CN111040056B - 荧光纳米粒子的制备及其在细胞表面糖检测中的应用 - Google Patents
荧光纳米粒子的制备及其在细胞表面糖检测中的应用 Download PDFInfo
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Abstract
本发明公开了一种甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法及其在细胞表面糖检测中的应用，包括通过可逆加成片段链转移聚合法合成甲基丙烯酸甘油酯亲水性刷，然后通过可逆加成片段链转移沉淀聚合法合成表面具有亲水性聚合刷的荧光纳米粒子并在其表面接枝上糖的识别原件——苯硼酸，制成细胞表面糖的荧光探针；本荧光纳米粒子表面的高亲水性聚合物刷具有抵抗蛋白的能力，在其表面接枝糖识别元件——苯硼酸使其对具有顺位二醇结构的糖有荧光响应，具有很好的选择性和特异性，能实现在复杂生物样品中对糖的特异性检测。该荧光纳米粒子制备工艺简单易行，易于规模化生产。
Description
技术领域
本发明涉及甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法及其在细胞表面糖检测中的应用。
背景技术
细胞表面的糖基参与细胞识别，并在生物信号传导以及肿瘤发生和转移过程中起到重要的作用。而不同的细胞表面的聚糖种类也不尽相同，相同的细胞在其不同的生长、分化过程中所表达的聚糖种类也会有较大的差别。其中及其有意义的聚糖变化体现在细胞发生癌变时会伴随其表面唾液酸的过表达现象。但是目前来看，糖分子结构复杂性，糖苷键的类型也有所不同，因此针对糖的研究困难重重，与同样是生物体重要组成部分的蛋白质、核酸比起来要滞后的多。近年来，相继发展了气相色谱法、高效液相色谱法、液质联用法以及酶联免疫法等，这些方法往往存在灵敏度低，特异性差或操作复杂，或仪器昂贵的缺点。
发明内容
本发明所要解决的技术问题是：克服现有技术存在的问题，提供一种甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子，该荧光纳米粒子选择性高，灵敏度高，其表面高分子刷的接枝能提高荧光纳米粒子的亲水性，可有效减少非特异性吸附，提高荧光纳米粒子在复杂生物样品检测中抵抗蛋白的能力，从而实现细胞表面糖的快速检测。本发明还提供该荧光探针的制备方法以及该探针在细胞表面糖检测中的应用。
由于荧光纳米材料粒径较小，能在血液中自由运动并通过血液运输运送到机体的各个器官，荧光纳米粒子本身具有荧光属性可以用于细胞的荧光成像，在其表面接上糖识别元件——苯硼酸，使其能与细胞表面糖特异性结合从而实现对细胞表面糖的监测。本发明首次制备了甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子并用于细胞表面糖的选择性检测，从而解决上述技术问题。
本发明提供的技术方案为：甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，包括通过可逆加成片段链转移聚合法合成甲基丙烯酸甘油酯亲水性刷，然后通过可逆加成片段链转移沉淀聚合法合成表面具有亲水性聚合刷的荧光纳米粒子并在其表面接枝上糖的识别原件——苯硼酸，制成细胞表面糖的荧光探针；
其具体包括以下步骤：
S1、甲基丙烯酸甘油单酯的制备：用10％的高氯酸水解甲基丙烯酸缩水甘油酯，得到亲水性单体甲基丙烯酸甘油单酯；
S2、聚甲基丙烯酸甘油酯的制备：将亲水性单体甲基丙烯酸甘油单酯、引发剂偶氮二异丁晴、小分子链转移剂二硫代苯甲酸异丙苯酯在甲醇中反应制成亲水性聚合物刷聚甲基丙烯酸甘油酯；
S3、荧光纳米粒子的制备：将聚甲基丙烯酸甘油酯、偶氮二异丁晴、二硫代苯甲酸异丙苯酯、荧光单体2-羟基乙基丙烯酸-9-蒽甲酸酯、功能单体甲基丙烯酸在乙腈和N,N-二甲基甲酰胺的混合溶剂中反应，得到表面具有亲水性聚合物刷的荧光纳米粒子；
S4、接枝苯硼酸的荧光纳米粒子的制备：将荧光纳米粒子、对氨基苯硼酸溶液、1-乙基-3-(3-二甲氨丙基)碳二亚胺盐酸盐在PBS缓冲液中反应使亲水性聚合物刷修饰的荧光纳米粒子表面接枝上苯硼酸。
进一步地，S1的具体过程为：
S1-1、将10％的高氯酸于20℃下滴加到甲基丙烯酸缩水甘油酯中搅拌反应2h，用氢氧化钾中和未反应的高氯酸，过滤沉淀，得澄清溶液；
S1-2、将澄清溶液用石油醚萃取五次，加氯化钠用乙酸乙酯萃取两次，旋蒸；
S1-3、再用无水MgSO4干燥过夜，产物用中性Al2O3过柱后，在室温真空干燥8h，收集剩余溶液，于-4℃冰箱封口保存。
进一步地，S2的具体过程为：
S2-1、将甲基丙烯酸甘油单酯、偶氮二异丁晴、二硫代苯甲酸异丙苯酯与甲醇先后加入到100mL单颈圆底烧瓶中，室温搅拌反应5min以使上述原料充分溶解；
S2-2、再用冰水浴用氩气饱和30min，封口后将反应瓶放在60℃油浴中，并搅拌反应48h；
S2-3、反应结束后用乙醚沉降两次，在25℃下真空干燥48h，得粉红色粉末状固体。
进一步地，S3的具体过程为：
S3-1、将聚甲基丙烯酸甘油酯、偶氮二异丁晴、二硫代苯甲酸异丙苯酯、荧光单体2-羟基乙基丙烯酸 -9-蒽甲酸酯、功能单体甲基丙烯酸、乙腈(6mL)和N,N-二甲基甲酰胺(4ml)先后加入25mL单颈圆底烧瓶中，室温搅拌5min使上述原料充分溶解；
S3-2、再用冰水浴通氩气30min，封口后将反应瓶置于60℃油浴中，搅拌反应48h；
S3-3、冷却至室温的荧光纳米粒子粗品转移至截留分子量为35000Da的透析袋中，以超纯水作为透析液，透析24h，收集透析袋内的溶液，冻干保存。
进一步地，S4的具体过程为：
S4-1、将荧光纳米粒子加入苯硼酸溶液轻搅2-3min后将EDC溶液加入上述反应混合物中，室温下反应4h；
S4-2、反应结束后，将反应液离心两次(8000rmb、4℃、10min)，取离心沉淀冻干48h得到浅粉色固体。
上述方法制备的甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子在细胞表面糖检测的应用，如肝癌细胞、正常肝细胞、宫颈癌细胞、宫颈上皮细胞等。
本发明公开了一种甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法及其应用，其可用于细胞表面糖含量的检测。
(1)本发明的荧光纳米粒子表面具有亲水性聚合物刷，有阻挡蛋白的功能，能实现在复杂生物样品中对糖的检测。
(2)本发明的荧光纳米粒子是光学性能稳定的纳米荧光材料，能进入到细胞，吸附在细胞表面，能实现对细胞表面糖的检测。
(3)本发明的荧光纳米粒子细胞毒性小，不影响细胞的正常生长。
(4)本发明的荧光纳米粒子在pH＝7.4的PBS缓冲溶液中呈蓝色荧光，随着细胞表面糖过表达，荧光纳米粒子在细胞表面大量聚集，在荧光共聚焦成像中显强蓝色荧光。
(5)该荧光纳米粒子制备工艺简单易行，易于规模化生产。
附图说明
图1为本发明实施的接枝苯硼酸荧光纳米粒子的接触角表征图。
图2为本发明实施的接枝苯硼酸荧光纳米粒子的光学性能图。
图3为本发明实施的接枝苯硼酸荧光纳米粒子与细胞成活率的关系图。
图4为本发明实施的接枝苯硼酸荧光纳米粒子与细胞在荧光共聚焦显微镜下的成像图。
具体实施方式
实施例一
甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，包括以下步骤：
S1、甲基丙烯酸甘油单酯的制备
将10％的高氯酸于20℃下滴加到甲基丙烯酸缩水甘油酯中搅拌反应2h，用氢氧化钾中和未反应的高氯酸，过滤沉淀，得澄清溶液，然后用石油醚萃取五次，加氯化钠用乙酸乙酯萃取两次，旋蒸，用无水 MgSO4干燥过夜，产物用中性Al2O3过柱后，在室温真空干燥8h，收集剩余溶液，于-4℃冰箱封口保存。
S2、聚甲基丙烯酸甘油酯的制备
将甲基丙烯酸甘油单酯、偶氮二异丁晴、二硫代苯甲酸异丙苯酯与甲醇先后加入到100mL单颈圆底烧瓶中，室温搅拌反应5min以使上述原料充分溶解。冰水浴用氩气饱和30min，封口后将反应瓶放在60℃油浴中，并搅拌反应48h。反应结束后，用乙醚沉降两次，在25℃下真空干燥48h，得粉红色粉末状固体。
S3、荧光纳米粒子的制备
将聚甲基丙烯酸甘油酯、偶氮二异丁晴、二硫代苯甲酸异丙苯酯、荧光单体2-羟基乙基丙烯酸-9-蒽甲酸酯、功能单体甲基丙烯酸、乙腈(6mL)和N,N-二甲基甲酰胺(4ml)先后加入25mL单颈圆底烧瓶中，室温搅拌5min使上述原料充分溶解。冰水浴通氩气30min，封口后将反应瓶置于60℃油浴中，搅拌反应48h。冷却至室温的荧光纳米粒子粗品转移至截留分子量为35000Da的透析袋中，以超纯水作为透析液，透析 24h，收集透析袋内的溶液，冻干保存。
S4、接枝苯硼酸的荧光纳米粒子的制备
将荧光纳米粒子加入苯硼酸溶液轻搅2-3min后将EDC溶液加入上述反应混合物中，在室温下反应4h。反应结束后，将反应液离心两次(8000rmb、4℃、10min)，取离心沉淀冻干48h得到浅粉色固体。
一、实施例一中制得接枝苯硼酸的荧光纳米粒子的亲水性能表征
称取10mg实施例一中制得的接枝苯硼酸的荧光纳米粒子，配成浓度为10mg/mL的PBS缓冲液 (0.1mM)，涂抹在玻片上阴干，测试接触角，所得接触角数据如附图1所示；
该实验结果表面接枝苯硼酸的荧光纳米粒子具有高亲水性。
二、实施例一制得接枝苯硼酸的荧光纳米粒子的光学性能表征
称取1mg实施例一中制得的接枝苯硼酸的荧光纳米粒子，配成浓度为1mg/mL的PBS缓冲溶液 (0.1mM)，作为母液；
荧光光谱测试：将30μL上述母液加入到一定量的PBS缓冲溶液(0.1mM)，使接枝苯硼酸的荧光纳米粒子的终浓度为0.1mg/mL，然后测试不同时间(0.5h、1h、2h、3h、4h、6h、8h、10h、12h)下的荧光发射光谱；荧光发射光谱测定以400nm激发，激发与发射的狭缝宽度为5nm/5nm；所得不同时间下荧光光谱图如附图2所示；
该实验结果表明，不同时间下接枝苯硼酸的荧光纳米粒子的荧光强度没有显著差异，证明该接枝苯硼酸的荧光纳米粒子光学性能稳定。
三、实施例一中制得接枝苯硼酸的荧光纳米粒子对细胞生长的影响
称取0.1mg实施例一中制备的接枝苯硼酸的荧光纳米粒子，配成浓度为0.1mg/mL的PBS缓冲液 (0.1mM)，作为母液。
取上述母液20μL加入到一定量的PBS缓冲液(0.1mM，pH7.4)，使接枝苯硼酸的荧光纳米粒子的终浓度分别为0μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL、200μg/mL。用上述不同浓度接枝苯硼酸的荧光纳米粒子分别在肝癌细胞、正常肝细胞、宫颈癌细胞、宫颈上皮细胞孵化24h，测试这四种细胞的存活率，得到接枝苯硼酸的荧光纳米粒子对细胞存活率的影响如附图3所示。
以上结果表明接枝苯硼酸的荧光纳米粒子几乎没有细胞毒性，不影响细胞的正常生长。
四、实施例一中制得接枝苯硼酸的荧光纳米粒子用于细胞表面糖检测。
称取50mg实施例一中制备的接枝苯硼酸的荧光纳米粒子，配成浓度为10mmol/L的PBS缓冲液 (0.1mM)，用上述缓冲液孵化肝癌细胞、正常肝细胞、宫颈癌细胞、宫颈上皮细胞4h，拍荧光共聚焦图像，成像结果如附图4所示。
以上结果表明，当细胞表面糖过表达时，接枝苯硼酸的荧光纳米粒子会吸附在细胞表面显明亮的蓝色荧光(即图中显示白色的部分)，证明该荧光纳米粒子可以用于细胞表面糖检测。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Claims (5)
1.甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，其特征在于，包括通过可逆加成片段链转移聚合法合成甲基丙烯酸甘油酯亲水性刷，然后通过可逆加成片段链转移沉淀聚合法合成表面具有亲水性聚合刷的荧光纳米粒子并在其表面接枝上糖的识别原件——苯硼酸，制成细胞表面糖的荧光探针；
其具体包括以下步骤：
S1、甲基丙烯酸甘油酯的制备：用10%的高氯酸水解甲基丙烯酸缩水甘油酯，得到亲水性单体甲基丙烯酸甘油酯；
S2、聚甲基丙烯酸甘油酯的制备：将亲水性单体甲基丙烯酸甘油酯、引发剂偶氮二异丁腈、小分子链转移剂二硫代苯甲酸异丙苯酯在甲醇中反应制成亲水性聚合物刷聚甲基丙烯酸甘油酯；
S3、荧光纳米粒子的制备：将聚甲基丙烯酸甘油酯、偶氮二异丁腈、二硫代苯甲酸异丙苯酯、荧光单体2-羟基乙基丙烯酸-9-蒽甲酸酯、功能单体甲基丙烯酸在乙腈和N,N-二甲基甲酰胺的混合溶剂中反应，得到表面具有亲水性聚合物刷的荧光纳米粒子；
S4、接枝苯硼酸的荧光纳米粒子的制备：将荧光纳米粒子、对氨基苯硼酸溶液、1-乙基-3-（3-二甲氨丙基）碳二亚胺盐酸盐在PBS缓冲液中反应使亲水性聚合物刷修饰的荧光纳米粒子表面接枝上苯硼酸。
2.根据权利要求1所述的甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，其特征在于，S1的具体过程为：
S1-1、将10%的高氯酸于20℃下滴加到甲基丙烯酸缩水甘油酯中搅拌反应2h，用氢氧化钾中和未反应的高氯酸，过滤沉淀，得澄清溶液；
S1-2、将澄清溶液用石油醚萃取五次，加氯化钠用乙酸乙酯萃取两次，旋蒸；
S1-3、再用无水MgSO4干燥过夜，产物用中性Al2O3过柱后，在室温真空干燥8h，收集剩余溶液，于-4℃冰箱封口保存。
3.根据权利要求1所述的甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，其特征在于，S2的具体过程为：
S2-1、将甲基丙烯酸甘油酯、偶氮二异丁腈、二硫代苯甲酸异丙苯酯与甲醇先后加入到100mL单颈圆底烧瓶中，室温搅拌反应5min以使上述原料充分溶解；
S2-2、再用冰水浴用氩气饱和30min，封口后将反应瓶放在60℃油浴中，并搅拌反应48h；
S2-3、反应结束后用乙醚沉降两次，在25℃下真空干燥48h，得粉红色粉末状固体。
4.根据权利要求1所述的甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，其特征在于，S3的具体过程为：
S3-1、将聚甲基丙烯酸甘油酯、偶氮二异丁腈、二硫代苯甲酸异丙苯酯、荧光单体2-羟基乙基丙烯酸-9-蒽甲酸酯、功能单体甲基丙烯酸、6mL的乙腈和4mL的N,N-二甲基甲酰胺先后加入25mL单颈圆底烧瓶中，室温搅拌5min使上述原料充分溶解；
S3-2、再用冰水浴通氩气30min，封口后将反应瓶置于60℃油浴中，搅拌反应48h；
S3-3、冷却至室温的荧光纳米粒子粗品转移至截留分子量为35000Da的透析袋中，以超纯水作为透析液，透析24h，收集透析袋内的溶液，冻干保存。
5.根据权利要求1所述的甲基丙烯酸甘油酯亲水性刷修饰的荧光纳米粒子的制备方法，其特征在于，S4的具体过程为：
S4-1、将荧光纳米粒子加入对氨基苯硼酸溶液轻搅2-3min后将EDC溶液加入上述反应混合物中，室温下反应4h；
S4-2、反应结束后，将反应液离心两次，离心条件为8000rpm、4℃、10min，取离心沉淀冻干48h得到浅粉色固体。
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| CN107880227A (zh) * | 2017-10-26 | 2018-04-06 | 江南大学 | 一种基于大分子自组装制备糖蛋白印迹荧光纳米粒子的方法 |
| CN108816202A (zh) * | 2018-06-27 | 2018-11-16 | 江苏大学 | 一种双重识别位点糖蛋白表面印迹纳米材料及其制备方法和应用 |
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| CN107880227A (zh) * | 2017-10-26 | 2018-04-06 | 江南大学 | 一种基于大分子自组装制备糖蛋白印迹荧光纳米粒子的方法 |
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