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.