量子点（CdSe）标记糖类分子的研究
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摘要
量子点由于其优良的荧光性能,目前已广泛应用于生物工程领域。CdSe量子点表面经过修饰后,表面带有活性功能基团,通过分子偶联作用,可以连接DNA,蛋白质等。目前在量子点的制备方法中,有机合成法是较成熟的,这种方法可以获得荧光效率高,颗粒均一的量子点。但是有机合成法中存在严重的问题,比如三辛基氧膦(TOPO)等具有较强的毒性,而且操作繁琐,制得的QDs表面有一层TOPO,TOPO不仅具有毒性,而且是疏水性物质,这样使得所制得的量子点不具有水溶性,对于QDs应用于生物学领域是不利的。
本实验中建立了量子点的水相合成法,并且通过糖类分子修饰量子点从而使得量子点可以通过细胞表面凝集素的作用而进入HepG2细胞中,讨论了在细胞毒性方面,量子点糖基化修饰的优势。
(1)水相合成量子点条件：400mgSe和400mgNaBH4在1ml水溶液中反应2h后,取0.25ml加入到90ml含有25mg的CdCl2·2.5H2O和0.025ml巯基乙酸的水溶液中,96℃回流2h制得量子点,将制得的量子点通过红外,紫外,电镜以及荧光等方面进行表征,结果证明获得了大小均一,颗粒分散的量子点,并且使用壳聚糖来包裹量子点,实现了量子点对纳米载药分子的标记。
(2)将量子点与9种糖类分子分别进入连接,并且将连接后的产物加到HepG2细胞培养液中,进行筛选,最终发现甘露糖修饰的量子点可以进入HepG2细胞中,这和文献报道的HepG2细胞表面甘露糖凝集素大量表达相一致,这一结果对于研究癌细胞表面凝集素,以及通过糖类分子来标记癌细胞提供有一定的依据。此外,甘露糖修饰的量子点对HepG2细胞没有表现出细胞毒性,而且仍可长时间的保持荧光特性。通过小鼠的荷瘤模型也发现,在体内实验中,甘露糖修饰的量子点也可以标记HepG2细胞。
(3)通过量子点以及甘露糖修饰的量子点分别同牛血清白蛋白作用,牛血清白蛋白通常被用做研究药物分子在体内的药物药理等研究,发现量子点被甘露糖修饰后,与BSA的结合明显降低,这对于量子点在体内毒性研究是有利的,在量子点表面降低毒性的修饰是一个补充。另外研究了量子点与牛血清白蛋白的作用,发现量子点可以引起BSA的猝灭,这个猝灭的过程是一个静态的猝灭过程,它们之间以摩尔比1：1结合,根据同步荧光光谱,可以得出,量子点主要与BSA的酪氨酸连接,而不与丝氨酸相作用。根据Foste偶极能量转移理论,可以计算出量子点与BSA的作用距离6.56nm,小于8nm,说明量子点与BSA的结合是个有效结合。这些对于研究量子点的体内毒性及量子点体内药理等,提供一定依据。
Quantum dots, as its excellent fluorescent properties, has been widely used in biological engineering. After the surface modification of CdSe quantum dots, the surface with active functional groups, by the molecular coupling effect, QDs can be connected to DNA, proteins. Currently in preparation methods, quantum dots, organic synthesis had many reports, this method can obtain high efficiency fluorescent particles homogeneous quantum dots. However, in organic synthesis, there are serious problems, such as TOPO has a strong toxicity, the obtained QDs surface layer of TOPO, TOPO is not only toxic, but also hydrophobic substances, which makes the prepared quantum point does not have a water-soluble, the QDs used in the field of biology at a disadvantage.
In this study, the QDs in the aqueous phase synthesis method was set up, and quantum dots by oligosaccharides molecular modification to make quantum dots can be entered HepG2 cells by cell surface lectin, and also on the toxicity in cells, QDs-Man have the advantage.
(1) Aqueous-phase synthesis of quantum dots conditions:400 mg Se and 400 mg NaBH4 was added in 1ml aqueous solution,2h latter, take 0.25 ml to 90 ml aqueous solution containing 25mg of CdCl2·2.5H2O and 0.025 ml thioglycolic acid,96℃,2h, QDs was obtained, the quantum dots was tested by infrared, ultraviolet, electron microscopy and fluorescence in areas such as characterization, the results was showed that the uniform size of particles dispersed quantum dots was obtained, and the use of chitosan to coat quantum dots, quantum dots marked the drug carried molecules.
(2) Quantum dots and 9 kinds of trisaccharides were connected, and the connection of products added to the HepG2 cell culture medium, then screened and found mannose modified quantum dots can entered the HepG2 cells, this result as the literature reported that HepG2 cell surface great expressed mannose lectin which could help carbohydrate molecules through cancer cells. In addition, it was found that mannose modified quantum dots on HepG2 cells showed no cytotoxicity, and still maintain a long fluorescence characteristics. It was also found mannose modification of quantum dots can also be labeled HepG2 cells byy in vivo mice tumor model.
(3) Mannose modified quantum dots and quantum dots were modified with bovine serum albumin, bovine serum albumin is often used as study drug molecules in vivo pharmacology study, it was found that quantum dots which are modified by mannose, its combination with BSA significantly reduced, it was beneficial for the quantum dots in vivo toxicity studies and a supplement of the quantum dot surface modification. In addition, the quantum dots connected with bovine serum albumin was studied, it was found that quantum dots can lead to quenching, and it was a static quenching process, the molar ratio of 1:1 between them to combine. According to synchronization fluorescence spectroscopy, it can be obtained that quantum dots mainly connected with tyrosine of BSA, but not with the serine. According to Foste dipole energy transfer theory, the distance ofquantum dots connected with BSA can be calculated, it was 6.56nm, less than 8nm, it showed the combination of quantum dots and the BSA was an effective combination. The result could provide some basis information for the study of quantum dots in vivo toxicity and in vivo pharmacology.
本实验中建立了量子点的水相合成法,并且通过糖类分子修饰量子点从而使得量子点可以通过细胞表面凝集素的作用而进入HepG2细胞中,讨论了在细胞毒性方面,量子点糖基化修饰的优势。
(1)水相合成量子点条件：400mgSe和400mgNaBH4在1ml水溶液中反应2h后,取0.25ml加入到90ml含有25mg的CdCl2·2.5H2O和0.025ml巯基乙酸的水溶液中,96℃回流2h制得量子点,将制得的量子点通过红外,紫外,电镜以及荧光等方面进行表征,结果证明获得了大小均一,颗粒分散的量子点,并且使用壳聚糖来包裹量子点,实现了量子点对纳米载药分子的标记。
(2)将量子点与9种糖类分子分别进入连接,并且将连接后的产物加到HepG2细胞培养液中,进行筛选,最终发现甘露糖修饰的量子点可以进入HepG2细胞中,这和文献报道的HepG2细胞表面甘露糖凝集素大量表达相一致,这一结果对于研究癌细胞表面凝集素,以及通过糖类分子来标记癌细胞提供有一定的依据。此外,甘露糖修饰的量子点对HepG2细胞没有表现出细胞毒性,而且仍可长时间的保持荧光特性。通过小鼠的荷瘤模型也发现,在体内实验中,甘露糖修饰的量子点也可以标记HepG2细胞。
(3)通过量子点以及甘露糖修饰的量子点分别同牛血清白蛋白作用,牛血清白蛋白通常被用做研究药物分子在体内的药物药理等研究,发现量子点被甘露糖修饰后,与BSA的结合明显降低,这对于量子点在体内毒性研究是有利的,在量子点表面降低毒性的修饰是一个补充。另外研究了量子点与牛血清白蛋白的作用,发现量子点可以引起BSA的猝灭,这个猝灭的过程是一个静态的猝灭过程,它们之间以摩尔比1：1结合,根据同步荧光光谱,可以得出,量子点主要与BSA的酪氨酸连接,而不与丝氨酸相作用。根据Foste偶极能量转移理论,可以计算出量子点与BSA的作用距离6.56nm,小于8nm,说明量子点与BSA的结合是个有效结合。这些对于研究量子点的体内毒性及量子点体内药理等,提供一定依据。
Quantum dots, as its excellent fluorescent properties, has been widely used in biological engineering. After the surface modification of CdSe quantum dots, the surface with active functional groups, by the molecular coupling effect, QDs can be connected to DNA, proteins. Currently in preparation methods, quantum dots, organic synthesis had many reports, this method can obtain high efficiency fluorescent particles homogeneous quantum dots. However, in organic synthesis, there are serious problems, such as TOPO has a strong toxicity, the obtained QDs surface layer of TOPO, TOPO is not only toxic, but also hydrophobic substances, which makes the prepared quantum point does not have a water-soluble, the QDs used in the field of biology at a disadvantage.
In this study, the QDs in the aqueous phase synthesis method was set up, and quantum dots by oligosaccharides molecular modification to make quantum dots can be entered HepG2 cells by cell surface lectin, and also on the toxicity in cells, QDs-Man have the advantage.
(1) Aqueous-phase synthesis of quantum dots conditions:400 mg Se and 400 mg NaBH4 was added in 1ml aqueous solution,2h latter, take 0.25 ml to 90 ml aqueous solution containing 25mg of CdCl2·2.5H2O and 0.025 ml thioglycolic acid,96℃,2h, QDs was obtained, the quantum dots was tested by infrared, ultraviolet, electron microscopy and fluorescence in areas such as characterization, the results was showed that the uniform size of particles dispersed quantum dots was obtained, and the use of chitosan to coat quantum dots, quantum dots marked the drug carried molecules.
(2) Quantum dots and 9 kinds of trisaccharides were connected, and the connection of products added to the HepG2 cell culture medium, then screened and found mannose modified quantum dots can entered the HepG2 cells, this result as the literature reported that HepG2 cell surface great expressed mannose lectin which could help carbohydrate molecules through cancer cells. In addition, it was found that mannose modified quantum dots on HepG2 cells showed no cytotoxicity, and still maintain a long fluorescence characteristics. It was also found mannose modification of quantum dots can also be labeled HepG2 cells byy in vivo mice tumor model.
(3) Mannose modified quantum dots and quantum dots were modified with bovine serum albumin, bovine serum albumin is often used as study drug molecules in vivo pharmacology study, it was found that quantum dots which are modified by mannose, its combination with BSA significantly reduced, it was beneficial for the quantum dots in vivo toxicity studies and a supplement of the quantum dot surface modification. In addition, the quantum dots connected with bovine serum albumin was studied, it was found that quantum dots can lead to quenching, and it was a static quenching process, the molar ratio of 1:1 between them to combine. According to synchronization fluorescence spectroscopy, it can be obtained that quantum dots mainly connected with tyrosine of BSA, but not with the serine. According to Foste dipole energy transfer theory, the distance ofquantum dots connected with BSA can be calculated, it was 6.56nm, less than 8nm, it showed the combination of quantum dots and the BSA was an effective combination. The result could provide some basis information for the study of quantum dots in vivo toxicity and in vivo pharmacology.
引文
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