2.2 Signal output
The signal of cell-free biosensors is mainly output in the form of optical or electrochemical signals. According to statistics, the optical reporter genes mainly include lux (bacterial luciferase),luc (firefly luciferase), gfp (green fluorescent protein), and lacZ (β-galactosidase) [42]. After the identification, the downstream reporter gene is activated and expressed in the cell-free system. Various output methods are available for monitoring the proteins expressed by the reporter genes, including fluorescence, luminescence, visible color changes, etc.
The most common method is to report the fluorescence of proteins based on GFP which is a simple, convenient and intuitive signal output method in laboratory operation, and it does not require reaction substrates (Fig. 3A). GFP expressed by the cell-free system could not glow by itself, and the fluorescence only can be generated by the light excitation. Therefore, the fluorescence output representing the gene expression level needs to be measured by means of absorbance detection. However, the obvious disadvantage of GFP is that the background signal is high, which makes the sensitivity of GFP low.
In addition, the signal can be output by the luciferase gene (lux/luc ) (Fig. 3B). The luciferase experiment is extremely sensitive. It has no signal background and has a wide linear range (up to 7-8 orders of magnitude). Luciferase needs to be mixed with luciferin and other required chemicals to produce fluorescence. Generally, luciferase assay reagent is added to the test system, and the luciferase level is measured in the plate reader after the reactant mixing. Moreover, the application of luciferase also has certain limitations, such as short half-life, poor repeatability, and certain requirements on oxygen, etc.
Enzymes catalyze substrate molecules that can produce strong exponential signals than fluorescent proteins. β-galactosidase (β-gal) can lyse synthetic substrates, resulting in color changes, fluorescence, or chemiluminescence, which allows the output of LacZ-based sensors to be adjusted for different conditions. However, β-gal has some dependence on the substrate. The colorimetric method based on the enzyme activity of β-gal can directly observe the color changes with the naked eye, and the most common substrate for colorimetric detection isO-nitrophenyl-d-pyrine galactoside. This signal output method is suitable for paper-based cell-free biosensors (Fig. 3C). Because the cell-free biosensors based on luciferases and β-gal have high sensitivity and short response time, so luc and lacZ are preferred reporter genes.
Except for the optical signal output, cell-free biosensors can also be output by electrochemical measurement. A variety of material electrodes combined with cell-free system and electrochemical signals can be generated, while the electrodes can capture the target. Capacitance-voltage and constant-capacitance measurements are employed to monitor the capacitance changes induced by surface modification steps for sensor preparation as well as interactions between receptor and its ligands [43]. Mousavi et al . [44] designed an extensible reporter enzyme system, in which a sensor based on a cell-free gene circuit cleaves specific DNA sequences in solution, generating electrochemical signals when the newly released strands are trapped on the surface of nanostructured microelectrodes (Fig. 3D). This method is in sharp contrast to the traditional optical reporter, which not only has limited multiplexing ability but also has a dependence on ventilation and reduced sensitivity in turbid solutions [45]. These limitations can be overcome by electrochemical measurements. The electrochemical system will enable the multiplexing of gene-based circuits for portable detection and will facilitate the development of electronic devices related to synthetic biology.