The concept behind FREE@POC is the development of a point-of-care diagnostic platform that is simple to use and relies on minimal equipment for control and operation, such as a smartphone. Specifically, it is proposed to use an isothermal amplification method that can operate close to room/body temperature, i.e. in the range of 25-37oC. Such a system would be classified as instrument- or electricity-free and would be ideal for operation not only close to the patient or the sample, but also in remote areas and the developing countries. In addition to low cost and complexity, we envision a system that would have the necessary sensitivity for detection even in crude samples. Overall, there are several challenges one has to overcome to produce a solution that is efficient in terms of detection capability and can also operate at the POC. Towards this end, all partners have put considerable effort in progressing the state of the art and producing novel tools of high performance and low cost. To maximize the project outputs and potential innovations by the end of the project, we followed several routes; these included various attempts to develop assays operating optimally at 25-37oC in terms of limit of detection as well as platforms and devices relying on basic instrumentation, with low technology/electricity dependency but with a high performance and detection capability.
All assays were developed upon extensive optimization of relevant steps, especially the sample pre-treatment for lysis at room temperature and under conditions compatible with the subsequent isothermal amplification. Regarding the HIV test, we demonstrated the detection of as little as 10 viral copies per reaction (or <1000 copies per ml of plasma) after isothermal (RPA) amplification at 37oC using our newly developed molecular lateral flow strip (LFS)-based technology. Such a high performance assay, leading to semi-quantitative HIV detection in patients’ plasma at the point of care is unique and has never been reported before. The same protocol and methodology were also applied to influenza detection, reaching a detection limit of 50 copies per reaction. Moreover, the HIV and influenza assays were validated by end users in a hospital environment, both in Europe and S. Africa, demonstrating the feasibility of the method. Finally, the universal and generic nature of the LFS molecular assay has been demonstrated during Xylella pathogens detection in the field using crude plant tissue. It is noted that the two assays developed for HIV and influenza detection are already commercially available and can be obtained for research purposes in the form of two kits.
Our newly developed and patented methodology, based on a molecular Lateral Flow Strip assay for amplicons’ detection and a 3D-printed LFS reader and relevant software, is an attractive solution for diagnostics at the POC using minimal instrumentation. The low cost of the reader (<70 euros), together with the simplicity in operation and rapid and semi-quantitative nature of the detection make the above innovation an attractive solution for global diagnostics. The method, currently at TRL=4-5, will form the main result for future exploitation and is the basis of FREE@POC business plan. Further innovations also produced within FREE@POC include a portable instrumentation which relies on the combination of paper microfluidics with an acoustic (SAW) biochip. This method also forms a generic platform suitable for nucleic acids detection but also antibodies and antigens. Such a flexible system has not been reported so far and a new patent application has been filed for this purpose. Overall, the project has resulted in several new approaches and methods, leading to two patent applications, two small pilot productions of HIV and influenza kits and a robust and attractive methodology for Xylella detection in the field using crude plant tissue.
