1 Introduction
Biosensors are analysis tools that take biological components as the
main functional elements and used for rapid detection of various
trace-level analytes. They generally consist of sensitive biorecognition
elements, transducers, and signal analysis systems [1]. The
sensitive element (enzyme, antibody, cell, etc. ) identifies and
binds the analytes, and then the transducer captures the interaction
between the sensitive material and the analyte and converts the analyte
into an identifiable signal (e.g., electricity signal). The quantity of
the analyte is further calculated through the signal analysis system.
Compared with traditional instrument-based analytical chemistry such as
gas chromatography (GC), high-performance liquid chromatography (HPLC),
biosensors are less costly, more specific, faster, easier to operate,
capable of on-line analysis, real-time identification, and simultaneous
output of results. So they are more sensitive and more convenient. With
the advantages of biosensors, they have been widely used in
environmental detection, food safety inspection, disease diagnosis,
biochemical analysis, and other fields [2-4].
Meanwhile, synthetic biology has propelled the development of biological
sensors. Synthetic biology has great potential in the application of
biosensors by designing genetic circuits and logic operations for
biological components, resulting in a variety of genetic devices and
biological modules [5]. It has promoted the construction of
customized biological function system, which can finally realize sensors
with customized programming sensitivity, specificity and dynamic range,
and expand the scope of sensor detection target [6, 7], providing
transformative tools for improving the performance of biosensors.
The design of synthetic biology is mainly dependent on living cells in
recent decades, which leads to the rapid development of cell biosensors.
Cell biosensors can use animal and plant cells, especially microbial
cells as basal cells (sensitive element). Cells can metabolize a variety
of compounds using intracellular enzymes with high stability and
activity. Cell biosensors have a long service life and high
reproducibility of experimental results [8]. Moreover, the
sensitivity and specificity of biosensors can be improved by the
introduction of corresponding genetic modules. Heavy metals, pesticides,
vitamins, and some macromolecules can be detected using cell biosensors.
For example, Hou et al. [9] specifically detected
bioavailability of cadmium, lead, and arsenic in contaminated soil using
a customized suite of multiple whole-cell Escherichia colisensors. Nevertheless, there are some shortcomings in cell biosensors
that need to be addressed. Since cell biosensors use live genetically
modified microorganisms (GMOs), if they are released into the
environment, there are certain unsafe factors. In order to prevent the
occurrence of this situation, it is usually impossible to apply these
GMO biosensors [10, 11]. Furthermore, the response time of
biosensors is prolonged because some analytes are hard to cross over the
cell membrane. Moreover, some analytes may be toxic to living cells, so
some cell-based biosensors are not suitable for detecting cytotoxic or
impermeable membrane analytes, such as organophosphates. They may also
be influenced by actual environmental samples as well as their own
physiological and chemical conditions [12]. The above disadvantages
lead to significant limitations for cell biosensors in applications.
However, the emergence of cell-free synthetic biology not only promotes
the construction of cell-free biosensors in synthetic biology but also
solves the limitations described above. Currently, the biosensor
platform based on cell-free protein synthesis (CFPS) system has been
developed [13]. The CFPS system is a mixed system of DNA templates,
transcriptional machinery, translational machinery, the necessary
substrates (including amino acids, energy substrates, cofactors, and
salts), and water[14]. CFPS system can be divided into crude extract
system and PURE system. The main difference between the two systems is
the source of the translational machinery. As the name implies, one is
from the crude extract, and the other one is from purified components.
Compared with the crude extract system, the PURE system is more stable;
however, the cost is higher, and the yield is also lower. The CFPS
system and DNA template can be assembled into a mature cell-free
biosensor. CFPS systems perform biological transcription and translation
in an open environment, exhibiting a variety of unique functions
superior to cellular systems and laying the foundation for biosensors to
introduce more complex genetic circuits. Due to the uniqueness of the
cell-free system, the biosensors based on the cell-free system have
obvious advantages, which are reflected in the aspects of higher safety,
higher tolerance, shorter response time, higher sensitivity, higher
stability, and higher selectivity. These advantages are summarized in
Table 1. Therefore, given these significant advantages, the application
of cell-free systems in biosensor engineering is growing rapidly. The
following contents focus on the design principle and workflow of
cell-free biosensors, as well as the development of cell-free biosensors
in environmental monitoring and biomedical applications.