**5. Conclusions**

DET-type bioelectrocatalysis is an ideal system that employs redox enzymes as biocatalysts for energy conversion under extremely mild conditions. The properties of redox enzymes lead to reactions with high specificity and high catalytic efficiency, which are suitable for various electrochemical devices, including biosensors, biofuel cells, and bioreactors. The major challenges for DET bioelectrocatalysis are the interfacial electron transfer between redox enzymes and electrodes. Carbon nanomaterials with high conductivity, large specific surface areas, and three-dimensional nanostructures have been widely utilized for the construction of bioelectrodes with the capability of DET.

In this review, we focused on the rational surface modification of carbon nanomaterials for improving DET-type bioelectrocatalysis based on an understanding of the effects of carbon nanomaterials on the interfacial electron transfer between redox enzymes and electrodes. Although a large specific surface area is important for increasing the enzyme loading, a suitable pore size distribution and specific surface chemistry of carbon nanomaterials have recently been proposed to play important roles in the promotion of interfacial electron transfer between redox enzymes and electrodes. Carbon nanomaterials, with hierarchical porosity that balance the enzyme adsorption, electron transfer, and mass transfer, are expected to be suitable for high-performance DET-type bioelectrocatalysis. By contrast, intricate interactions between redox enzymes and carbon nanomaterials play an important

role in the orientation of redox enzymes. From this perspective, rational surface modification based on an understanding of the interaction between enzymes and the specific modifier to control the orientation of the redox enzymes for improved DET-type bioelectrocatalysis has been developed during the past decades. Typically, charged compounds, polycyclic aromatics, and substrate mimics have usually been utilized for the construction of functionalized carbon nanomaterials for controlling the orientation of redox enzymes. Unlike the random surface properties of native enzymes, recently developed engineered enzymes with specific sites that can be immobilized onto modified carbon nanomaterials with controlled orientations have also attracted increased attention. To further understand the situation of redox enzymes in carbon nanomaterials, various techniques other than the use of electrochemical methods alone are highly desired. In particular, QCM-D and ATR-IR spectroscopy have been utilized to investigate the adsorption, conformational change, and orientation distribution of redox enzymes in carbon nanomaterials.

Although significant progress has been made in the past decades, several important issues regarding the DET-type bioelectrocatalysis are still waiting to be tackled. Firstly, detail mechanism of DET reaction is still instinct. Advanced technology, for example coupling-technique as well as single molecule analysis, is desired to deeply understand the process of electron transfer between a redox enzyme and electrode surfaces. Further development in theoretical discussion on this issue is also expected. Secondly, redox enzyme that capable of DET-type bioelectrocatalysis is limited in number. Native redox enzymes have usually large and sophisticated three-dimensional molecular structures. Besides to electrode surface modification discussed in this review, protein engineering to reform redox enzymes, for example shortening the distance between active site and enzyme surfaces by elimination of the domains that are not related to the electron transfer, is proposed to improve the performance of DET-type bioelectrocatalysis effectively. Thirdly, stability is continued to be an important issue in DET-type bioelectrocatalysis for practical applications. Strategies including optimization of enzyme immobilization approaches, tuning enzyme properties and using suitable electrode materials have been proposed recently to improve the stability and increase the lifetime of redox enzymes at electrode surfaces for rapid interfacial electron transfer.

**Author Contributions:** H.X.: Original manuscript preparation and revision; J.Z.: Revision and discussion. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is partially supported by Guangdong Basic and Applied Basic Research Foundation (No. 2019A1515111183) and President Foundation of Institute of Fruit Tree Research, Guangdong Academy of Agricultural Science (No. 202005).

**Conflicts of Interest:** The authors declare no conflict of interest.
