CN106226285B - 一种金为核多巴胺为壳的“拉曼静默区”基底及其制备方法和应用 - Google Patents
一种金为核多巴胺为壳的“拉曼静默区”基底及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物大分子分离分析技术领域,具体为以金为核多巴胺为壳的“拉曼静默区”基底及其制备方法和应用。本发明制备过程包括:用水热法直接还原金纳米粒子,离心后重溶于硼砂溶液,加入水热法制备的“拉曼静默区”分子(E)‑2‑((4‑(乙炔基)亚苄基)氨基)乙基硫醇;然后加入微量的硝酸银,随后将多巴胺加入,使其原位还原银纳米粒子;通过多巴胺的自聚合,产生稳定的“拉曼静默区”的拉曼基底。实验表明,该新型拉曼探针分子在金纳米粒子的增表面增强作用下实现对细胞内部优异的拉曼成像效果,本发明的基底材料无模板,低成本,低毒性,将其应用于活细胞成像,操作简单,新颖便捷,实用高效。
Description
技术领域
本发明属于生物大分子分离分析技术领域,具体涉及“拉曼静默区”探针分子及其制备方法与在细胞内拉曼成像中的应用。
背景技术
表面增强拉曼散射是在一些金、银、铜等贵金属的粗糙表面,待测分子的拉曼散射信号由于受到表面等离子共振(LSPR)作用被几何倍数(103‐109)放大。同时由于拉曼光谱的灵敏度较高,以及较好的波谱形状,目前被广泛应用高灵敏性分析研究的各个领域。另外由于拉曼信号可以稳定于多种化学环境中,同时图谱分析中可以显示被测分子的结构信息,为单分子检测提供了有力的技术支持,目前纳米材料技术与理论的不断发展的不断完善,表面增强拉曼散射的基底的方法也被不断开发,为表面增强拉曼的应用提供了更为宽广的应用范围。
目前在使用的拉曼探针分子多为(1000 -1800 cm-1),此阶段的拉曼探针分子不利于细胞静默区的成像,将碳碳三件、氮氮三键、碳氮三键引入探针分子之中为拉曼成像奠定一定的基础,本实验合成的(E)-2-((4-(苯乙炔基)亚苄基)氨基)乙硫醇(2200cm-1)是将炔基分子引入探针分子的制备中,是较好的细胞“拉曼静默区”的分子,可用于对多种类型的细胞进行成像,为细胞静默区成像提供了一定的基础。
表面增强拉曼散射成像是利用表面增强拉曼光谱的拉曼特征峰,然后通过软件进行拟合得到的模拟图像。其本质实际上是特定拉曼峰的强度分布图。相比于传统的荧光成像,由于表面增强拉曼探针不会像荧光那样容易淬灭和受到背景自发荧光的干扰;这就使其成像灵敏度便远远超过荧光成像。目前表面增强拉曼散射成像在细胞成像中尤其是细胞“拉曼静默区”的应用还比较少。将拉曼静默区探针分子引入拉曼细胞内部成像中的研究还需要进一步探索。因此,发制备出高效率、高灵敏度的“拉曼静默区”表面增强拉曼散射基底尤为重要。
为达到对肿瘤细胞内部进行成像,本发明设计并制备了以金为核多巴胺还原硝酸银为壳的细胞“拉曼静默区”表面增强拉曼散射基底,金纳米粒子对探针分子的信号大大增强,其还原的银纳米粒子进一步的增强效果为拉曼细胞内部的成像提供了可能。将其材料与细胞内部作用之后,实验结果可以实现良好的细胞成像图,说明材料对细胞检测及其临床应用具有较好的前景。
发明内容
本发明的目的在于提供一种高灵敏度的金为核多巴胺还原硝酸银为壳的拉曼静默区的细胞基底及其制备方法和在细胞拉曼成像中的应用。
本发明提供的以金纳米粒子为核、多巴胺还原银纳米粒子为壳的“拉曼静默区”的表面增强拉曼散射基底(简称金为核多巴胺为壳的“拉曼静默区”基底),其制备的基本过程如下:首先,通过水热法合成表面增强拉曼散射探针(E)-2-((4-(乙炔基)亚苄基)氨基)乙硫醇,此探针分子的特征吸收峰在2100 cm-1,由于细胞中天然生物分子在1800 cm-1至2800cm-1区间没有拉曼信号,故其数值是检测细胞的优异峰段,是典型细胞的“拉曼静默区”,用此分子作为表面增强拉曼拉曼散射探针并对细胞呈像,具有无背景干扰、易分辨等无可比拟的优势;为了更好的利用表面增强拉曼散射探针,加入多巴胺与硝酸银,利用多巴胺还原银纳米粒子,从而形成细胞“拉曼静默区”的”热点效应”,制备得到较好的表面增强拉曼散射并可对癌细胞内部成像的拉曼基底。此材料可以高灵敏对癌细胞内部进行拉曼成像。
本发明提出的金为核多巴胺为壳的“拉曼静默区”基底的制备方法,具体步骤如下:
(1)(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇)的合成:在通有氩气的条件下,向150mL的三口烧瓶中加入2-巯基已胺200-300 mg,4-乙炔基苯甲醛300-400 mg,干燥的无水硫酸镁50-150 mg, 以及无水四氢呋喃10-30 mL;加料完成之后将反应体系置于常温下搅拌,反应过程中用薄板层析监测反应,洗脱剂为石油醚和乙酸乙酯,石油醚︰乙酸乙酯=(15-25)︰1(优选20︰1)(体积比);反应结束后向体系中加入二氯甲烷稀释反应液,将反应液倒入250 mL单口烧瓶中,将烧瓶置于旋转蒸发仪中进行旋蒸,出去溶剂,溶剂旋蒸干后向残留物中加入一定量的硅胶制样,使用梯度洗脱过柱,使用流动相为石油醚和乙酸乙酯,石油醚︰乙酸乙酯=(15-25)︰1(优选20︰1)(体积比),洗脱剂500-1000 mL,更换流动相为石油醚︰乙酸乙酯=(0.8-1.2)︰1(优选1︰1)(体积比),分离后得到(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇;
(2)水浴法合成金纳米粒子:取1-3 mL浓度为8-10 mg/mL的高氯金酸溶液及90-100 mL的去离子水,混合均匀,放入120-140℃的油浴中加热,控制其大约每秒钟回流一次,此时向其中加入600-800 uL浓度为1-3%的柠檬酸三钠,保持回流10-30min,冷却至常温,得到尺寸均一的金纳米粒子,将其储存在干净的玻璃瓶中,放于4-8℃保存;
(3)多巴胺还原的银层的修饰:取步骤(2)中合成的金纳米粒子,采用转速6000-8000 rpm对其进行离心5-10min,吸取上层清液,重溶在硼砂溶液中,向其中加入 10-20 uL浓度为 8-10 mM的(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇,混合均匀后加入50-100uL的多巴胺溶液,最终得到金为核多巴胺还原银纳米粒子为壳的拉曼基底。
本发明的金为核多巴胺还原硝酸银为核的细胞内部“拉曼静默区”的表面增强拉曼散射基底,当对不同种类的细胞时均可以呈现拉曼散射成像中。例如,将材料与子宫颈瘤细胞作用可以实现对细胞内部的拉曼散射成像。
细胞拉曼成像的操作步骤为:首先在20mm的直径玻璃表面皿中加入约105的细胞密度的细胞,培养12-24h 后,取10-30 μL金为核硝酸银为壳的“拉曼静默区”的表面增强拉曼散射基底,加入玻璃皿中,然后将其玻璃培养皿放于培养箱中,与细胞作用20-24 h后,再吸取培养基,用PBS洗涤三次后,用2-4%的多聚甲醛溶液,在25-37℃中反应12-18min(优选15 min),最后用PBS 洗涤二到三次,并加入100-300uL的PBS ,放于4℃的冰箱中保存,用于表面增强拉曼散射成像。
本发明合成的波谱在1800cm-1-2800cm-1的细胞拉曼静默区的分子,可扩展到细胞中的任意拉曼静默区的成像范围,此材料均可用于对细胞的拉曼静默区的成像。此应用可以进一步的扩展到组织切片中,为鉴定组织切片提供了实验基础,具有较大的临床应用前景。
通过上述步骤成功实现了以金纳米粒子为核多巴胺还原硝酸银纳米粒子为壳的细胞“拉曼静默区”表面增强拉曼散射探针,材料的进一步表征通过透射电子显微镜(TEM)、表面增强拉曼散射光谱(SERS)、紫外吸收光谱(UV)、核磁图像说明。
实验表明,该表面增强拉曼散射探针对细胞内部成像具有较好的效果,相比单一贵金属纳米粒子的表面增强拉曼散射,金银纳米粒子产生的热点效应对拉曼信号分子可以提升接近几个甚至几十个数量级的强度,在维持细胞活性的前提下,将材料与活细胞作用24 h 后,在2100 cm-1 ((E)-2-((4-(乙炔基)亚苄基)氨基)乙硫醇)峰谱处实现对活细胞的内部拉曼成像。本发明无模板,低成本,低毒性,将其应用于活细胞内部成像,新颖便捷,实用高效,具有巨大的临床应用前景。
附图说明
图1(E)-2-((4-(乙炔基)亚苄基)氨基)乙硫醇分子的制备流程图示。
图2标签浓度对拉曼信号强度的波谱图。
图3硝酸银浓度对拉曼信号强度的波谱图。
图4金纳米粒子、金为核多巴胺还原硝酸银为壳的TEM图像。其中,(a)为金球,(b)为多巴胺还原硝酸银壳金为核拉曼基底的TEM图像。
图5金球与多巴胺还原硝酸银的紫外对比。其中,上方为金球紫外,下方为多巴胺还原硝酸银的纳米粒子的紫外图。
图6细胞拉曼图。(a)人神经胶质瘤细胞的明场图,(b)人神经胶质瘤细胞的拉曼成像图。
具体实施方式
实施例1:人宫颈癌细胞的金为核多巴胺还原硝酸银为壳的表面增强拉曼散射细胞内部成像基底的合成
向250 mL的三口烧瓶中取1-3 mL浓度为8-10 mg/mL的高氯金酸溶液及90-100 mL的去离子水,混合均匀,放入120-140℃的油浴中加热,控制其大约每秒钟回流一次,此时向其中加入600-800 uL浓度为1-3%的柠檬酸三钠,保持回流10-30min,冷却至常温,得到尺寸均一的金纳米粒子,将其储存在干净的玻璃瓶中,放于4-8℃保存;
取合成好的金纳米粒子,采用8000 rpm, 5min 对其进行离心,吸取上层清液,重溶在硼砂溶液中,向其中加入 10 uL 10 mM的(E)-2-((4-(乙炔基)亚苄基)氨基)乙硫醇,混合均匀后加入 5 uL 10 mM的硝酸银,最后加入60 uL 1 mg/mL 多巴胺。采用醌基共价偶联的方法将表皮生长因子受体抗体(anti-EGFR)修饰于银壳表面,合成的材料可用于对细胞内部的拉曼成像。
实施例2:金为核多巴胺还原硝酸银为壳的拉曼探针对神经胶质瘤细胞U251内表面增强拉曼成像的应用
首先在20mm的直径玻璃表面皿中加入约105的细胞密度的细胞,培养12- 24 h后,取10μL金为核多巴胺还原硝酸银为壳的静默区的表面增强拉曼散射材料,加入玻璃皿中,然后将其玻璃培养皿放于细胞培养箱中与细胞作用12 -24 h, 再吸取培养基,用PBS洗涤二到三次后,用4%的多聚甲醛溶液,在37℃培养箱中反应15 -30 min, 最后用PBS 洗涤二到三次,并加入100 -200uL的PBS 放于4℃的冰箱中保存,用于表面增强拉曼散射成像。
Claims (6)
1.一种金为核多巴胺为壳的“拉曼静默区”基底的制备方法,其特征在于,具体步骤为:
(1)表面增强拉曼散射探针(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇)的合成:在通有氩气的条件下,向150mL的三口烧瓶中加入2-巯基已胺200-300 mg,4-乙炔基苯甲醛300-400 mg,干燥的无水硫酸镁50-150 mg, 以及无水四氢呋喃10-30 mL;加料完成之后将反应体系置于常温下搅拌,反应过程中用薄板层析监测反应,洗脱剂为石油醚和乙酸乙酯,石油醚:乙酸乙酯=(15-25):1;反应结束后向体系中加入二氯甲烷稀释反应液,将反应液倒入250 mL单口烧瓶中,将烧瓶置于旋转蒸发仪中进行旋蒸,出去溶剂,溶剂旋蒸干后向残留物中加入一定量的硅胶制样,使用梯度洗脱过柱,使用流动相为石油醚和乙酸乙酯,石油醚:乙酸乙酯=(15-25):1,洗脱剂500-1000 mL,更换流动相为石油醚:乙酸乙酯=(0.8-1.2):1,分离后得到(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇,即表面增强拉曼散射探针;
(2)水浴法合成金纳米粒子:取1-3 mL浓度为8-10 mg/mL的高氯金酸溶液及90-100 mL的去离子水,混合均匀,放入120-140℃的油浴中加热,控制其每秒钟回流一次,此时向其中加入600-800 uL浓度为1-3%的柠檬酸三钠,保持回流10-30min,冷却至常温,得到尺寸均一的金纳米粒子,将其储存在干净的玻璃瓶中,放于4-8℃保存;
(3)多巴胺还原的银层的修饰:取步骤(2)中合成的金纳米粒子,采用转速6000-8000rpm对其进行离心5-10min,吸取上层清液,重溶在硼砂溶液中,向其中加入 10-20 uL浓度为 8-10 mM的(E)-2-((4-(乙炔基)亚苄基)氨基)乙基硫醇,混合均匀后加入5 uL浓度为10mM的硝酸银,最后加入50-100 uL的多巴胺溶液,最终得到金为核多巴胺还原银纳米粒子为壳的拉曼基底。
2.由权利要求1所述制备方法中步骤(1)所制备得到的表面增强拉曼散射探针。
3.由权利要求1所述制备方法制备得到的金为核多巴胺为壳的“拉曼静默区”基底。
4.如权利要求3所述的金为核多巴胺为壳的“拉曼静默区”基底在肿瘤细胞拉曼成像中的应用。
5.根据权利要求4所述的应用,其特征在于,细胞内部拉曼成像的操作步骤为:首先在20mm的直径玻璃表面皿中加入105的细胞密度的细胞,培养12-24h 后,取10-30 μL金为核多巴胺为壳的“拉曼静默区”基底,加入玻璃皿中,然后将玻璃培养皿放于培养箱中,与细胞作用20-24 h,再吸取培养基,用PBS洗涤三次后,用2-4%的多聚甲醛溶液,在25-37℃中反应12-18 min,最后用PBS 洗涤二到三次,并加入100-300uL的PBS ,放于4℃的冰箱中保存,用于表面增强拉曼散射成像。
6.根据权利要求4或5所述的应用,其特征在于,用于多种吞噬肿瘤细胞,并对成功吞噬材料的细胞实现拉曼成像。
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