Based on the understanding of the regulatory mechanism of prokaryotic transcription factors in prokaryotes, the research group of the Institute of Microbiology, Chinese Academy of Sciences, for the first time, used the prokaryotic aTF as a recognition element in combination with an Alpha signal amplification system to construct a detection platform for small molecules of induction chemistry. This is of great significance in areas such as food safety.
The sensitive detection of small molecules is an important requirement in the fields of clinical diagnosis, environmental monitoring and food safety. Therefore, it is of great significance to develop efficient chemical small molecule detection methods. Among them, the specific identification component is the core and foundation for realizing the detection. The traditional detection method is to identify the identification component and the object to be detected by using antibodies, enzymes, etc., and then realize the detection by amplifying the identification signal. The whole development process often faces problems such as long cycle, difficulty, and unsatisfactory signal output.
In response to the above problems, Yang Keqian, a research group of the Institute of Microbiology, Chinese Academy of Sciences, based on years of understanding of the regulatory mechanism of allosteric transcription factors (aTF), firstly used prokaryotic aTF as a recognition element in vitro to bind to Alpha (amplified luminescent proximityimousousassays) signals. Amplify the system and build a detection platform for sensing chemical small molecules (pictured).
Prokaryotic aTF contains a DNA binding domain and a small molecule recognition domain, and the binding of small molecules can dissociate aTF from a specific DNA sequence. Therefore, the core design of the platform is to immobilize biotin-labeled aTF recognition sequence DNA and aTF on Donor microbeads and Acceptor microbeads, respectively. When there is no small molecule to be detected, the Donor microbead and the Acceptor microbead are adjacent by aTF-DNA interaction. The Donor microbead can diffuse into the Acceptor microbead by absorbing a large amount of singlet oxygen (1O2) generated by the excitation light, making the Acceptor The bead emits light; when the small molecule to be detected exists, the small molecule binds to the aTF to dissociate from the DNA, thereby separating the Donor microbead from the Acceptor microbead, and the singlet oxygen (1O2) cannot diffuse from the Donor bead to the Acceptor. The beads make the Acceptor beads not illuminate. Thus, the small molecule concentration achieves high sensitivity detection by alpha signal amplification. Yang Keqian's research team used this concept to establish a universally applicable testing platform, and developed the most sensitive detection method for the detection of clinical markers uric acid and antibiotic residual oxytetracycline.
Due to the presence of aTFs in prokaryotic organisms that induce various small molecules, combined with this detection platform and a large number of aTF resources, highly sensitive, novel detection methods and kit development can be performed for different target small molecules. At present, this achievement has applied for relevant patents and published related articles (Chem.Commun. cover article). In the published article, the microbiology institute Li Shanshan was the first author, researcher Yang Keqian and associate researcher Wang Weishan (member of the Youth Promotion Association) as a co-author.
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