Abstract:

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.

Key words: capillary electrophoresis (CE), immobilized enzyme microreactor, immobilized enzyme, DNA-directed immobilization