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.