基于DNA定向固定化技术构建毛细管固定化酶微反应器的研究进展

生物酶影响着物质代谢和质能转换等生命活动，生物体内某些酶的活性变化会导致疾病的发生。发展新型的酶分析方法对深刻理解生物代谢过程、疾病诊断和药物研发等具有重要意义。毛细管电泳（CE）具有分离效率高、分析速度快、操作简单和样品消耗少以及可与多种检测手段联用等优点，在酶分析研究中越来越受到关注。CE酶分析主要包括离线和在线两种模式，其中，固定化酶微反应器与毛细管电泳联用（CE-IMER）的在线酶分析已经成为主要的酶分析方法之一。CE-IMER充分结合了固定化酶和CE的优势，将游离酶固定在毛细管内，不仅可以显著提高酶的稳定性和重复使用性，而且可以实现纳升规模溶液的自动化酶分析，进而显著降低酶分析成本。目前已有大量方法制备IMER用于CE酶分析，然而如何构建性能良好、可再生使用、酶固载量大、自动化程度高的CE-IMER一直是该领域重点研究的问题。DNA定向固定化技术（DDI）可以充分利用DNA分子的碱基互补配对（A-T，C-G），在温和的生理条件下特异性固定生物大分子。由于短链双螺旋DNA分子具有较强的机械刚性和物理化学稳定性，通过DDI将酶固定在载体表面，有利于降低传质阻力，提高酶与底物的接触能力，进而促进酶促分析过程。该文主要综述了利用DDI构建新型IMER在CE酶分析中的应用现状，并对其未来发展进行了展望。

Advances in capillary-based immobilized enzyme microreactor based on DNA-directed immobilization

Life processes such as metabolism and energy conversion are catalyzed by biological enzymes. The changes of enzymatic activity in organisms can lead various diseases. Thus, it is imperative to develop novel methods of analyzing enzymatic activities for gaining deeper insights into metabolic processes, disease diagnosis, and drug development. Capillary electrophoresis (CE) has the advantages of high separation efficiency, fast analysis speed, and simple operation; moreover, it requires less sample and can be combined with a variety of detection methods. Therefore, CE has attracted increasing attention for enzyme analysis. Enzyme analysis based on CE mainly includes off-line mode and on-line mode. In the off-line mode, the enzyme and substrate are incubated outside the capillary, and then the product is introduced into the CE for analysis. In the on-line mode, the capillary is not only used as a separation channel, but also as an enzyme reaction site. Therefore, the on-line mode facilitates all steps of enzymatic hydrolysis, separation, and detection within a capillary. In the on-line mode, homogeneous analysis method, electrophoretically mediated microanalysis (EMMA), and heterogeneous analysis method, immobilized enzyme microreactor (IMER), were developed. The on-line enzyme analysis method of IMER combined with capillary electrophoresis (CE-IMER) was developed into a mainstream enzyme analysis method. CE-IMER combines the advantages of immobilized enzyme and CE. By immobilizing the free enzyme in capillary, it can not only significantly improve the stability and reusability of enzyme, but also enables the automatic enzyme analysis at nanoscale. This can significantly reduce the cost of enzyme analysis. Although, there are numerous methods to prepare new IMER for enzyme analysis by CE, preparing CE-IMER with good performance, reusability, large enzyme loading, and high degree of automation is the focus of research in this field. DNA-directed immobilization (DDI) makes use of the complementary base pairs (A-T, C-G) of DNA molecules to specifically immobilize biomacromolecules under mild physiological conditions. The enzyme can be immobilized on the carrier surface by DDI and the short double helix DNA molecules possess strong mechanical strength and physicochemical stability. This can form an enzyme microarray, reduce the resistance of mass transfer, improve the contact between enzyme and substrate, and promote the enzymatic analysis process. Compared with the traditional immobilization methods of adsorption, crosslinking, encapsulation, and covalent bonding, DDI can be operated under mild physiological conditions. Further, this can significantly reduce the influence of the immobilization process on the activity, conformation, and stability of the enzyme. Meanwhile, the reversible immobilization process of DDI can regenerate the surface of the carrier, thereby significantly reducing the economic and time cost of IMER preparation. Therefore, DDI is an ideal method to prepare IMER. In this article, the preliminary research and progress of our research group in the field of IMER preparation by DDI technology are presented. At present, the research on the preparation of novel IMER based on DNA nanotechnology, such as DDI, is in the initial stage and there is much scope for development and research. Based on the previous studies, we can focus on the following aspects: (1) building a more efficient catalytic IMER cascade reaction system by immobilizing target enzymes in specific regions of the capillary based on DDI; (2) aiming at the problems existing in the preparation of IMER, such as stability, enzymatic activity, and enzyme immobilization capacity, while taking advantages of DNA structure and nanomaterials to prepare novel IMERs to promote the wide application of CE-IMER in enzyme analysis.