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

We describe a sensitive and selective method for thedeterminationof tetracycline content in foods using a riboswitch sensor. The sensoris based on a cell-free expression system that can be lyophilizedto produce paper-based sensors or tube-based sensors for long-termstorage. The riboswitch constructed using artificially screened tetracyclineRNA aptamers was cloned into the pET-28a(+) vector of Escherichia coli TOP 10. The expression of the greenfluorescent protein was positively correlated with the concentrationof tetracyclines. The binding of tetracyclines to the aptamer domainresults in a conformational change in the riboswitch secondary structure,resulting in the exposure of the ribosome binding site thereby promotingexpression. The detection limits of the prepared sensor for the detectionof tetracycline, oxytetracycline, chlortetracycline, and doxycyclinewere 0.47, 0.079, 0.084, and 0.43 mu M, respectively. Moreover,the 1 mu M tetracyclines allow for qualitative detection in milksamples by the naked eye. The work provides a proof-of-principle forriboswitch design to address global health and food safety.