Kecskemeti Adam, Bako Jozsef, Csarnovics Istvan, Csosz Eva, Gaspar Attila
Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary.
Department of Biomaterials and Prosthetic Dentistry, University of Debrecen, Nagyerdei krt. 98., Debrecen, 4032, Hungary.
Anal Bioanal Chem. 2017 May;409(14):3573-3585. doi: 10.1007/s00216-017-0295-9. Epub 2017 Mar 15.
Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.
本文描述了一种含有固定化胰蛋白酶的微流控装置（MD），用于快速高效的蛋白水解。通过非特异性蛋白质吸附将胰蛋白酶固定在MD的疏水性聚二甲基硅氧烷（PDMS）通道壁上。对牛血清白蛋白（BSA）样品进行肽图分析，以评估吸附在PDMS表面的胰蛋白酶的稳定性。通过毛细管区带电泳（CZE）获得BSA样品的肽图，迁移时间的相对标准偏差（RSD%）低于1%。在约50秒的接触时间内，能有效消化多种分子量范围较宽（15 - 70 kDa）的蛋白质（血红蛋白、肌红蛋白、溶菌酶和BSA）。分离峰的数量与蛋白质完全胰蛋白酶消化中形成的预期肽段数量高度相关。对BSA和人血清进行了肽质量指纹图谱分析。胰蛋白酶保持其活性2小时；在此期间，该MD可用于多次消化。该装置的主要特性包括简单的通道模式、简单的固定化程序、可再生性和一次性使用；所有这些特点使该MD成为最简单但适用的酶促微反应器之一。图形摘要：开发一种包括蛇形通道作为蛋白质消化酶反应器的微流控装置。
