Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith:: Integrating solid-phase extraction and enzymatic digestion for peptide mass mapping
Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith:: Integrating solid-phase extraction and enzymatic digestion for peptide mass mapping
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DOI：
10.1021/ac034108j
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发表时间：
2003-10-15
影响因子：
7.4
通讯作者：
Fréchet, JMJ
中科院分区：
文献类型：
--
作者：
Peterson, DS;Rohr, T;Fréchet, JMJ
制备了具有固相萃取器和酶促微反应器双重功能的微流控装置，并对其操作进行了演示。该装置是由一个25毫米长的多孔聚（甲基丙烯酸丁酯-二甲基丙烯酸乙酯）单体在50微米内制备而成。毛细管。这种带9-12微米针尖的毛细管被用作纳米电喷雾发射器，将该装置与质谱仪耦合。然后通过掩膜照射进行光接枝，选择性地功能化单体的20mm长部分，引入反应性聚（2-乙烯基-4,4-二甲基内酯）链，以便随后附着胰蛋白酶，从而创建具有高蛋白水解活性的酶促微反应器。另5mm未改性疏水单体作为微固相萃取器（muSPE）。双功能装置分别用于两个不同的流动方向；肌红蛋白的浓缩，从其稀释的溶液中吸收，然后洗脱和消化或消化，然后浓缩。两个方向的操作提供了相同的序列覆盖。不同体积的肌红蛋白溶液从2到20 μ l加载到设备上。在最高负载下，几乎达到了80%的非常高的序列覆盖率。尽管萃取器单元的长度很短，但该装置在消化-固相萃取方向上运行，也可以分离出大多数含有未消化蛋白质和肽的峰。
Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-mum-i.d. capillary. This capillary with a pulled 9-12-mum needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (muSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 muL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.