Functionalized magnetic micro- and nanoparticles:: Optimization and application to μ-chip tryptic digestion
Functionalized magnetic micro- and nanoparticles:: Optimization and application to μ-chip tryptic digestion
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DOI：
10.1002/elps.200500587
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发表时间：
2006-05-01
期刊：
影响因子：
2.9
通讯作者：
Viovy, Jean-Louis
中科院分区：
文献类型：
--
作者：
Bilkova, Zuzana;Slovakova, Marcela;Viovy, Jean-Louis
描述了用于μ芯片应用的易于更换的蛋白酶微反应器的制备。利用包被有聚(N-异丙基丙烯酰胺)、聚苯乙烯、聚(甲基丙烯酸2-羟乙酯-共-乙二醇二甲基丙烯酸酯)、聚(甲基丙烯酸缩水甘油酯)、[(2-氨基乙基)羟亚甲基]二膦酸或固定化胰蛋白酶的海藻酸的磁性颗粒进行异相消化。通过磁场梯度允许在微通道中固定的限制对特性进行了优化。为了获得最高的消化效率，通过垂直于通道方向的不均匀外部磁场来组织亚微米球体。测定了固定在μ芯片毛细管中的酶反应器（μ芯片固定磁性酶反应器（IMER））的动力学参数。蛋白水解反应器的能力由分子量范围为 4.3 至 150 kDa 的五种模型（糖）蛋白证明。使用 SDS-PAGE、HPCE、RP-HPLC 和 MS 研究了各种构象蛋白质的消化效率。 mu-chip IMER 系统与高分子量（糖）蛋白的完全和有限蛋白水解的兼容性得到了证实。它为自动化、高通量蛋白质组 mu 芯片设备开辟了道路。
The preparation of an easily replaceable protease microreactor for mu-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in mu-chip capillary (mu-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the mu-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic mu-chip devices.