3 Improvement of cell-free biosensors in practical application
To keep up with the development of practical
applications, the cell-free biosensor is required to be more portable,
stable, and cost-efficient. These requirements have led to continuous
improvement of cell-free biosensors. Generally, cell-free biosensor
reagents are often refrigerated or frozen during storage or
transportation. This method of storage is not convenient, which may
hinder the field deployment. Therefore, the researchers develop the
lyophilization technology to improve the storage stability of cell-free
biosensors. The cell-free system ensures that it remains active after
lyophilization and does not adversely affect the efficiency. By
freeze-drying, the cold-chain storage problems can be overcome to
stabilize the long-term and higher temperature storage of the cell
extract, which can last up to three months to a year and reduce the
necessary storage space. Moreover, the stability of cell-free biosensors
can be improved in other ways. For example, Karig et al. [46]
also investigated using the non-reducing disaccharide trehalose to
protect cell-free components during oven drying. Moreover, components
can be encapsulated in the liposomes [47] or polymer substrates to
maintain the system stability [48]. However, the decrease of the
activity of the cell-free system after lyophilization is the main
improvement target that needs to be developed in the
future.
To facilitate the field monitoring, biosensors need improving the
portability. Cell-free systems can be freeze-dried with appropriate
supports (e.g., microtubules and microporous plates). Beyond that, the
cell-free system also can be introduced onto a cheap matrix simple paper
[26], where the freeze-drying and the signal amplification can
reduce the chance of contamination and false positives. Because of the
small size of the freeze-dried reagent required for the paper-based
cell-free biosensor, this greatly reduces the cost and facilitates the
field testing.
Furthermore, there may be some differences in the cell-free systems
prepared in different batches, so it is necessary to standardize the
cell-free systems. PURE systems can be developed to improve sensor
standards. The PURE system for cell-free protein synthesis reduces the
level of contaminated proteases, nucleases, and phosphatases, and
provides a more precise method of preparation, a higher reproducibility,
and better flexibility of the modular system. Because there is no
metabolic side effect of amino acid library like in crude extract-based
cell-free system, so the PURE system is more stable [49]. The PURE
system has been commercialized to make them available for a variety of
applications, with strict quality control. However, the commercial PURE
system is expensive, and the production cost of the PURE system is very
high, so the general sensor design still uses the crude extract system.
Therefore, in order to standardize the cell-free biosensors, more time
and energy should be spent on the development of the PURE system to
improve the efficiency and reduce the cost in the future. At present,
there is some lag in commercial applications of cell-free biosensors,
which will be the focus of future development.