Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, China.
Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China.
Talanta. 2019 May 15;197:539-547. doi: 10.1016/j.talanta.2019.01.080. Epub 2019 Jan 25.
Immobilized enzyme micro-reactors (IMERs) are of vital importance in developing miniaturized bioanalytical systems and have promising applications in various biomanufacturing. An inherent limitation in designing IMERs is the one-dimensional cylindrical geometry of micro-channels that offers limited exposed surface area for molecular reorganization and enzyme immobilization. In this study, we report a robust capillary-IMER based on a three dimensional porous layer open tubular (3D-PLOT) column which is prepared by an easy-to-control surface modification strategy via single-step in situ biphasic reaction. The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels, showing the beneficial effects for enzyme immobilization to enhance reaction efficiency and shorten analysis time. Taking trypsin as a model enzyme, enzymatic activities of immobilized enzyme are analyzed. We compare enzyme assays using the proposed 3D-PLOT-IMER with those using normal capillary-IEMR without surface modification as well as free trypsin. The 3D-PLOT-IMER exhibits excellent stability and inter/intra-day reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate enzyme assay. The feasibility of the proposed method for potential application in biological analysis is demonstrated by coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins, cell extraction and living Hela cells. Our study show that the surface modification with the proposed 3D-porous layer is a simple and efficient approach for enzyme immobilization, and could be widely suitable for different kinds of IMERs.
固定化酶微反应器（IMER）在开发微型生物分析系统中至关重要，并且在各种生物制造中具有广阔的应用前景。在设计 IMER 时，一个固有的限制因素是微通道的一维圆柱几何形状，其为分子重排和酶固定化提供的暴露表面积有限。在本研究中，我们报告了一种基于三维多孔层开管（3D-PLOT）柱的稳健毛细管-IMER，该柱是通过易于控制的表面改性策略通过一步原位双相反应制备的。具有高度均匀的多孔几何形状和狭窄的孔分布的 3D-PLOT 柱可以极大地提高微通道的表面积与体积比，显示出对酶固定化的有益效果，以提高反应效率并缩短分析时间。以胰蛋白酶为模型酶，分析固定化酶的酶活性。我们将使用提议的 3D-PLOT-IMER 进行的酶测定与未进行表面改性的普通毛细管-IEMR 以及游离胰蛋白酶进行的酶测定进行了比较。3D-PLOT-IMER 表现出出色的稳定性和日内/日间重现性，这些特性表明所提出的 IMER 可用于准确的酶测定，具有可靠性。通过将 3D-PLOT-IMER 与纳升 LC-MS/MS 系统耦合，用于标准蛋白质的在线消化、细胞提取和活的 Hela 细胞，证明了该方法在生物分析中的潜在应用的可行性。我们的研究表明，使用提议的 3D 多孔层进行表面改性是一种简单而有效的酶固定化方法，并且可以广泛适用于不同类型的 IMER。
