Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase
Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase
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DOI：
10.1039/c1an15137a
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发表时间：
2011-01-01
期刊：
影响因子：
4.2
通讯作者：
Paull, Brett
中科院分区：
文献类型：
--
作者：
Alwael, Hassan;Connolly, Damian;Paull, Brett
原位制备的乙烯二甲基丙烯酸酯多孔聚合物整料内20 μ L的聚丙烯吸液管尖端，通过表面接枝甲基丙烯酸酯锚网站，结合报告。金纳米颗粒（AuNPs）固定到整料孔表面上，利用azlactone化学和覆盖验证使用场发射扫描电子显微镜。刺桐凝集素（ECL）通过生物功能接头固定在所附的AuNPs上。由于ECL的特异性，ECL修饰的尖端成功地应用于半乳糖基化蛋白（去唾液酸化转铁蛋白）与非半乳糖基化蛋白（核糖核酸酶B）的富集。反相毛细管HPLC用于验证开发的微萃取相的效率和选择性，其导致由于AuNP增强的表面积而导致萃取回收率增加类似于95%。ECL修饰的尖端的进一步特异性用具有不同末端糖结构的非糖基化和糖基化蛋白质的复杂混合物证明。最后，将凝集素亲和相应用于加标半乳糖基化糖蛋白的大肠杆菌细胞裂解物，以成功证明基质耐受性。
The in situ preparation of ethylene dimethacrylate porous polymer monoliths within 20 mu L polypropylene pipette tips, bound via surface grafted methacrylate anchor sites, is reported. Gold nano-particles (AuNPs) were immobilised onto the monolith pore surface utilising azlactone chemistry and coverage verified using field emission scanning electron microscopy. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a bio-functional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialylated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of similar to 95% due to the AuNP enhanced surface area. Further specificity of the ECL-modified tip was demonstrated with a complex mixture of non-glycosylated and glycosylated proteins with differing terminal sugar structures. Finally, the lectin affinity phase was applied to a galactosylated glycoproteins spiked Escherichia coli cell lysate to successfully demonstrate matrix tolerance.