水溶性CdTe量子点的光稳定性研究及其应用
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摘要
随着生物医学研究和显微镜技术的发展,对新一代荧光探针的研究展现出越来越多的必要性和发展潜力。水溶性半导体荧光量子点由于其良好的光学特性,如高量子效率、尺寸可调的荧光发射波长、宽带吸收与窄带荧光、光稳定性良好、双光子吸收截面大等,被认为是非常重要的一种荧光标记材料,并且广泛地应用于细胞标记技术、蛋白示踪、DNA阵列检测技术、免疫荧光标记方法、生物活体及组织检测等方向的研究。本文围绕硫醇包裹水溶性的碲化镉量子点在活体细胞内的光稳定性行为展开,研究了CdTe量子点在细胞内的光稳定性影响因素,逐步确定了量子点光淬灭过程的步骤和机理,并基于光淬灭机理发展出量子点结合肽箐用于光动力治疗以及用表面处理的方法增加量子点光稳定性两方面的应用。主要有以下四个方面的结果:
1.在细胞内,量子点远比叶绿素和FITC,但相对浓度较低时和绿荧光蛋白相近。发现量子点在细胞内不同浓度的区域,其光稳定性也不同,即浓度效应。并进一步从溶液和细胞两方面确定了氧气在载光淬灭中的作用。
2.通过实验研究了量子点在水溶液中光淬灭过程的机理。拉曼光谱证明光照后量子点表面Cd-S键脱离,动态散射实验确定了光照后量子点聚集,叠氮纳影响荧光淬灭时间确定单态氧参与的光氧化过程,从而最终确认了量子点光淬灭的全过程。
3.由直接和间接两种方法确定了量子点单态氧量子产额为1%。将量子点与肽箐结合,使量子点复合物的单态氧量子产额增加到15%,同时保留量子点光学性质,使之能够用于光动力治疗。
4.通过表面处理的方法,增加了CdTe量子点的量子产额以及光稳定性。通过单量子点的研究,证实表面处理的方法能使量子点的光稳定增加3倍左右。
With the development of biomedical application and the microscope technology, the study of new type fluorescence probes become more reasonable and have bright future.Water-soluble semiconductor fluorescent quantum dots(QDs) are considered as a very important bio-labeling material owing to their outstanding optical properties such as size-tunable emission,broad absorption and narrow symmetric emission,high photo-stability,large 2-photon absorption cross section,and etc.This thesis focused on the photostability of thiol-capped CdTe QDs.We studied the effects of the photostability of QDs,conculed the theory of photobleaching process.Base on the theory,we developed two application:1) link QD and phthalocyanines to improve the singlet oxygen quantum yield for photodynamic therapy;2) using surface-treatment to improve photostability of QDs.The main results are listed as follows:
1.The photostability of thiol-capped CdTe quantum dots(QDs) in Euglena gracilis (EG 277) and human embryonic kidney(HEK 293) cells was studied.The photobleaching for the cellular QDs is dependent both on the irradiation power density and the QD local concentration.The photostability of cellular QDs is better than that of chlorophyll and of green fluorescence protein(GFP) and is much better than that of FITC when the local concentration of QDs is not too low. The photobleaching of cellular QDs was remarkably reduced in the nitrogen treated EG 277 cells,indicating that photobleaching in living cells mainly results from photo-oxidation.The effect of photo-oxidation on QD photobleaching was further confirmed by comparing the situations in oxygen treated and nitrogen treated QD aqueous solutions.The photobleaching rate is related to the irradiation power density and the local density of QDs.The higher irradiation power density and oxygen abundance and lower QD concentration will result in a higher photobleaching rate.
2.The process and mechanism of photochemical instability of thiol-capped CdTe quantum dots(QDs) in aqueous solution were experimentally studied.After laser irradiation,the corresponding Raman bands of the Cd-S bond decreased obviously, indicating bond breaking and thiol detachment from the QD surfaces.Meanwhile, a photoinduced aggregation of QDs occurred with the hydrodynamic diameter increased to hundreds of nanometers from an initial 20 nm,as detected with dynamic light scattering measurements.The bleaching of the photoluminescence of QDs under laser irradiation could be attributed to the enhanced nonradiative transfer in excited QDs caused by increased surface defects due to the losing of thiol ligands.Singlet oxygen(1O2) was involved in the photooxidation of QDs,as revealed by the inhibiting effects of 1O2 quenchers of histidine or sodium azide (NaN3) on the photobleaching of QDs.The linear relationship in Stern-Volmer measurements between the terminal product and the concentration of NaN3 demonstrated that 1O2 was the main pathway of the photobleaching in QD solutions.
3.By means of the direct detection of near-infrared 1270 nm,we found that the water-soluble thiol-capped CdTe QDs can photoproduce 102 in deuterated water with a low quantum yield(QY) of 1%.When sulfonated aluminum phthalocyanines(AISPc's) were connected to these QDs,forming water-soluble QD-Pc composites,the 1O2 QY of the composites increased to 15%under the excitation of 532 nm.QD-Pc composites can fully utilize visible region light excitation to effectively produce 1O2,which may facilitate the applications of QD-Pc composites in broad areas.
4..The surface treatment of thiol-capped CdTe quantum dots(QDs) was carried out with a small amount of sodium borohydride(NaBH4) in aqueous solution at room temperature.The treatment effectively enhanced the photostability of QDs and increased the photoluminescence(PL) efficiency by a factor of two as against the original ones.By measuring the PL trajectories of single QDs with a total internal reflection fluorescence microscope,the photostable lifetimes were determined to be 15.2±5.9 s for surface-treated QDs,and 5.8±1.9 s for the original ones, respectively.
1.在细胞内,量子点远比叶绿素和FITC,但相对浓度较低时和绿荧光蛋白相近。发现量子点在细胞内不同浓度的区域,其光稳定性也不同,即浓度效应。并进一步从溶液和细胞两方面确定了氧气在载光淬灭中的作用。
2.通过实验研究了量子点在水溶液中光淬灭过程的机理。拉曼光谱证明光照后量子点表面Cd-S键脱离,动态散射实验确定了光照后量子点聚集,叠氮纳影响荧光淬灭时间确定单态氧参与的光氧化过程,从而最终确认了量子点光淬灭的全过程。
3.由直接和间接两种方法确定了量子点单态氧量子产额为1%。将量子点与肽箐结合,使量子点复合物的单态氧量子产额增加到15%,同时保留量子点光学性质,使之能够用于光动力治疗。
4.通过表面处理的方法,增加了CdTe量子点的量子产额以及光稳定性。通过单量子点的研究,证实表面处理的方法能使量子点的光稳定增加3倍左右。
With the development of biomedical application and the microscope technology, the study of new type fluorescence probes become more reasonable and have bright future.Water-soluble semiconductor fluorescent quantum dots(QDs) are considered as a very important bio-labeling material owing to their outstanding optical properties such as size-tunable emission,broad absorption and narrow symmetric emission,high photo-stability,large 2-photon absorption cross section,and etc.This thesis focused on the photostability of thiol-capped CdTe QDs.We studied the effects of the photostability of QDs,conculed the theory of photobleaching process.Base on the theory,we developed two application:1) link QD and phthalocyanines to improve the singlet oxygen quantum yield for photodynamic therapy;2) using surface-treatment to improve photostability of QDs.The main results are listed as follows:
1.The photostability of thiol-capped CdTe quantum dots(QDs) in Euglena gracilis (EG 277) and human embryonic kidney(HEK 293) cells was studied.The photobleaching for the cellular QDs is dependent both on the irradiation power density and the QD local concentration.The photostability of cellular QDs is better than that of chlorophyll and of green fluorescence protein(GFP) and is much better than that of FITC when the local concentration of QDs is not too low. The photobleaching of cellular QDs was remarkably reduced in the nitrogen treated EG 277 cells,indicating that photobleaching in living cells mainly results from photo-oxidation.The effect of photo-oxidation on QD photobleaching was further confirmed by comparing the situations in oxygen treated and nitrogen treated QD aqueous solutions.The photobleaching rate is related to the irradiation power density and the local density of QDs.The higher irradiation power density and oxygen abundance and lower QD concentration will result in a higher photobleaching rate.
2.The process and mechanism of photochemical instability of thiol-capped CdTe quantum dots(QDs) in aqueous solution were experimentally studied.After laser irradiation,the corresponding Raman bands of the Cd-S bond decreased obviously, indicating bond breaking and thiol detachment from the QD surfaces.Meanwhile, a photoinduced aggregation of QDs occurred with the hydrodynamic diameter increased to hundreds of nanometers from an initial 20 nm,as detected with dynamic light scattering measurements.The bleaching of the photoluminescence of QDs under laser irradiation could be attributed to the enhanced nonradiative transfer in excited QDs caused by increased surface defects due to the losing of thiol ligands.Singlet oxygen(1O2) was involved in the photooxidation of QDs,as revealed by the inhibiting effects of 1O2 quenchers of histidine or sodium azide (NaN3) on the photobleaching of QDs.The linear relationship in Stern-Volmer measurements between the terminal product and the concentration of NaN3 demonstrated that 1O2 was the main pathway of the photobleaching in QD solutions.
3.By means of the direct detection of near-infrared 1270 nm,we found that the water-soluble thiol-capped CdTe QDs can photoproduce 102 in deuterated water with a low quantum yield(QY) of 1%.When sulfonated aluminum phthalocyanines(AISPc's) were connected to these QDs,forming water-soluble QD-Pc composites,the 1O2 QY of the composites increased to 15%under the excitation of 532 nm.QD-Pc composites can fully utilize visible region light excitation to effectively produce 1O2,which may facilitate the applications of QD-Pc composites in broad areas.
4..The surface treatment of thiol-capped CdTe quantum dots(QDs) was carried out with a small amount of sodium borohydride(NaBH4) in aqueous solution at room temperature.The treatment effectively enhanced the photostability of QDs and increased the photoluminescence(PL) efficiency by a factor of two as against the original ones.By measuring the PL trajectories of single QDs with a total internal reflection fluorescence microscope,the photostable lifetimes were determined to be 15.2±5.9 s for surface-treated QDs,and 5.8±1.9 s for the original ones, respectively.
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