2 Principle and workflow of cell-free biosensors
The design principle of most cell-free biosensors is to select the appropriate recognition mechanism and reporter gene based on the analytes, and then clone the encoded gene into the cell-free expression vector. Furthermore, select the appropriate cell-free protein synthesis system to constitute a mature cell-free biosensor with the coding template DNA. Cell-free biosensors can detect a variety of analytes, including ions, antibiotics, amino acids as well as nucleic acids of viral pathogens. The workflow of cell-free biosensor (Fig. 1) can be divided into two parts, the recognition and response of analytes by specific identification mechanisms, and the output of readable signals through reporter gene expression. First, accurate recognition of analytes is the key to cell-free biosensors. When analytes are added to the cell-free system, the sensor’s recognition mechanism is regulated by specific molecular structure and binding [15, 16]. Second, the downstream reporter gene is activated to be transcribed and translated into reporter proteins in a cell-free system. According to the presentation mode of different reporting proteins, the detection results are output by optical and other readable signals [17].

2.1 Recognition-response mechanisms

With the emergence of multiple analytes, some analytes are difficult to be recognized and detected by traditional recognition mechanisms (such as enzymes, antibodies, and PCR technology), which has led to the development of recognition mechanisms to use diversified molecular structures as recognition elements. The following contents mainly introduce the recognition mechanism of cell-free biosensors based on different molecular structures, including transcription factors, CRISPR-Cas, toehold switches, and adaptors [18].