Periodic Reporting for period 1 - FreeATPOC (Towards an instrument-free future of molecular diagnostics at the point-of-care)
Periodo di rendicontazione: 2020-01-01 al 2021-12-31
Molecular diagnostics is still primarily a lab-based method. Emerging technologies for application at the point of care (POC)or need, i.e. where a patient or a sample is, have started gaining a big part of the molecular diagnostics market.
Interestingly, available methods are divided in two groups: sophisticated instruments for ultra-sensitive temperature regulated enzymatic amplification of nucleic acids and extremely simple paper-based immuno-diagnostic kits. Complexity, the main disadvantage of the former, results in their poor translation to several applications at the POC. Simplicity, often accompanied by failures, of the latter is the reason for their slow adoption even in resource limited areas. Our vision is an
instrument-free approach that combines the advantages of both groups, i.e. a method that does not use heaters or modules that require electricity, infrastructure and maintenance, but, still, takes advantage of enzymatically-amplified nucleic acids detection. To achieve the above, novel diagnostic tools for genetic amplification based on ligases, polymerases and restriction enzymes operating at ambient temperature will be developed, combined with quantitative smartphone
colorimetric/UV detection. The global applicability of the new approach will be demonstrated during infectious disease (Influenza and HIV) testing in human samples (blood and swabs) and plant pathogens (Xylella fastidiosa) in plant-tissues. In all cases, we aim for a time-to-result of less than 60 min, demonstrated sensitivities down to the clinically/field relevant values and a final cost in the order of $1 per assay. Combination of Free@POC concept with newly developed predictive models will expand its utility to monitoring disease outbreaks and their spreading. This new generation of instrument-free molecular diagnostics is expected to revolutionize nucleic acid analysis at the POC but also in applications beyond resulting in substantial societal as well as economic benefits.
Interestingly, available methods are divided in two groups: sophisticated instruments for ultra-sensitive temperature regulated enzymatic amplification of nucleic acids and extremely simple paper-based immuno-diagnostic kits. Complexity, the main disadvantage of the former, results in their poor translation to several applications at the POC. Simplicity, often accompanied by failures, of the latter is the reason for their slow adoption even in resource limited areas. Our vision is an
instrument-free approach that combines the advantages of both groups, i.e. a method that does not use heaters or modules that require electricity, infrastructure and maintenance, but, still, takes advantage of enzymatically-amplified nucleic acids detection. To achieve the above, novel diagnostic tools for genetic amplification based on ligases, polymerases and restriction enzymes operating at ambient temperature will be developed, combined with quantitative smartphone
colorimetric/UV detection. The global applicability of the new approach will be demonstrated during infectious disease (Influenza and HIV) testing in human samples (blood and swabs) and plant pathogens (Xylella fastidiosa) in plant-tissues. In all cases, we aim for a time-to-result of less than 60 min, demonstrated sensitivities down to the clinically/field relevant values and a final cost in the order of $1 per assay. Combination of Free@POC concept with newly developed predictive models will expand its utility to monitoring disease outbreaks and their spreading. This new generation of instrument-free molecular diagnostics is expected to revolutionize nucleic acid analysis at the POC but also in applications beyond resulting in substantial societal as well as economic benefits.
The concept behind FREE@POC is the development of point-of-care diagnostic platforms that are simple to use and rely only on a smartphone for control and operation. 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 sample-to-be-tested, 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 and the risk to produce something less efficient in terms of detection capability remains high. 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 innovation produced at the end of the project, we followed several routes; these included both attempts to develop platforms operating at 25-37oC, which inevitably areless efficient in terms of limit of detection and platforms relying on very basic instrumentation, resulting in minimal technology/ electricity dependency but showing high performance and a low limit of detection. Regarding the former, we demonstrated the detection of as little as 100 viral copies during amplification at 37oC, using a torch and a dye. This work will continue during years 3 and 4 and if successful, it will produce innovation in molecular diagnostics, bioimage analysis and software development. In parallel, during Year 1 we developed a 3D-printed device that performs real time quantitative LAMP and costs less than 80 euros during lab-production. This prototype is currently in the market, certified as per IVDD for SARS-CoV-2 and flu testing. The innovation falls within the FREE@POC work since the first validation studies were performed during Year 1 of the project (2020). One more innovation, also employing a very simple instrumentation is currently under development; it relies on the combination of paper microfluidics with an acoustic biochip to form a generic platform suitable for Ab, nucleic acids and antigen detection. Such a flexible system has not been reported so far and will be patented as well for future exploitation. The above are expected to contribute significantly in advancing of the state of the art in the field of diagnostics and produce useful tools for application in the field. The fact that we are targeting both human and plant-borne pathogens is also enhancing the impact of the work; potential applications could be found in the healthcare, but also agro/food safety, enhancing the impact of the project results in the developing and developed countries.
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 innovation produced at the end of the project, we followed several routes; these included both attempts to develop platforms operating at 25-37oC, which inevitably are less efficient in terms of limit of detection and platforms relying on very basic instrumentation, resulting in minimal technology/ electricity dependency but showing high performance and a low limit of detection. Regarding the former, we demonstrated the detection of as little as 100 viral copies during amplification at 37oC, using a torch and a dye. This work will continue during years 3 and 4 and if successful, it will produce innovation in molecular diagnostics, bioimage analysis and software development. In parallel, during Year 1 we developed a 3D-printed device that performs real time quantitative LAMP and costs less than 80 euros during lab-production. This prototype is currently in the market, certified as per IVDD for SARS-CoV-2 and flu testing. The innovation falls within the FREE@POC work since the first validation studies were performed during Year 1 of the project (2020). One more innovation, also employing a very simple instrumentation is currently under development; it relies on the combination of paper microfluidics with an acoustic biochip to form a generic platform suitable for Ab, nucleic acids and antigen detection. Such a flexible system has not been reported so far and will be patented as well for future exploitation. The above are expected to contribute significantly in advancing of the state of the art in the field of diagnostics and produce useful tools for application in the field. The fact that we are targeting both human and plant-borne pathogens is also enhancing the impact of the work; potential applications could be found in the healthcare, but also agro/food safety, enhancing the impact of the project results in the developing and developed countries.