Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, Viikinkaari 5E, 00014, Helsinki, Finland.
Laboratory of Physical Chemistry, Åbo Akademi University, Porthaninkatu 3-5, 20500, Turku, Finland.
Anal Bioanal Chem. 2019 Apr;411(11):2339-2349. doi: 10.1007/s00216-019-01674-9. Epub 2019 Mar 21.
We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic α-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research. Graphical abstract.
我们介绍了有序、高纵横比的硫醇-烯微柱阵列的快速复制成型，用于实现微流控固定化酶反应器 (IMER)。通过利用过量硫醇-烯组成物中丰富的游离表面硫醇，我们能够通过硫醇-金相互作用使原始的硫醇-烯微柱官能化金纳米颗粒 (GNPs)，并使这些微柱官能化金纳米颗粒与蛋白水解 α-糜蛋白酶 (CHT)。微柱阵列通过 PDMS 软光刻复制，该方法促进了硫醇-烯的固化，而无需光引发剂，因此可以直接键合，并很好地控制表面化学（游离表面硫醇的数量）。通过在富含烯丙基的硫醇-烯表面上进行的硫醇-金相互作用的特异性研究，以及在不同流速和反应温度下使用缓激肽水解作为模型反应对 CHT-IMER 的稳健性研究，证明了其稳健性。结果表明，产物转化率随着流速的降低（停留时间的增加）而增加，并且在将 IMER 加热至生理温度时也会增加。由于通过 GNPs 将有效的酶固定在微柱阵列上，因此在使用质谱检测缓激肽水解产物之前，无需对反应溶液进行进一步纯化，也不会观察到通常与传统毛细管填料相关的堵塞问题。IMER 的活性至少稳定 1.5 小时（连续使用），这表明所开发的方案可能为实施 IMER 技术提供一种稳健的新方法，用于蛋白质组学研究。示意图。
