CN111793496A - 一种表面氨基功能化的纳米晶材料和其在细胞免疫标记中的用途 - Google Patents
一种表面氨基功能化的纳米晶材料和其在细胞免疫标记中的用途 Download PDFInfo
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Abstract
本申请涉及生物荧光标记领域,尤其涉及一种表面氨基功能化的纳米晶材料及其制备方法和在细胞免疫标记中的用途和试剂盒。一种表面氨基功能化的纳米晶材料,该纳米晶材料的分子式为NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2。该纳米晶能够与生物分子相连接,且生物相容性好、单分散性好和荧光强度高,很好地应用于细胞的免疫标记和荧光成像。
Description
技术领域
本申请涉及生物荧光标记领域,尤其涉及一种表面氨基功能化的纳米晶材料及其制备方法和在细胞免疫标记中的用途。
背景技术
胃癌(gastric carcinoma)是起源于胃黏膜上皮的恶性肿瘤,在我国各种恶性肿瘤中发病率居首位。绝大多数胃癌属于腺癌,早期无明显症状,或出现上腹不适、嗳气等非特异性症状,常与胃炎、胃溃疡等胃慢性疾病症状相似,易被忽略,因此,目前我国胃癌的早期诊断率仍较低。癌抗原50是一种非特异性的广谱肿瘤标志物,主要用于胰腺癌、结肠/直肠癌、胃癌的辅助诊断。
钼酸盐作为稀土离子发光的基质材料,化学稳定性好,发光强度高;在紫外区有宽而强的电荷转移带,与其它基质相比,Eu3+离子在约395 nm和465 nm处的f-f跃迁激发带显著增强。这两个激发带分别与目前应用的紫外光和蓝光激发的白光发射二极管(LED)芯片相匹配。Eu3+离子掺杂的钼酸盐化合物是一种很有希望应用在白光LED上的红色荧光材料。同时这也为发展新型的近紫外光和可见光激发的稀土荧光探针提供了机会。
目前,大多采用固相法制备钼酸盐发光材料,用该法得到的钼酸盐具有良好的结晶度,作为光学材料,显示出很好的发光性能。但是固相反应存在不可避免的高温、高能耗、工艺复杂等缺点,而且制备的颗粒尺寸较大,形状不规整。液相法反应条件温和,在控制晶体的形貌以及分散性方面具有较大优势。其中水热合成法能够在较低的温度下得到较好的结晶产物。有文献报道在油酸和油胺的混合表面活性剂中,利用水热法合成了均匀的、单分散的和NaLa(MoO4)2和NaLa(MoO4)2:Eu3+双棱锥型的纳米晶。基于实验结果,作者提出了油酸和油胺共配位控制形成均匀四面体结构的双棱锥形纳米晶的生长机制。
尽管钼酸盐纳米材料的合成与应用研究取得了一系列进展但通过这些方法制备的粉体还具有一些局限性反应产物需要较高的温度和长时间的保温;或者所制备产物具有相对较大的颗粒尺寸、颗粒易团聚且形貌不均一等。在发展新型稀土钼酸盐荧光探针方面还有很多需要解决的问题。如生物探针要求发光颗粒粒径要小,发光强度要高,单分散性要好。目前,合成100 nm以下的钼酸盐球形纳米粒子还是一个挑战,对颗粒表面进行功能化修饰实现与生物分子的连接还未见报道。
发明内容
为了解决上述技术问题,本申请的目的是提供一种表面氨基功能化的纳米晶材料,该纳米晶材料表面包覆了二氧化硅,氨基化修饰后可与通过与生物分子的连接来标记细胞。该纳米晶颗粒具有单分散性好、生物相容性好和荧光强度高的优点,同时具有可以在紫外光和近红外光激发下多色发光的优势。
为了实现上述目的,本申请采用了以下的技术方案:
一种表面氨基功能化的纳米晶材料,该纳米晶材料的分子式为NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2。
优选,该纳米晶材料为水溶性纳米晶材料,由二氧化硅包覆NaLuMo2O8:Yb/Tm/Ce/Eu纳米晶构成。
优选,NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2纳米晶的分子式为NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2。
进一步,本申请提供了所述纳米晶材料的制备方法,该方法包括以下的步骤:
1)1毫摩尔硝酸钠NaNO3,0.645毫摩尔硝酸镥Lu(NO3)3∙5H2O,0.2毫摩尔硝酸铈Ce(NO3)3∙5H2O,0.005毫摩尔硝酸铥Tm(NO3)3∙5H2O,0.05毫摩尔硝酸铕Tb(NO3)3∙5H2O以及3毫摩尔柠檬酸溶于8毫升去离子水中,搅拌均匀;
2)搅拌20分钟后,加入2毫摩尔钼酸钠Na2MoO4和30毫升聚乙二醇,搅拌1小时后,转移到高压水热釜中,于180℃加热6小时;产物用乙醇和去离子水的混合液洗涤3-5次;
3)20 mg NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶加入70 mL异丙醇中,超声分散20min,加入5 mL水和3 mL的25%的NH4OH,置于30℃水浴中。搅拌下,滴加0.02 mL正硅酸四乙酯,搅拌反应3 h后,将0.2 mLγ-氨丙基三乙氧基硅烷溶于20 mL异丙醇中,缓慢加入到上述反应液中,30 min滴完,继续搅拌反应1 h。通过离心分离,无水乙醇洗涤,50℃干燥2 h后得氨基修饰的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米颗粒。
进一步,本申请提供了所述纳米晶材料在胃癌细胞免疫检测中的应用。
进一步,本申请提供了一种胃癌细胞免疫检测试剂盒,该试剂盒包括所述纳米晶材料。
进一步,本申请提供了所述纳米晶材料在细胞免疫标记和成像中的应用。
进一步,本申请提供了一种细胞免疫标记荧光探针,该荧光探针包括所述纳米晶材料。
本申请首先通过一种溶剂热法制备出四方相NaLuMo2O8:Yb/Tm/Ce/Eu纳米晶,纳米晶由于表面存在柠檬酸配体,能够很好地分散在水溶液中。然后在纳米晶的表面包覆二氧化硅进行氨基化修饰后获得NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2纳米颗粒,该纳米晶能够与生物分子相连接,且生物相容性好、单分散性好和荧光强度高,很好地应用于细胞的免疫标记和荧光成像。
附图说明
图1(a)和(b)分别为NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu的XRD图谱和透射电子显微镜图,(c)为NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2透射电子显微镜图,其中插图为样品的电子衍射照片。
图2 (a)NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶在980纳米近红外激光器激发条件下的上转换发射谱,(b)NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶在紫外激发条件下的下转换发射谱,(c)产物在紫外与近红外激光器双波长激发条件下的发射谱,(d)产物的发光强度随敏化离子Ce3+的变化关系曲线。
图3不同浓度的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米粒子与MGC-803细胞共培养。MTT实验结果显示各组间OD值无显著差异。“0”组为对照组。
图 4 NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米粒子对MGC-803细胞的荧光标记。(a)明场下的MGC-803细胞照片,(b)纳米粒子与MGC-803细胞共培养之后,在 980 nm 激发下的发光照片,(c)纳米粒子的上转换发光照片与细胞照片复合。
具体实施方式
1.实验部分
1.1 主要仪器和试剂:
硝酸钠(99.0%),硝酸镥(99.9%),硝酸铈(99.9%),硝酸镱(99.9%),硝酸铥(99.9%),硝酸铕(99.9%),钼酸钠(98.0%),柠檬酸(99.5%)与乙二醇(99.8%)购买于Sigma-Aldrich公司;无水乙醇、正硅酸四乙酯(TEOS)、γ-氨丙基三乙氧基硅烷(KH-550)、磷酸缓冲溶液(PBS)N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)购买于国药集团化学试剂有限公司;二甲基亚砜(DMSO,纯度>99.5%),噻唑兰(MTT,纯度 98%)购买于北京鼎国昌盛生物技术有限公司;羊抗人IgG(anti-CA50)购买于北京奥博星生物技术有限公司;MGC-803细胞购买于IBCB;RPMI1640细胞培养基购买于Gbico。
1.2 NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米晶的制备
以NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶为例,1毫摩尔硝酸钠(NaNO3),0.645毫摩尔硝酸镥(Lu(NO3)3∙5H2O),0.2毫摩尔硝酸铈(Ce(NO3)3∙5H2O),0.005毫摩尔硝酸铥(Tm(NO3)3∙5H2O),0.05毫摩尔硝酸铕(Tb(NO3)3∙5H2O)以及3毫摩尔柠檬酸溶于8毫升去离子水中,搅拌20分钟后,加入2毫摩尔钼酸钠(Na2MoO4)和30毫升聚乙二醇,搅拌1小时后,转移到高压水热釜中,于180℃加热6小时。产物用乙醇和去离子水的混合液洗涤3-5次。20 mg NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶加入70 mL异丙醇中,超声分散20 min,加入5 mL水和3 mL的25%的NH4OH,置于30℃水浴中;搅拌下,滴加0.02 mL正硅酸四乙酯;搅拌反应3 h后,将0.2mLγ-氨丙基三乙氧基硅烷溶于20 mL异丙醇中,缓慢加入到上述反应液中,30 min滴完,继续搅拌反应1 h;通过离心分离,无水乙醇洗涤,50℃干燥2 h后得氨基修饰的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu @SiO2纳米颗粒。
不同浓度或种类的离子掺杂样品,通过改变前驱溶液中相应的离子浓度或种类来实现。
1.3 表征仪器
X射线衍射图谱 (Bruker D8 Advance,Cu-Kα (λ=1.5405 Å)),透射电子显微镜 (TEM,FEI Tecnai G2 F20) ,光谱仪(FLUROHUB-B, HORIBA JOBIN YVON),紫外灯功率为50W,980nm近红外激光器功率范围为0-2w,电感耦合等离子体原子发射仪(PerkinElmer Optima3300DV)。
X射线衍射样品的制备:将烘干的纳米晶铺满样品支架的凹槽;
透射电子显微镜样品的制备:将每次合成的全部纳米晶溶于4毫升乙醇溶液中,超声5分钟后,滴3-6滴液体于超薄碳膜上。
1.4胃癌细胞(MGC-803)培养和毒性测试
将盖玻片置于培养皿中,倒入需要传代的MGC-803细胞和RPMI1640培养基,然后将培养皿放入CO2培养箱中(5% CO2,37℃),孵育24 h,待显微镜下观察细胞生长饱满、均匀分布时,将生长有细胞的培养皿取出备用。
将MGC-803细胞与50,100,200 μg/mL三种浓度的纳米粒子共培养,每个浓度设3个复孔,并设置调零孔及对照孔,24小时之后观察其细胞数量及形态,均与对照组细胞无明显差别。吸净孔内培养基,每孔重新加入培养基以及MTT溶液共培养4小时后,加入DMSO,摇床低速振荡10分钟。检测各孔OD值。
1.5 NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米颗粒与抗体的连接
将2 mg表面修饰有氨基的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米颗粒溶于2 mL10 mmol·L-1 pH7.4的PBS溶液中,超声分散均匀,加入100μL 0.1mg·mL-1 NHS和100μL0.1mg·mL-1 EDC,室温活化30 min,然后加入400 μL 0.1mg·mL-1羊抗人IgG(anti-CA50)的抗体溶液,在37℃恒温振荡器中反应1 h即可得到NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米颗粒与抗体的结合物,即后续用于MGC-803细胞标记试验的免疫探针。
1.6 细胞的荧光显微镜成像
将连接抗体的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米颗粒滴于有细胞生长的盖玻片上,然后在CO2培养箱中(5% CO2,37℃),孵育1 h。显微镜成像前用PBS缓冲溶液洗涤,冲洗盖玻片去除未与细胞连接的纳米颗粒。然后将盖玻片置于荧光显微镜下观察。
2. 数据分析与讨论
NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu产物的X射线衍射图谱如图1a所示,所有衍射峰均与标准PDF卡片JCPDS 54-0896号一一对应,且无多余的衍射峰,表明本申请得到的产物为纯四方相。透射电子显微镜分析结果如图1b所示,表明产物为四方块状,分散性很好。单分散纳米颗粒在溶液中分散性好,能够与金属离子大面积接触,有利金属离子的检测。图1c可看出包裹二氧化硅后的样品的形貌清晰可见,呈现类球形,平均尺寸大约41 nm,这样的小尺寸足够使纳米粒子用于生物荧光探针的应用。右上角的插图为样品的电子衍射花样,可以看出样品为多晶粉末,此纳米晶具有良好的结晶性。
在980nm近红外光激发条件下,产物在蓝光区域表现出很强的上转换发光(图2a),对应于Tm3+离子1G4→3H6的跃迁;在254nm波长的紫外光激发条件下,产物在红光区域表现出很强的下转换发光,对应于Eu3+离子的f→f跃迁(图2b)。基于此,本申请将紫外氙灯与近红外激光器同时照射样品,产物能够同时表现出蓝光和红光发射(图2c)。随着敏化离子Ce3+离子浓度从10 增到20 mol%,纳米晶的吸收能力增大,因此发光强度显著增强,当Ce3+离子浓度超过20 mol%,敏化离子与激活离子之间的无辐射交叉弛豫几率增大,导致发光强度下降(图2d)。
如图3所示,对照组OD值为 0.475;50,100,200 μg/mL组OD均值分别为 0.460,0.431,0.430。组间无显著差异。可以确定,NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2纳米粒子在无红外光激发状态下对MGC-803细胞几乎无生物毒性作用。
如图4所示,(a)图为明场下的细胞照片,细胞状态良好,(b)图为纳米粒子与MGC-803细胞共培养之后,在980 nm激发下的上转换发光照片,肉眼看上去发明亮的蓝色光,并且很好的对细胞进行了标记,从(c)图中看出上转换发光与细胞轮廓能很好的重合,说明所制备的样品能够成功对MGC-803细胞进行体外荧光标记。
3. 结论
本申请首先通过溶剂热法制备出水溶性NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶,然后在其表面包覆了二氧化硅进行修饰。研究结果表明包裹了二氧化硅的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶表面氨基化后可以与CA50抗原的抗体等生物分子相连接,纳米晶颗粒分散性好,对细胞几乎无毒性作用,且在紫外光和近红外光激发下都能发荧光,从而能够应用于标记细胞的免疫探针制备中。这种荧光探针具有生物相容性好、细胞毒性小和荧光强度高等优点,具有很好的应用前景。
以上为对本申请实施例的描述,通过对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本申请。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的。本文中所定义的一般原理可以在不脱离本申请的精神或范围的情况下,在其它实施例中实现。因此,本申请将不会被限制于本文所示的这些实施列,而是要符合与本文所公开的原理和新颖点相一致的最宽的范围。
Claims (8)
1.一种表面氨基功能化的纳米晶材料,其特征在于,该纳米晶材料的分子式为NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2。
2.根据权利要求1所述的一种表面氨基功能化的纳米晶材料,其特征在于,该纳米晶材料为水溶性纳米晶材料,由二氧化硅包覆NaLuMo2O8:Yb/Tm/Ce/Eu纳米晶构成。
3.根据权利要求1或2所述的一种表面氨基功能化的纳米晶材料,其特征在于,NaLuMo2O8:Yb/Tm/Ce/Eu@SiO2纳米晶的分子式为NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu@SiO2。
4.权利要求1-3任意一项权利要求所述的一种表面氨基功能化的纳米晶材料的制备方法,其特征在于,该方法包括以下的步骤:
1)1毫摩尔硝酸钠,0.645毫摩尔硝酸镥,0.2毫摩尔硝酸铈,0.005毫摩尔硝酸铥,0.05毫摩尔硝酸铕以及3毫摩尔柠檬酸溶于8毫升去离子水中,搅拌均匀;
2)搅拌20分钟后,加入2毫摩尔钼酸钠和30毫升聚乙二醇,搅拌1小时后,转移到高压水热釜中,于180℃加热6小时;产物用乙醇和去离子水的混合液洗涤3-5次;
3)20 mg NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu纳米晶加入70 mL异丙醇中,超声分散20min,加入5 mL水和3 mL的25%的NH4OH,置于30℃水浴中;搅拌下,滴加0.02 mL正硅酸四乙酯;搅拌反应3 h后,将0.2 mLγ-氨丙基三乙氧基硅烷溶于20 mL异丙醇中,缓慢加入到上述反应液中,30 min滴完,继续搅拌反应1 h;通过离心分离,无水乙醇洗涤,50℃干燥2 h后得氨基修饰的NaLuMo2O8:10Yb/0.5Tm/20Ce/5Eu @SiO2纳米颗粒。
5.权利要求1-3任意一项权利要求所述纳米晶材料在胃癌细胞免疫检测中的应用。
6.一种胃癌细胞免疫检测试剂盒,其特征在于,该试剂盒包括权利要求1-3任意一项权利要求所述纳米晶材料。
7.权利要求1-3任意一项权利要求所述纳米晶材料在细胞免疫标记和成像中的应用。
8.一种细胞免疫标记荧光探针,其特征在于,该荧光探针包括权利要求1-3任意一项权利要求所述纳米晶材料。
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