A cell-free biosensor based on strand displacement amplification and hybridization chain reaction for fluorescence detection of tetracycline

Tetracycline (TC) is a broad-spectrum antibiotic used to treat diseases and in other fields. In the current research, a universal biosensing platform was developed based on strand displacement amplification (SDA) and hybridization chain reaction (HCR), which could facilitate the rapid detection of TC and is suitable for various substrates. The allosteric transcription factor (aTF) TetR was selected as the recognition element to control SDA and HCR. When TC target are present in solution, competitive binding with aTF led to the release of aTF from the primers, and the nicking endonuclease successfully bound to the recognition site and initiated the SDA reaction, leading to the release of the SDA product (SP), which triggered the autonomous cross-opening of Hairpin 1 (H1) and Hairpin 2 (H2). G-quadruplex subunits co-localized at the nick of the formed duplex complex self-assembled into several ThT/G-quadruplex complexes. The activated G-quadruplex folded into G-dimer and self-assembled with ThT to emit a strong fluorescence. Under optimized conditions, the fluorescent biosensor showed a linear relationship ranging from 20 to 1000 ng/mL toward TC with the detection limit of 17.16 ng/mL, which is significantly lower than the national limit standard (100 µg/kg) of China. Furthermore, the proposed biosensor exhibited good performance in the TC analysis of different matrices, such as tap water, Yellow River, Huangpu River, Mudan River, Songhua River, milk, honey, and chicken samples. The present study proposed a robust biosensor for real-life analysis, thus highlighting the great potential for the development of small-molecule biosensors in other fields.