水溶性壳聚糖基聚集诱导发光荧光探针在生物检测领域的应用

发布时间：2018-03-12 16:13

  本文选题：聚集诱导发光　切入点：壳聚糖　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：荧光探针或化学分子荧光传感器是一类能够选择性结合特定物质的分子体系,通过紫外吸收、荧光发射光谱或氧化还原电位等变化来实现对靶向分子定性或定量检测,具有操作简便、灵敏度高等优点,被广泛应用于生命科学、环境科学等领域。然而,传统的荧光分子往往存在不可忽视的缺点,如随浓度变化的stokes位移导致荧光强度的非线性变化、高浓度或聚集时发生荧光淬灭现象等,大大限制了其应用范围。而具有聚集诱导发光(AIE)特性的荧光分子针对性地解决了这一难题,并因其敏感度高、光谱随浓度变化无漂移等优异特性使之在荧光检测各领域得到极大应用。在具有AIE特性荧光探针被广泛探究的同时,水溶性AIE探针的需求日益迫切。壳聚糖富含氨基、羧基,是自然界仅次于纤维素的第二大天然可再生高分子材料,具有生物可降解性、生物相容性、易与金属离子络合等优良性能,在生物医药、环境检测等领域具有广泛应用。本论文将四苯基乙烯(TPE)荧光分子标记在壳聚糖及其衍生物分子链上,制得具有AIE特性的壳聚糖基荧光分子,继而拓展其在生物、环境监测领域中的应用。主要研究成果如下:1)采用均相反应法在乙酸/甲醇溶剂体系中对壳聚糖进行琥珀酰化改性,继而标记荧光分子,成功制备TPE-NSCS,并以之为检测探针应用于细胞长周期示踪。1HNMR、荧光光谱(FL)、表面Zeta电位、TEM等测试结果表明成功制备表面负电性的水溶性佳、稳定性好的AIE探针TPE-NSCS;用已制得的TPE-NSCS对宫颈癌细胞(HeLa)进行染色,MTT结果证明其无细胞毒性,荧光显微镜照片及共聚焦显微镜(CLSM)图片表明随着染色时间增加,探针成功进入细胞内部,成像效果良好,且可长周期示踪至30代。2)利用2,3-环氧丙基三甲基氯化铵对壳聚糖进行季铵化改性,继而标记荧光分子,成功制备TPE-TMC,并以之为检测探针应用于细胞长周期示踪。1HNMR、FL光谱、表面Zeta电位、TEM等测试结果表明成功制备表面正电性的水溶性佳、稳定性好的AIE探针TPE-TMC;用已制得的TPE-TMC对HeLa进行染色,MTT结果证明其无细胞毒性,荧光显微镜照片及CLSM图片表明随着染色时间增加,探针快速进入细胞内部,成像效果良好,且可长周期示踪至25代。该优异性能使之在药物代谢检测、细胞示踪等领域具有良好而广泛的应用前景。3)成功制得具有AIE特性的TPE-CS,并以之为荧光探针研究了其与Cu(Ⅱ)之间相互作用。采用FL光谱、紫外吸收光谱、TEM等分析方法对其作用过程进行表征,FL光谱表明,TPE-CS与不同金属离子相互作用其荧光强度变化不同,且唯有与Cu(Ⅱ)作用呈现荧光光强减弱趋势,且降低的荧光强度与Cu(Ⅱ)浓度在一定范围内呈现良好的线性关系,可用于定量检测Cu(Ⅱ)。
[Abstract]:Fluorescence probe or chemical molecular fluorescence sensor is a kind of molecular system which can selectively bind to a specific substance. It can detect the target molecule qualitatively or quantitatively by the changes of ultraviolet absorption, fluorescence emission spectrum or redox potential. With the advantages of simple operation and high sensitivity, it has been widely used in life science, environmental science and other fields. However, traditional fluorescent molecules often have disadvantages that can not be ignored. For example, the shift of stokes with concentration leads to the nonlinear change of fluorescence intensity, and the phenomenon of fluorescence quenching occurs at high concentration or aggregation, etc. The fluorescence molecules with the characteristic of agglomeration induced luminescence (AIEs) have solved this problem pertinently, and because of its high sensitivity, The spectral properties such as no drift with concentration make it widely used in various fields of fluorescence detection. While the fluorescence probe with AIE characteristics has been widely explored, the demand of water-soluble AIE probe is increasingly urgent. Chitosan is rich in amino, carboxylic groups. Nature is the second largest natural renewable polymer material after cellulose, with biodegradability, biocompatibility, easy to complex with metal ions and other excellent properties in biomedicine, In this paper, the fluorescent molecular markers of tetraphenylethylene tetrachloride (TPE) were labeled on the molecular chain of chitosan and its derivatives, and chitosan based fluorescent molecules with AIE properties were prepared, and then their biological properties were expanded. Applications in the field of environmental monitoring. The main research results are as follows: (1) the succinylation of chitosan in acetic acid / methanol solvent system was carried out by homogeneous reaction method, and then fluorescent molecules were labeled. TPE-NSCSs were successfully prepared and used as detection probes for cell long cycle tracer. 1HNMRs, fluorescence spectra and Zeta potential Tem. The results showed that the water solubility of surface negative electricity was good. The stable AIE probe TPE-NSCS, which was stained with TPE-NSCS, showed no cytotoxicity. Fluorescence microscopy and confocal microscopy images showed that the probe successfully entered the cell interior with the increase of staining time. The imaging effect is good, and can be traced to 30 generations. 2) Quaternary ammonium chloride was used to modify chitosan and then label fluorescent molecules. TPE-TMC was successfully prepared and used as a probe for cell long period tracer. 1HNMRFL spectra. The results of Zeta potential Tem on the surface showed that the water solubility of the positive surface was good. The stable AIE probe TPE-TMC.The HeLa was stained with TPE-TMC and proved to be non-cytotoxic. Fluorescence microscope and CLSM images showed that the probe quickly entered the cell interior with the increase of dyeing time, and the imaging effect was good. And can be traced for a long period to 25 generations. TPE-CSs with AIE properties were successfully prepared and their interaction with Cu2 (鈪，

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