Broad-Spectrum Detection of Tetracyclines by Riboswitch-Based Cell-Free Expression Biosensing

We describe a sensitive and selective method for the determination of tetracycline content in foods using a riboswitch sensor. The sensor is based on a cell-free expression system that can be lyophilized to produce paper-based sensors or tube-based sensors for long-term storage. The riboswitch constructed using artificially screened tetracycline RNA aptamers was cloned into the pET-28a(+) vector of Escherichia coli TOP 10. The expression of the green fluorescent protein was positively correlated with the concentration of tetracyclines. The binding of tetracyclines to the aptamer domain results in a conformational change in the riboswitch secondary structure, resulting in the exposure of the ribosome binding site thereby promoting expression. The detection limits of the prepared sensor for the detection of tetracycline, oxytetracycline, chlortetracycline, and doxycycline were 0.47, 0.079, 0.084, and 0.43 μM, respectively. Moreover, the 1 μM tetracyclines allow for qualitative detection in milk samples by the naked eye. The work provides a proof-of-principle for riboswitch design to address global health and food safety.