CN104459154A - 一种新型人工模拟酶的合成及对糖基检测方法的构建 - Google Patents
一种新型人工模拟酶的合成及对糖基检测方法的构建 Download PDFInfo
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Abstract
本发明基于石墨烯家族人工模拟酶对双氧水的催化作用以及探针分子刀豆蛋白和细胞表面糖基的结合作用，构建了以人工模拟酶代替天然过氧化物酶的方法灵敏检测肿瘤细胞表面的糖基表达。发明的人工模拟酶由石墨烯、铁卟啉及金纳米棒构成，三重复合物之间的协同作用大大增强了其催化活性。利用静电吸附作用在人工模拟酶表面固定探针分子刀豆蛋白，其和细胞表面糖基的特异性结合作用可以捕获肿瘤细胞。根据人工模拟酶对底液3,3',5,5'-四甲基联苯胺和双氧水催化信号的强度实现肿瘤细胞表面糖基的检测。本发明通过简单方法合成新型人工模拟酶，与辣根过氧化物酶等天然类过氧化物酶相比，稳定性提高，催化活性大大增强，提高了检测的灵敏度。
Description
技术领域
本发明涉及一种新型三重人工模拟酶的合成及以其作为催化剂的肿瘤细胞表面糖基检测方法的构建。
背景技术
几乎所有真核细胞的表面都含有丰富的聚糖。糖基的功能复杂而多样，参与了绝大部分真核生物的生命过程，与某些疾病尤其是肿瘤的发生、转移密切相关。细胞表面的糖基作为细胞信息功能的重要承担者，不仅是酶、凝集素、细菌等的识别分子，同时影响着细胞之间的识别、联络以及信号传导等过程。因而可以通过研究癌细胞表面的糖基在细胞分化时的作用为癌细胞的某些恶性行为提供有效的分子机制。糖基化的改变不仅能够为癌症提供行之有效的检测和治疗方法，而且还能作为预后性症状的指示。因而实现细胞表面聚糖的原位、实时监测能够为深入了解糖基化的变化在细胞的點附、迁移及恶性肿瘤的形成中的作用，对某些疾病尤其是癌症的早期诊断和研究具有非常重要的临床意义。
近年来发现，某些纳米材料具有与天然酶类似的催化活性，作为人工模拟酶引起了人们的极大兴趣。例如最初发现的四氧化三铁纳米粒子具有与辣根过氧化酶 (HRP) 相似的催化活性, 能够催化酶底物如3,3’,5,5’-四甲基联苯胺 (TMB) 等与双氧水发生氧化还原反应，生成蓝色的氧化产物。随后陆续发现，很多纳米材料均具有模拟酶活性，包括过渡金属纳米材料、贵金属纳米材料、碳纳米材料等。与天然酶相比，纳米材料具有稳定性高、催化活性高及廉价易得等优点，特别是避免了天然酶的不稳定和易变性特征，增加了其在过程催化和酶促动力学领域应用的前景，因而这些具有模拟酶活性的纳米材料在分析化学中具有重要的意义。
本专利创新性地制备了一种基于石墨烯、铁卟啉及金纳米棒的三重纳米复合物，制备的三重纳米复合物作为一种新型人工模拟酶，具有极佳的类过氧化物酶催化性质，且其表面带有大量正电荷，有利于探针刀豆蛋白的修饰。进一步结合刀豆蛋白和细胞表面糖基的亲合作用，实现肿瘤细胞表面糖基的检测。通过比较不同细胞作用下人工模拟酶对底物TMB与双氧水的催化反应生成蓝色的氧化产物的颜色的深浅，可评估细胞表面糖基的表达。
发明内容
本发明的目的是设计合成基于石墨烯、铁卟啉及金纳米棒的三重纳米材料人工模拟酶，基于此人工模拟酶对底液3,3',5,5'-四甲基联苯胺和双氧水的催化作用以及探针分子刀豆蛋白和细胞表面糖基的结合作用，构建了以人工模拟酶代替天然过氧化物酶的方法灵敏检测肿瘤细胞表面的糖基表达。石墨烯家族类过氧化物酶由石墨烯、铁卟啉及金纳米棒构成，三重复合物之间的协同作用大大增强了此人工模拟酶的催化活性。本发明进一步利用静电吸附作用在人工模拟酶表面固定探针分子刀豆蛋白，利用刀豆蛋白和细胞表面糖基的特异性结合作用，可以结合肿瘤细胞。微孔板上首先固定一层刀豆蛋白探针分子，然后通过三明治夹心结构结合肿瘤细胞及表面修饰刀豆蛋白的人工模拟酶材料。根据人工模拟酶对底液3,3',5,5'-四甲基联苯胺和双氧水催化信号的强度实现肿瘤细胞表面糖基的检测。本发明通过简单方法合成新型人工模拟酶，与辣根过氧化物酶等天然类过氧化物酶相比，稳定性提高，催化活性大大增强，提高了检测的灵敏度。
本方法所用仪器装置为：LB 942 微孔板（berthold, Germany）；DF-101S集热式恒温加热磁力搅拌器(巩义市英峪予华仪器厂)；pHS-25型酸度计(上海雷磁仪器厂)；BS124S电子天平(北京赛多利斯系统有限公司)；SZ-93自动双重纯水蒸馏器(上海亚荣生化仪器厂)；KQ-50B型超声波清洗器(昆山市超声仪器有限公司)；电热恒温干燥箱。
所用试剂为：羧基化修饰的石墨烯分散液(南京先丰纳米材料科技有限公司)；NaBH4 及抗坏血酸 (上海化学试剂有限公司)；1-(3-二甲基氨丙基)碳二亚胺盐酸盐 (EDC) 、N-羟基丁二酰亚胺 (NHS) 及刀豆蛋白均购自Sigma公司。实验所用试剂均为分析纯，水均为二次蒸馏水。
附图说明
图1为三重人工模拟酶的透射电镜表征图；
图2为以三重人工模拟酶为催化剂检测细胞表面糖基过程的示意图；
图3为三重人工模拟酶为催化剂与传统人工模拟酶的催化性能比较，(a) 为空白对照，(b)为金纳米棒、铁卟啉及石墨烯的混合液，(c)为发明前的双重人工模拟酶，(e) 为本发明所合成的三重人工模拟酶。
图4为三重人工模拟酶为催化剂检测细胞表面糖基。
具体实施方式
以下为实施本发明的具体示例，其作用在于进一步阐明本发明的内容，使阅读者更容易理解，但不构成对本发明要求的保护范围的限定或限制。
实施例一 三重人工模拟酶的制备
10 g石墨烯（GO）溶解于100 mL 干燥的四颈瓶中，加入40 mL 超纯水。搅拌超声30 min 后，加入60 μL 氨水溶液和10 mg 铁卟啉（hemin），接着加入10μL 联氨溶液。上述混合液水浴中加热到60 ℃，保持4 h。产物经10000 rpm 离心25 min后水洗数次，经真空干燥后得到双重人工模拟酶（H-RGO）。
三重人工模拟酶（H-RGO-Au）在H-RGO的基础上通过NaBH4及抗坏血酸存在下HAuCl4的还原制得。首先制备金纳米棒的种子溶液，将1 mL 0.12 mM HAuCl4和1 mL 0.048 M CTAB溶液混合，在不断搅拌中加入0.12 mL 新鲜配制的2.4 mM NaBH4。此时溶液颜色由黄色变为棕黄色。种子溶液在使用前放置在27 ℃ 环境中2 h。
生长液的制备：将50 mL 0.2 mM HAuCl4 和50 mL 0.048 M十六烷基三甲基溴化铵 (CTAB)溶液混合，然后于室温条件下温柔加入2.5 mL 0.96 mM AgNO3溶液和3.4 mg H-RGO。 之后，加入0.7 mL 0.01M 抗坏血酸，此时溶液颜色从暗黄色变为无色。接着加入0.12 mL之前配制好的种子液，于27 ℃条件下反应20 min。上述溶液被移至30 ℃条件下反应12 h。反应产物经8500 rpm 离心25 min后去除上清液，沉淀重新分散于超纯水中。
实施例二 刀豆蛋白连接的三重人工模拟酶（ConA-H-RGO-AuNRs）的制备
因为刀豆蛋白的等电点在4.5-5.5之间，而合成的三重人工模拟酶表面带有大量正电荷，刀豆蛋白和三重人工模拟酶之间的结合可以通过静电吸附作用完成。具体操作步骤如下：100 μL 溶于PBS缓冲液中的H-RGO-AuNRs 和100 μL 2.0 mg mL-1 刀豆蛋白（Con A）溶液混合，室温下搅拌1.5 h，然后10000 rpm 离心20 min。之后用PBS洗涤，重新分散于200 μL PBS中。
实施例三 细胞表面糖基的比色法检测
如图2所示，羧基修饰的微孔板用新鲜配制的含有30 mg mL-1 EDC和15 mg mL-1 NHS的咪唑盐酸缓冲溶液处理30 min，加入10 μL Con A 溶液反应12 h。用3% 牛血清白蛋白（BSA）于37 ℃条件下反应30 min去除非特异性吸附。
200 μL 含有1mM Ca2+ 和1mM Mn2+ 的K562细胞悬浊液滴到经上述方法处理的微孔板上，37 ℃条件下孵育90 min。经冲洗和干燥后，加入10 μL刀豆蛋白连接的三重人工模拟酶（ConA-H-RGO-AuNRs）在相同条件下继续反应90 min。实验过程中，在肿瘤细胞的存在下通过刀豆蛋白和细胞表面糖基的特异性作用，具有催化性能的人工模拟酶引入到微孔板表面。从而催化底物TMB和双氧水的反应，使本来无色的溶液显蓝色。
其实验结果：
如图3所示，传统的人工模拟酶材料催化性能不高，而本发明中制备的三重人工模拟酶材料催化性能大大增加。这主要是由于三重人工模拟酶材料中石墨烯、铁卟啉和金纳米棒之间的协同增强作用所致。
经肿瘤细胞作用后，合成的人工模拟酶可以催化底物TMB和双氧水的反应，使本来无色的溶液显蓝色。收集微孔板中催化后的溶液放置于样品管中，可明显观察到溶液显蓝色，而作为对照的未经细胞作用的微孔板中的溶液仍然呈无色，如图4所示。
Claims (5)
1.一种新型人工模拟酶的合成及对糖基检测方法的构建，其特征在于：利用静电吸附作用在人工模拟酶表面固定探针分子刀豆蛋白，利用刀豆蛋白和细胞表面糖基的特异性结合作用，可以结合肿瘤细胞；电极上首先固定一层刀豆蛋白探针分子，然后通过三明治夹心结构结合肿瘤细胞及表面修饰刀豆蛋白的人工模拟酶材料；根据人工模拟酶对底液3,3',5,5'-四甲基联苯胺和双氧水电化学催化信号的强度实现肿瘤细胞表面糖基以及肿瘤细胞的定量检测。
2.按照权利要求1所述一种新型人工模拟酶的合成及对糖基检测方法的构建，其特征在于新型三重人工模拟酶的构建：合成方法中引入石墨烯、铁卟啉及金纳米棒，构建三重人工模拟酶。
3.按照权利要求1所述一种新型人工模拟酶的合成及对糖基检测方法的构建，其特征在于以三重人工模拟酶为催化剂：利用静电吸附作用在人工模拟酶表面固定探针分子刀豆蛋白，利用刀豆蛋白和细胞表面糖基的特异性结合作用，在微孔板上构建刀豆蛋白/肿瘤细胞/刀豆蛋白修饰的人工模拟酶材料的三明治夹心结构，根据人工模拟酶对底液3,3',5,5'-四甲基联苯胺和双氧水催化信号的强度实现肿瘤细胞表面糖基的检测。
4.按照权利要求1所述一种新型人工模拟酶的合成及对糖基检测方法的构建，其特征在于对催化信号的检测，催化剂为三重人工模拟酶，催化底物为3,3',5,5'-四甲基联苯胺和双氧水。
5.按照权利要求1所述一种新型人工模拟酶的合成及对糖基检测方法的构建，其特征在于利用新型三重人工模拟酶的强催化作用和高稳定性，提高检测性能。
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