Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

Abstract

In recent years, CE‐integrated immobilized enzyme reactors (IMERs) for single‐enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE‐integrated IMER, using DNA‐directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE‐integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE‐integrated IMERs are potentially useful in a wide range of biochemical research applications.

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

“…In line with previous work performed in 2013 [86], this time frame also witnessed the simultaneous analysis of two enzymes in one run either offline [61,87] or inline [53,77,80].…”

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

“…Other than spherical nanoparticles, surfaces of other shapes may also be used for immobilizing DNA-guided multienzyme systems to adapt different application scenarios. For example, Li et al built a capillary electrophoresis-integrated immobilized enzyme reactor (CE-IMER), in which the ssDNA strands for directing the immobilization of complementary ssDNA-coupled GOx and HRP were fixed on the inner surface of a capillary (Figure 4B,E) (Li et al, 2020).…”

Co‐Immobilization of GOD & HRP on Y‐Shaped DNA Scaffold and the Regulation of Inter‐Enzyme Distance

“…Using them as carriers to directionally coimmobilize multiple enzymes has shown great potential for the precise positioning of enzyme molecules. [10,22,23] Many kinds of literature have reported that various DNA assemblies constructed by DNA origami can accurately position multiple enzymes. [24,25,26] Recently, a DNA tetrahedral scaffold [27] as well as a switchable trident-shaped DNA nanoactuator [28] has been prepared to co-immobilize GOD and HRP.…”