生物功能纳米纤维的制备方法

摘要

本文描述了一种制备具有可特异性结合蛋白质功能基团的纳米纤维的高效方法。该方法首先需要制备带有适当功能基团的聚合物。通过静电纺丝技术将该功能化聚合物加工成纳米纤维。采用共聚焦显微镜研究纳米纤维与蛋白质结合的有效性。

摘要

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, ¹H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

方案

1. α,ω-二羟基聚合物[HO-PEO-P2MS-PEO-OH]的合成

1. 按照图1所示组装聚合反应器。本实验所用反应器包括一个100 ml圆底双颈烧瓶（Chemglass），两个带旋塞的流量控制接头（Chemglass）和一根聚四氟乙烯（Teflon）搅拌棒。接头A（图1）用于使超纯氮气（UHP Nitrogen）持续通过系统，以防止空气和水分进入惰性环境。接头B（图1）用于将溶剂、单体和引发剂注入反应烧瓶中。
2. 使用钠金属干燥200 ml四氢呋喃（THF），以二苯甲酮作为指示剂，在干燥氮气氛围下至少干燥6小时。
3. 将10 ml 2-甲氧基苯乙烯在氢化钙中干燥24小时。
4. 使用异丙醇与液氮混合物制备并维持一个-78 °C的低温浴。
5. 在氮气氛围下向聚合反应烧瓶中加入25 ml THF（见图1），并在整个聚合过程中始终保持反应器处于氮气氛围中。
6. 将100 ml圆底烧瓶放入上述低温混合液中。
7. 向反应烧瓶中加入2 ml（0.27 mmol/ml）引发剂溶液。
8. 注入第一种单体，即2-甲氧基苯乙烯（4 ml）至反应烧瓶中。
9. 让反应持续进行40分钟。
10. 加入1 ml第二种单体，环氧乙烷。
11. 使聚合反应....

讨论

在本报告中，我们介绍了一种制备生物功能化纳米纤维的强有力方法。该纳米纤维被修饰上可特异性结合模型蛋白的功能基团。本文所报道的实验步骤与方法具有普适性，可用于制备携带任意所需功能基团的纳米纤维。阴离子活性聚合是一种强大的技术，可用于合成结构可控的聚合物，并通过共价键连接任意数量的功能分子或功能基团，这些功能基团可特异性识别特定的目标生物标志物。对于单体2-methoxystyrene而言，阴离子活性聚合技术已十分成熟。² 静电纺丝是一种多功能技术，通过改变电压以及调节待纺溶液的浓度，可轻松控制纤维的尺寸。⁵ 纳米纤维表现出电阻型I-V特性，因此有望作为生物传感器中的活性组分发挥作用 即 该报道的方法在开发生物标志物检测装置方面具有广阔前景。^6,7

第一种单体2-甲氧基苯乙烯的聚合在40分钟内完成100%，i.e. 100%的单体转化为聚合物，而第二种单体的聚合较慢，需要2天时间才能完成聚合。也就是说，单体一的聚合速度比单体二快。单体一没有剩余，但在2天结束时仍有一些未反应的单体二，.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
钠金属	Sigma-Aldrich	282065
二苯甲酮	Sigma-Aldrich	239852
2-甲氧基苯乙烯	Sigma-Aldrich	563064
四氢呋喃	Sigma-Aldrich	178810
氯苯	Sigma-Aldrich	319996
单壁碳纳米管	Sigma-Aldrich	704113
聚苯乙烯	Sigma-Aldrich	81416
硅晶圆	Silicon Quest Int’l	720200
蔡司场发射扫描电子显微镜	Carl Zeiss Inc.	Ultra 60
配有 Bausch & Lomb MicroZoom II 高性能显微镜的探针台	Bausch and Lomb
徕卡扫描共聚焦系统	Leica Microsystems	TCS SP2
亚飞安远程源表	Keithley Instruments	6430
自动量程数字万用表	Keithley Instruments	175A
注射泵	Chemyx Inc.	Fusion 200
蔡司光学显微镜	Carl Zeiss Inc.	Zeiss/Axiotech