毛细管电泳监视肽接枝壳聚糖膜实时
Summary
Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.
Abstract
自由溶液的毛细管电泳（CE）在溶液中，通过电场的应用分离分析物，通常电荷的化合物。相对于其他的分析分离技术，如色谱法，CE是便宜，耐用和有效不需要样品制备（对于一些复杂的天然基质或聚合物样品）。 CE是快速和可用于实时跟踪（ 例如 ，化学反应动力学）的混合物的演进，作为用于分离的化合物中观察到的信号是直接正比于它们在溶液中的数量。
这里，CE的效率表现出用于监测肽的共价接枝到用于随后的生物医学应用的壳聚糖薄膜。脱乙酰壳多糖的抗微生物和生物相容特性使其用于生物医学应用，如细胞生长底物的有吸引力的材料。共价嫁接肽RGDS（精氨酸 – 甘氨酸 -天冬氨酸 – 丝氨酸）到脱乙酰壳多糖膜的表面旨在改善细胞附着。从历史上看，色谱法和氨基酸分析已经被用于提供接枝肽的量的直接测量。然而，通过CE提供样品制备的快速分离和不存在下使肽接枝过程同样准确但实时监控。 CE是能够分离和量化反应混合物的不同组成部分：（非接枝）肽和化学偶联剂。在这种方式中使用的CE导致下游应用改进的薄膜。
通过固态NMR（核磁共振）光谱的壳聚糖膜进行了表征。这种技术是比较耗时，并且不能在实时应用，但产生的肽的直接测量，从而验证了CE技术。
Introduction
自由溶液的毛细管电泳（CE）是分隔在根据它们的电荷对摩擦比1,2解化合物的技术。电荷-尺寸比通常在文献中所提到的，但这种简化并不适用于聚电解质，其中包括在这项工作中的多肽，并且也显示出不适合小有机分子3。 CE不同于其它分离技术，因为它不具有固定相，仅一个背景电解质（通常是缓冲液）。这使得该技术将在其分析大范围样本的复杂基质4，如植物纤维5，发酵酿造6接枝到合成聚合物7，食品样品8和难溶肽9无需繁琐的样品制备和能力强劲纯化。这是一个复杂的聚电解质具有的溶解问题（S特别显著UCH壳聚糖10和结冷胶11），因此，存在如凝集或在溶液中沉淀，并已成功地分析没有样品过滤。此外，早餐谷类食品糖的分析涉及的早餐麦片样品颗粒注入样品中水8沉淀。这还延伸至支链聚电解质或共聚物12,13的分析。大量的工作也已在CE技术的发展专门为蛋白质的蛋白质组学14，天然或合成的肽15和蛋白质和肽16的微芯片的分离的手性分离的分析完成。由于分离和分析发生在毛细管，样品只有小体积和溶剂被用于使CE为具有比其它分离技术包括色谱法5,6,17较低的运行成本。由于通过CE分离速度快，它允许monito反应动力学的环。这表现在肽的接枝改进细胞粘附18脱乙酰壳多糖膜的情况下。
脱乙酰壳多糖是从几丁质的N- -deacetylation衍生的多糖。脱乙酰壳多糖膜可用于各种生物医学应用，例如生物粘合剂19和细胞生长基板18,20，由于脱乙酰壳多糖的生物相容性21。细胞附着到特定的细胞外基质蛋白，如纤连蛋白，胶原和层粘连蛋白，直接关系到细胞22的存活。值得注意的是，不同的细胞类型往往需要连接到不同的细胞外基质蛋白生存和正常功能。结果表明细胞附着于壳聚糖膜，通过纤维连接蛋白23的嫁接得到加强;然而，制备，纯化和这样大蛋白质的接枝是在经济上不可行。或者一系列小肽甲肝È被证明能够模仿大胞外基质蛋白的性质。例如，肽如纤连蛋白模拟物的RGD（精氨酸-甘氨酸-天冬氨酸）和RGDS（精氨酸-甘氨酸-天冬氨酸-丝氨酸）已经被用于促进和增加细胞附着24。 RGDS到壳聚糖膜的共价嫁接导致已知附加到纤维连接蛋白在体内细胞18提高细胞附着。代较大的蛋白质喜欢和较小的肽具有相同的功能提供了一个显著成本降低纤维连接蛋白。
这里，如先前发表18进行肽接枝到壳聚糖。正如以前证明，这种方法通过使用偶联剂EDC盐酸（1-乙基-3-（3-二甲氨基丙基）碳化二亚胺）和NHS（N-羟基）官能的RGDS的羧酸是提供简单而有效的接枝接枝到壳聚糖薄膜。这种接枝方法的两个优点是，它不需要壳聚糖或肽的任何修改，并且在水性介质中进行的，以最大程度地与未来细胞培养应用18,20的兼容性。作为耦合剂和所述肽可以充电，CE是用于反应动力学的分析的合适方法。重要的是，经由CE中的反应动力学的分析，可以在接枝反应进行实时监控，并且因此能够既优化和量化接枝度。
虽然它不是常规必要，在CE分析的结果可以被离线由肽接枝到利用固体NMR（核磁共振）光谱25,26的脱乙酰壳多糖膜的直接测量验证以证明共价接枝的的肽到薄膜18。然而，随着固态NMR光谱法相比，实时分析由设置CE能够实时肽消耗，从而以评估反应的动力学的能力的定量。
上述方法是简单，并且允许肽的接枝到与接枝的程度的间接定量脱乙酰壳多糖膜的实时分析。所表现出的方法可以扩展到不同的化学反应，只要反应物或待分析可充电产品的实时定量评估。
Protocol
Representative Results
Discussion
这里所描述的协议的简单性使得它非常适合于广泛的应用。但是，需要特别注意支付给下面的关键步骤。
正确的CE仪器准备
它以分离已知的标准之前立即未知样品的分离（以及在一系列分离的结束时），检查在毛细管和仪器在当天的有效性是重要的。这个标准可以是低聚丙烯酸酯27或已知在宽范围的迁移时间，得到多个峰的任何样品…
Disclosures
The authors have nothing to disclose.
Acknowledgements
MG, MO’C and PC thank the Molecular Medicine Research Group at WSU for Research Seed Funding, as well as Michele Mason (WSU), Richard Wuhrer (Advanced Materials Characterisation Facility, AMCF, WSU) and Hervé Cottet (Montpellier) for discussions.
Materials
| Water | Millipore | All water used in the experiment has to be of Milli-Q quality | |
| Chitosan powder (medium molecular weight) | Sigma-Aldrich | 448877 | lot MKBH1108V was used. Significant batch-to-batch variations occur with natural products such as polysaccharides |
| Acetic acid – Unilab | Ajax Finechem | 2-2.5L GL | laboratory reagent |
| Dimethylsulfoxide | Sigma-Aldrich | D4540 | laboratory reagent, slightly hazardous to skin, hazardous if ingested |
| Sodium hydroxide | Sigma-Aldrich | 221465 | laboratory reagent, corrosive |
| RGDS | Bachem | H‐1155 | peptide, bought from Auspep Pty Ltd |
| 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide | Sigma-Aldrich | D80002 | Irritant to skin |
| N-hydroxysuccinimide | Sigma-Aldrich | 130672 | Irritant to skin |
| Sodium chloride | Ajax Finechem | 466-500G | laboratory reagent |
| Potassium chloride – Univar | Ajax Finechem | 384-500G | analytical reagent, slight skin irritant |
| Disodium hydrogen phosphate – Unilab | Ajax Finechem | 1234-500G | laboratory reagent, slight skin irritant |
| Potassium dihydrogen phosphate – Univar | Ajax Finechem | 4745-500G | analytical reagent, slight skin irritant |
| Oligoacrylate standard | custom made | See reference for synthetic protocol: Castignolles, P.; Gaborieau, M.; Hilder, E. F.; Sprong, E.; Ferguson, C. J.; Gilbert, R. G. Macromol. Rapid Commun. 2006, 27, 42-46 | |
| Boric acid | BDH AnalR, Merck Pty Ltd | 10058 | Corrosive |
| Hydrochloric acid – Unilab | Ajax Finechem | A1367-2.5L | laboratory reagent, corrosivie |
| Fused silica tubing | Polymicro (Molex) | TSP050375 | Flexible fused silica capillary tubing with standard polyimide coating, 50 µm internal diameter, 363 µm outer diameter |
| Agilent 7100 CE | Agilent Technologies | G7100CE | Capillary electrophoresis instrument |
| Orbital shaker | IKA | KS260 | |
| Electronic balance | Mettler Toledo | MS204S | |
| Milli-Q Synthesis | Millipore | ZMQS5VF01 | Ultrapure water filtration system |
| Parafilm | Labtek | PM966 | Parrafin wax |
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