CN110982098B - 锡/蛋白质纳米杂化膜及其应用 - Google Patents

锡/蛋白质纳米杂化膜及其应用 Download PDF

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CN110982098B
CN110982098B CN201910439761.XA CN201910439761A CN110982098B CN 110982098 B CN110982098 B CN 110982098B CN 201910439761 A CN201910439761 A CN 201910439761A CN 110982098 B CN110982098 B CN 110982098B
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杨鹏
巴萨姆·赛义夫
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Abstract

本发明公开了一种锡/蛋白质纳米杂化膜及其应用，该杂化膜是通过Sn2+还原蛋白质二硫键来诱导蛋白质自组装形成的锡/蛋白质复合纳米薄膜，所述的蛋白质为溶菌酶、牛血清白蛋白、胰岛素或β‑乳球蛋白。本发明杂化膜的制备方法简单、快速，易实现大面积的制备，且其具有良好的生物相容性，对细胞无明显毒性，具有较好的化学稳定性和机械稳定性、光学透明度，以及较好的粘附性和柔性，可粘附到各种基材表面。本发明杂化膜对大肠杆菌、金黄色葡萄球菌和白色念珠菌具有很好的抑制效果，同时具有良好的催化性能，可作为光催化材料在可见光照射下光催化分解水产氢，还可作为电催化材料制备导电聚合物膜。

Description

锡/蛋白质纳米杂化膜及其应用
技术领域
本发明涉及一种多功能锡掺杂的二维蛋白质纳米膜，以及该膜在微图案化、抗菌和光/电催化中的多种用途。
背景技术
多肽和蛋白质的自组装用于创建结构明确、稳定性好的二维(2D)纳米级组件，这对生物学和技术都具有重要意义。与基于肽的系统相比，蛋白质具有更大的结构和化学复杂性，因此，将蛋白质组装成超分子结构通常并不简单，而且比肽的过程更具挑战性。尤其，将其组装成一种具有更大尺度且均匀的宏观蛋白质2D材料，与一般大尺寸的自然物体相比，仍然是一个挑战。此外，低组装效率和速度阻碍了可规模化生产。
金属离子通过缔合作用对自然界中超过三分之一的天然蛋白质起着至关重要的作用，这种固有的金属原子对蛋白质的功能起着催化、调节或结构上的关键作用。因此，这些金属蛋白从光合作用和药物代谢到呼吸和水氧化等许多关键的细胞过程中发挥着至关重要的作用。然而，大多数的策略相对复杂，效率低，不适合大规模的工程应用。实际上，自然界中广泛存在的金属与蛋白质之间良好的相互作用，在合成体系中是不容易被(尤其是重金属离子)。这种限制通常导致设计和合成金属/蛋白质杂化材料的难度大，效率低。
发明内容
本发明的目的是提供一种利用金属定向诱导蛋白质自组装形成的锡/蛋白质纳米杂化膜，并为该杂化膜提供新的应用。
针对上述目的，本发明所采用的锡/蛋白质纳米杂化膜是Sn2+诱导蛋白质自组装形成的锡/蛋白质复合纳米薄膜，所述的蛋白质为溶菌酶、牛血清白蛋白、胰岛素、β-乳球蛋白中任意一种。
上述锡/蛋白质纳米杂化膜的制备方法为：将含蛋白质的4-羟乙基哌嗪乙磺酸缓冲溶液与SnCl2混合，并用盐酸调节混合液的pH为1～2，在气液界面或固液界面形成的锡/蛋白质复合纳米薄膜。
上述制备方法中，所述混合液中蛋白质的浓度为5～50mg/mL，优选蛋白质的浓度为10～20mg/mL。
上述制备方法中，所述混合液中SnCl2的浓度为2～40mg/mL，优选SnCl2的浓度为5～15mg/mL。
本发明锡/蛋白质纳米杂化膜作为光刻胶的应用。
本发明锡/蛋白质纳米杂化膜作为抗菌材料的应用。
本发明锡/蛋白质纳米杂化膜作为光催化材料光催化分解水产氢的应用。
本发明锡/蛋白质纳米杂化膜作为电催化材料制备导电聚合物膜的应用。
本发明的有益效果如下：
1、本发明利用Sn2+有效地还原蛋白质的二硫键来诱导蛋白质快速自组装(在几秒钟内发生)，得到面积可控、功能多样的锡/蛋白质纳米杂化膜。所得锡/蛋白质纳米杂化膜的厚度可调，具有较好的粘附性，可粘附到各种基材表面，且具有柔性，以及较好的化学稳定性和机械稳定性、光学透明度。
2、本发明锡/蛋白质纳米杂化膜具有生物相容性，对细胞无明显毒性，且具有很好的抗菌效果，对大肠杆菌、金黄色葡萄球菌和白色念珠菌的杀菌率高达99％。
3、本发明锡/蛋白质纳米杂化膜具有良好的催化性能，可作为光催化材料在可见光照射下光催化分解水产氢，还可作为电催化材料制备导电聚合物膜。
4、本发明锡/蛋白质纳米杂化膜的制备方法简单，快速，易实现大面积制备。
附图说明
图1是实施例1形成的锡/溶菌酶纳米杂化膜的照片。
图2是实施例1形成的锡/溶菌酶纳米杂化膜的X射线光电子能谱图。
图3是实施例1形成的锡/溶菌酶纳米杂化膜的原子力显微镜图。
图4是采用细胞计数Kit-8(CCK-8)分析实施例4形成的锡/溶菌酶纳米杂化膜对细胞毒性图。
图5是实施例9中粘附有锡/溶菌酶纳米杂化膜的硅片光刻后的扫描电镜图。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
在室温下，将100mg溶菌酶加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.08g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8，5min内在气液界面清晰地漂浮着一层锡/溶菌酶纳米杂化膜(见图1～3)。所得锡/溶菌酶纳米杂化膜的厚度为50nm，具有较好的柔性，杨氏弹性模量为0.2GPa，硬度为0.02GPa。
实施例2
在室温下，将200mg溶菌酶加入盛有10mL 10mmol/L pH为7.4 4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.08g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8，5min内在气液界面清晰地形成锡/溶菌酶纳米杂化膜。
实施例3
在室温下，将50mg溶菌酶加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.08g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8，5min内在气液界面清晰地形成锡/溶菌酶纳米杂化膜。
实施例4
在室温下，将200mg溶菌酶加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的培养皿中，然后加入0.04g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8，5min内在气液界面清晰地形成锡/溶菌酶纳米杂化膜。
将所得锡/溶菌酶纳米杂化膜转移到钛片上，用氮气吹干，得到表面粘附有锡/溶菌酶纳米杂化膜的钛片。在紫外光照射下，分别将表面粘附有锡/溶菌酶纳米杂化膜的钛片和空白钛片用体积浓度为70％的乙醇水溶液浸泡2h消毒；将骨髓间充质干细胞以1×104个细胞/cm2接种于96孔板中，然后分别将消毒后的表面粘附有锡/溶菌酶纳米杂化膜的钛片和空白钛片放入96孔板中，加入500μL DMEA培养液，在37℃、5％CO2的细胞培养箱内培养，用DMEA培养液作为对照，培养液每隔一天更换一次。分别培养至3、5、7天后，在96孔板的各孔板中加入100μL新的培养液和10μL CCK-8溶液，然后于37℃、5％CO2的环境中孵育4小时。最后，用分光光度法在酶标仪上测定450nm处培养液的吸光度。由图4可见，该膜对细胞无明显毒性。
实施例5
在室温下，将200mg溶菌酶加入10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液中，然后加入0.04g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8后，滴加到FTO玻璃表面，静置2min，在FTO玻璃表面形成锡/溶菌酶纳米杂化膜，用超纯水洗净，氮气吹干。
将表面形成锡/溶菌酶纳米杂化膜的FTO玻璃分别浸泡在乙醇、正己烷、石油醚、HCl水溶液(pH为2和3)或NaOH水溶液(pH为9和13)中，实验结果显示，浸泡30min后膜仍能够稳定存在，说明形成的锡/溶菌酶纳米杂化膜化学稳定性好。
另外，将表面形成锡/溶菌酶纳米杂化膜的FTO玻璃用3M胶带反复撕拉三次，膜未剥落，说明形成的锡/溶菌酶纳米杂化膜粘附性较好。
实施例6
在室温下，将200mg牛血清白蛋白加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.15g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.8，5min内在气液界面清晰地形成锡/牛血清白蛋白纳米杂化膜。
实施例7
在室温下，将400mg胰岛素加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.30g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.5，5min内在气液界面清晰地形成锡/胰岛素纳米杂化膜。
实施例8
在室温下，将500mgβ-乳球蛋白加入盛有10mL 10mmol/L pH为7.4的4-羟乙基哌嗪乙磺酸缓冲溶液的10mL培养皿中，然后加入0.40g SnCl2，混合均匀，所得混合液用盐酸调节pH为1.5，5min内在气液界面清晰地形成锡/β-乳球蛋白纳米杂化膜。
实施例9
锡/溶菌酶纳米杂化膜作为光刻胶的应用
将实施例1得到的锡/溶菌酶纳米杂化膜粘附到硅片上后放在光掩模和ITO玻璃板之间固定好，在1000W高压汞灯下用波长365nm、光强12000μw/cm2的紫外线辐照1小时。紫外线照射后，直接在超纯水中冲洗30秒(冲洗降解的分子)，并用氮气吹干。由图5可见，经光刻后锡/溶菌酶纳米杂化膜在硅片表面形成微图案。
实施例10
锡/溶菌酶纳米杂化膜作为抗菌材料的应用
将储存在低温的大肠杆菌、金黄色葡萄球菌和白色念珠菌的菌液分别稀释106倍、107倍后，均匀的涂布在细菌固体培养基上，37℃下培养12h，然后从固体培养基上挑选一个单菌落置于液体培养基中震荡过夜。所得菌液通过离心沉淀的方法除去培养基后重悬在0.9％的氯化钠水溶液中。向终浓度为2×107cell/cm2的细菌悬浮液中加入实施例1得到的锡/溶菌酶纳米杂化膜，在37℃孵育2h后，用PBS缓冲溶液洗下存活的细菌微生物，同时以没有添加锡/溶菌酶纳米杂化膜的细菌悬浮液作为对照。实验果显示，锡/溶菌酶纳米杂化膜对大肠杆菌、金黄色葡萄球菌和白色念珠菌的杀菌率均高达99％以上。
实施例11
锡/溶菌酶纳米杂化膜的光催化性能
在耐热的玻璃密闭气体再循环系统中，将0.03g实施例1得到的锡/溶菌酶杂化膜分散于10mL三乙醇胺和90mL蒸馏水的混合液，催化反应保持真空条件，利用在300W氙灯(λ>420nm)作为照射光源，产生的氢气量用气相色谱法测定。100分钟时可产生0.75μmol H2。
实施例12
锡/溶菌酶纳米杂化膜的电催化性能
将实施例4得到的锡/溶菌酶纳米杂化膜转移到FTO玻璃片上，用氮气吹干，得到粘附有锡/溶菌酶纳米杂化膜的FTO玻璃。以粘附有锡/溶菌酶纳米杂化膜的FTO玻璃为工作电极，Pt线为对电极，Ag/AgCl为参比电极，在5mL 0.2mol/L pH为5.3的Na2SO4水溶液中加入吡咯单体(使体系中的浓度为50mol/L)后，采用循环伏安法，在-1～1.8V之间进行循环伏安扫描，扫描速率为100mV s-1。实验结果显示，在电位为0.9V下，在粘附有锡/溶菌酶纳米杂化膜的FTO玻璃上沉积了一层黑色聚吡咯薄膜。
另外，在不施加任何额外电压的情况下，直接将实施例4得到的锡/溶菌酶纳米杂化膜浸泡在50mmol/L氯化铜水溶液中，然后在室温下，暴露于50mmol/L吡咯水溶液中汽化30h，最终也会在锡/溶菌酶纳米杂化膜上形成黑色的聚吡咯膜。

Claims (9)

1.一种锡/蛋白质纳米杂化膜，其特征在于：该杂化膜是Sn2+诱导蛋白质自组装形成的锡/蛋白质复合纳米薄膜，所述的蛋白质为溶菌酶、牛血清白蛋白、胰岛素、β-乳球蛋白中任意一种；
所述的锡/蛋白质纳米杂化膜的制备方法为：将含蛋白质的4-羟乙基哌嗪乙磺酸缓冲溶液与SnCl2混合，并用盐酸调节混合液的pH为1～2，在气液界面或固液界面形成的锡/蛋白质复合纳米薄膜。
2.根据权利要求1所述的锡/蛋白质纳米杂化膜，其特征在于：所述混合液中蛋白质的浓度为5～50mg/mL。
3.根据权利要求1所述的锡/蛋白质纳米杂化膜，其特征在于：所述混合液中蛋白质的浓度为10～20mg/mL。
4.根据权利要求2所述的锡/蛋白质纳米杂化膜，其特征在于：所述混合液中SnCl2的浓度为2～40mg/mL。
5.根据权利要求3所述的锡/蛋白质纳米杂化膜，其特征在于：所述混合液中SnCl2的浓度为5～15mg/mL。
6.权利要求1所述的锡/蛋白质纳米杂化膜作为光刻胶的应用。
7.权利要求1所述的锡/蛋白质纳米杂化膜作为抗菌材料的应用。
8.权利要求1所述的锡/蛋白质纳米杂化膜作为光催化材料光催化分解水产氢的应用。
9.权利要求1所述的锡/蛋白质纳米杂化膜作为电催化材料制备导电聚合物膜的应用。
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