Diagnosis is an important part of public health, and a key step in addressing any disease is determining whether it is present. Diagnostic development has two main goals, one is to improve clinical performance, such as increasing the sensitivity, specificity and accuracy of quantification, and the other is to improve analytical characteristics, such as reducing cost, improving portability, simplifying workflow, shortening the time to obtain results, and the ability to adapt to pollutants. With the development of synthetic biology, synthetic biological devices, including whole-cell activity detection, engineered cell-free nucleic acid sensors, and a combination of using natural disease biology and reconstructing enzyme function, have been successfully applied to non-infectious diseases (such as cancer and coronary artery disease), infectious diseases (such as Ebola, Zika, tuberculosis, malaria, HIV and SARS-CoV-2), and other aspects of public health (such as regular blood analyte quantification).

Figure 1: Cell-free diagnostics based on synthetic biology

However, the infrastructure for diagnosis is severely lacking in most parts of the world. In a study conducted by the World Health Organization in ten countries on three continents, only 1% of health centres and clinics were considered competent for basic diagnostic tests. While synthetic biology has created novel diagnostic methods based on synthetic gene networks, they are overwhelmingly limited to laboratory use and require expensive equipment and materials.

In recent years, the development of cell-free expression systems has made it possible to engineer RNA elements as multifunctional diagnostic tools. Cell-free expression systems, which contain all the components needed for gene expression because manipulation in living cells can cause excessive toxicity and other deleterious problems, have also been used to study fundamental biochemical processes. Important advances in the deployment of synthetic gene circuits (genetically engineered networks linked by transcriptional and post-transcriptional regulation) outside the laboratory have also demonstrated that the freeze-dried cell-free expression system embedded in a porous matrix (such as paper) can largely retain its function for at least 1 year, even when stored at room temperature. The synthetic gene circuit can be embedded in a piece of paper, enabling storage or shipping at room temperature, and reactivated by rehydration after sample administration. This can be applied to almost any design, including cell-free testing of antibiotics, heavy metals, sedative drugs, and biologically active small molecules.

Figure 2: Paper-based synthetic gene networks

An engineered RNA regulatory factor called "Toehold Switch" is particularly suitable for use as a biomedical diagnostic for specific nucleic acids. These "Toehold Switches" can be used in conjunction with cell-free expression systems to create highly portable, paper-based nucleic acid diagnostics. The combination of isothermal nucleic acid amplification, a modular "Toehold Switch", and a cell-free system that can be stably expressed on paper provides an adaptable, sensitive, and stable diagnostic platform that can detect specific nucleic acids at the point of care at a total cost of only $0.10-$1.00 per test. And because the output of the "Toehold Switch" can be any protein, there is almost unlimited multiplexing capability. The system is easily adaptable to fluorescence, bioluminescence, colorimetric, and bioelectrical outputs, and can build complex gene networks for multi-input logic. Moreover, the characteristics of the cell-free expression system itself enable it to express anti-infective agents (such as nanobodies or bacteriolysins). The paper-based "Toehold Switch" for Zika virus diagnosis is currently undergoing collaborative research at five national sites in North and South America, and nearly 300 clinical samples have been processed so far.

In recent years, antibodies targeting intracellular proteins have great application prospects in the development of new therapeutic interventions for various diseases, especially antibodies that can cross the cell membrane, which have potential applications in controlling disease-related intracellular protein-protein interactions. Given the large number of cytosolic proteins and complex interactions that may be associated with disease development, the discovery of antibodies that target intracellular proteins requires repeated expression and evaluation of candidate antibodies. However, current cell-based expression methods do not provide sufficient capacity to produce and assay cytosol-penetrating antibodies, while cell-free protein synthesis systems can provide optimal conditions for the production of functional antibodies by cytosol permeation assays. Cell-free synthesis is therefore an effective option for the production and detection of cell-penetrating antibodies. Compared to E. coli production, smaller reaction volumes, minimal laboratory settings, and high-speed production of more functional antibodies justify the use of cell-free protein synthesis as an advantageous method for developing cell-penetrating antibodies.

Cell-free expression system has a wide application prospect in clinical diagnosis. Whether it is tumor markers or other disease markers, cell-free expression system can synthesize specific proteins in real time, and realize the early detection and quantitative analysis of diseases by detecting its expression level. This is very important to improve the accuracy and efficiency of disease diagnosis. The cell-free expression system can avoid problems such as cell contamination, and is more flexible in terms of expression efficiency and functional regulation. These advantages make the cell-free expression system more reliable and stable in clinical diagnosis.

As an emerging technology, cell-free expression systems are emerging in the field of clinical diagnostics. Its simplicity, efficiency and stability have brought many new possibilities for clinical diagnosis. Its use in synthetic biology also illustrates how collaboration between academia and industry is accelerating the pace of technological innovation and industrial application. For example, the CRISPR-based collateral dissection, first proposed in 2017, allowed the research team to create two biotech companies within a year, with a total capital of $12.42 billion, and by 2020, obtain FDA approval for two SARS-CoV-2 diagnostics.

In addition, a number of functional vaccines against the newly discovered SARS-CoV-2 virus were also developed, tested in clinical trials, received emergency approval and deployed within 10 months. It is believed that under the continuous research and optimization, the cell-free expression system will bring more innovations for clinical diagnosis and make great contributions to people's health.

As a professional cell-free protein expression biotechnology company, Perotin Biology has a professional technical team composed of national high-level leading talents, returned doctors and other talents. Relying on the independent research and development and unique cell-free protein expression technology platform, Perotin Biology specializes in the research and development of peptides, recombinant proteins, genetically engineered antibodies, recombinant vaccines and macromolecular protein drugs, at the same time for the vast number of biomedical enterprises and research institutions to provide cell-free protein expression products, protein raw materials reagents and customized services.

The cell-free protein expression kit developed by the company only needs to add protein gene (PCR product or circular plasmid), and protein can be produced in 1 hour at the earliest, and the protein yield can reach up to 3 mg/mL!

References

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