Study on the modification of amino acid oxidase based on the expansion of gene codon and the new biorthogonal reaction "s-click"

On October 5, the Journal of angelwandte chemistry international edition published a research article titled "s-click reaction for isotropic orientation of oxidates on electronics to promote electron transfer at low potentials" by Wang Jiangyun, Institute of Biophysics, Chinese Academy of Sciences In this paper, the amino acid oxidase was modified based on the gene codon expansion and the new biorthogonal reaction "s-click" developed by the research group, and the rapid, real-time and accurate electrochemical detection of amino acids was successfully realized Amino acids are important physiological and biochemical metabolism and cell signaling molecules Abnormal amino acid metabolism leads to many serious diseases, so real-time amino acid analysis is of great significance to medicine, diagnosis and life science With the continuous development of life science, the importance of amino acids in cell metabolism and basic pathology is increasing in international journals, such as tryptophan metabolism, which is closely related to mental diseases, immune diseases and immune escape of cancer cells In addition, the metabolism of tryptophan, glycine, arginine, serine has an important relationship with the development of tumor, and has become an important target of precise drugs At present, the detection of amino acids mainly includes spectrum, liquid chromatography, enzyme-linked color reaction, etc., which are difficult to realize the real-time and dynamic analysis of amino acids in human body fluids Because of the lack of a fast, real-time, sensitive and accurate analysis method of amino acids in body fluids, it not only limits the further research on the relationship between different amino acids and human health, but also restricts the further research on the relationship between different amino acids and human health Missed the opportunity of early warning of health status by monitoring this important physiological and metabolic basis material One of the key points of improving enzyme electrochemical biosensor (EEB) is to improve the electron transfer between enzyme and electrode The use of electronic mediator in EEB is a common method to improve the electron transfer, but the use of electronic mediator usually leads to an excessive potential increase relative to the original redox potential of the enzyme, and redox medium is usually non selective, which not only promotes the electron transfer between the electrode and the protein, but also promotes the electron transfer of various interfering molecules In addition, there are many limitations in the application of electronic mediators in the field of in vivo analysis The enhancement of the electron transfer of enzyme electrode by nano materials has made a great contribution to the realization of the third generation biosensor, but the random orientation of enzyme relative to the electrode surface results in the great change of the electron transfer efficiency In this study, a novel biorthogonal "s-click" reaction was developed to specifically couple the sulfhydryl group of TF with the chlorobenzene group of the linker (bodipy373), while the linker through π - π Stacking is assembled on the surface of carbon material to realize the site coupling of different amino acid oxidase on the electrode surface of carbon material (as shown in Figure 2) The site-specific coupling system (s-click, figure 2C) created by the team has better reaction activity and biocompatibility compared with the current mainstream coupling system based on azido alkyne "click chemistry", which is more in line with the needs of developing wearable devices The tryptophan oxidase electrode prepared based on the technology shows a higher and more uniform enzyme load, and also shows a very low catalytic potential of tryptophan direct bioelectrocatalysis in the electrochemical test (Fig 3) Using the amino acid bioelectrochemical sensor based on gene codon expansion, the team further carried out accurate real-time in situ dynamic detection of tryptophan and glycine in blood samples and cancer cell culture system (Figure 4) Wang Jiangyun, researcher of the Institute of Biophysics, is the corresponding author of the paper, and Xia Lin, associate researcher, is the first author of the paper This work was supported by NSFC, key R & D plan, key deployment project of Chinese Academy of Sciences and "three famous" project of Shenzhen (BIOON Com)