Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.
Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.
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DOI：
10.1039/b408222b
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发表时间：
2004-11
期刊：
影响因子：
6.1
通讯作者：
Huiling Wu;Jianjun Zhai;Yuping Tian;Haojie Lu;Xiaoyan Wang;Weitao Jia;Baohong Liu;Pengyuan Yang;Yunmin Xu;Honghai Wang
中科院分区：
文献类型：
--
作者：
Huiling Wu;Jianjun Zhai;Yuping Tian;Haojie Lu;Xiaoyan Wang;Weitao Jia;Baohong Liu;Pengyuan Yang;Yunmin Xu;Honghai Wang
报告了两种聚（二甲基硅氧烷）（PDMS）微流控酶反应器的设计和表征以及它们与 MALDI TOF 和 ESI MS 结合的分析实用性。使用PDMS铸造技术制造了与微通道和不锈钢管（SST）集成的微流体装置，并用于酶反应器的制备。基于丙烯酸的紫外接枝聚合，在PDMS表面引入羧基进行化学改性。分别使用活化剂1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)/N-羟基琥珀酰亚胺(NHS)和偶联剂聚(二烯丙基二甲基氯化铵)(PDDA)对胰蛋白酶进行共价物理固定。通过测量接触角、衰减全反射光谱的红外吸收、AFM 成像和电泳图来研究胰蛋白酶固定化过程的特性和成功。微流控酶反应器的一个创新特点是可以通过嵌入式 SST 电极和可更换尖端进行在线蛋白质分析。即使在 EDC/NHS 制造的装置为 3.5 µL min(-1) 的快速流速和 PDDA 制造的装置为 0.8 µL min(-1) 的快速流速下，实验室制造的装置也能很好地消化多种模型蛋白质，分别提供 5 s 和 20 s 的极短停留时间。此外，实验室制造的设备不易受记忆效应影响，一周内可运行至少 50 次，而不会出现明显的活动损失。此外，降解的PDDA制成的装置可以通过HCl溶液的简单处理来再生。这些功能是用于蛋白质分析的微流体装置最需要的。
The design and characterization of two kinds of poly(dimethylsiloxane)(PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride)(PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 microL min(-1) for the EDC/NHS-made device and 0.8 microL min(-1) for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.