Fig. 1. The workflow of cell-free biosensors.
Fig. 2. Recognition-response mechanisms of cell-free biosensor systems. (A) After the ligand is identified, reporter genes can be activated by binding activated transcription factors or releasing inhibitory transcription factors. Unknown ligands can be converted into detectable ligands by using metabolic enzymes before the identification. (B) Cell-free biosensors use NASBA-CRISPR/Cas9 cleavage system to identify different strains of virus. (C) Nucleic acid ligands are used to identify thrombin. When thrombin is absent, the reporter protein is successfully expressed, but the translation is blocked in the presence of thrombin. (D) When samples containing amino acids are added, incomplete CFPS is activated, leading to successful expression of the reporter protein.
Fig. 3. Signal output of cell-free biosensor systems. (A) The reporter gene gfp is expressed in the CFPS system and output in the form of optical signal. (B) The reporter gene luc is expressed in the CFPS system and output in the form of optical signal. (C) The reporter gene lacZ is expressed in a paper-based cell-free system, and the color change on the measured paper can be easily detected by the naked eye. (D) The biosensor based on the cell-free genetic circuit captures the analyte through the nanostructured microelectrode to generate the electrochemical signal output.