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].