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.