Liu Junyan, Lin Shuang, Qi Dawei, Deng Chunhui, Yang Pengyuan, Zhang Xiangmin
Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
J Chromatogr A. 2007 Dec 28;1176(1-2):169-77. doi: 10.1016/j.chroma.2007.10.094. Epub 2007 Nov 4.
An easily replaceable microchip enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized superparamagnetic nanoparticles. Magnetic nanoparticles with small size (50 nm in diameter) and strong magnetism were synthesized. At first, amine-functionalized magnetic nanoparticles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde (-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The prepared magnetic nanoparticles were then locally packed onto the glass microchip by the application of a strong magnetic field using a magnet to form an on-chip magnetic nanoparticles packing bed. Capability of the proteolytic microreactor was demonstrated by cytochrome c, bovine serum albumin and myoglobin as model proteins. The digestion products were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with sequence coverage of 83%, 43% and 79% observed, respectively. Complete protein digestion was achieved in a short time (10 s) under the flow rate of 5 microL/min. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. It is easy to replace the nanoparticles and make the new microreactor. It takes less than 1 min under the condition of extra magnetic to form a new packing bed. The packing bed can be used for at least five times without any treatments. Additionally, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. After a database search, six proteins were identified. This opens a route for its further application in bottom-up proteomic analysis.
基于带有固定化胰蛋白酶的超顺磁性纳米颗粒的玻璃微芯片，制备了一种易于更换的微芯片酶微反应器。合成了具有小尺寸（直径50nm）和强磁性的磁性纳米颗粒。首先，通过简便的一锅法策略制备了具有高磁响应性和优异分散性的胺官能化磁性纳米颗粒。然后，通过用戊二醛处理合成的胺官能化磁性纳米颗粒，使磁性纳米颗粒用大量醛基（-CHO）官能化。最后，通过醛基与胰蛋白酶的胺基反应，将胰蛋白酶固定在醛官能化磁性纳米颗粒上。然后使用磁铁通过施加强磁场将制备的磁性纳米颗粒局部填充到玻璃微芯片上，以形成芯片上的磁性纳米颗粒填充床。以细胞色素c、牛血清白蛋白和肌红蛋白作为模型蛋白，证明了蛋白水解微反应器的能力。使用基质辅助激光解吸/电离飞行时间质谱对消化产物进行表征，观察到的序列覆盖率分别为83%、43%和79%。在5μL/min的流速下，短时间（10s）内实现了蛋白质的完全消化。这些结果有望为蛋白质水解分析开辟新的可能性，以及磁性纳米颗粒的新应用。更换纳米颗粒并制造新的微反应器很容易。在额外磁场条件下，不到1分钟即可形成新的填充床。该填充床无需任何处理即可至少使用五次。此外，由于磁性纳米颗粒的制备和表面功能化成本低且可重复，磁性纳米颗粒的制备方法和应用途径可能在蛋白质组研究中具有很大潜力。该微反应器还成功应用于大鼠肝脏提取物的反相液相色谱馏分的分析。经过数据库搜索，鉴定出六种蛋白质。这为其在自下而上的蛋白质组分析中的进一步应用开辟了一条途径。
