applied to the detection of pH variation in live cells.

AIE 效应 pH 响应性 i-motif 结构 荧光探针 细胞成像

AIE effect, pH responsiveness i-motif structure fluorescent probes cell imagin

中文

联合培养

南方科技大学

李盼. 功能性DNA双亲性嵌段聚合物的合成与应用[D]. 深圳. 哈尔滨工业大学,2018.