Periodic Reporting for period 1 - Smartpolymer (Developing smart polymer compositions for highly sensitive multiplex PCR assays)
Période du rapport: 2019-08-01 au 2021-07-31
The project Smartpolymer is dedicated to the development of smart polymer compositions for highly sensitive PCR assays that are able to detect multiple pathogens.
Nowadays being confronted to a huge world sanitary crisis caused by COVID-19 virus this is clear for everybody that we need further and further to develop fast, sensitive and easy-to-perform tests for detection of different pathogens. These are all kind of viruses like Coronaviruses, Influenza viruses, plurality of bacteria responsible for water and food contamination, causing such dangerous diseases as Pneumonia, bacterial meningitis, a lot of sexually transmitted diseases and so on. Thefore, the important mission is to develop ultrafast PCR machines and tests, able to detect all of these pathogens in a fast, user-friendly and highly sensitive way.
PCR, polymerase chain reaction, is a molecular diagnostic method based on rapid production of millions to billions of copies of DNA or RNA of bacteria or virus, allowing to detect ultra-small quantities of pathogens in a sample. PCR technique greatly overtops traditional petri-dish culture methods due to its superior fastness (the analysis takes several hours instead of several days), ability to detect viruses and non-growable bacteria. However, culturing methods still remain in use because of their reliability, efficiency, sensitivity, and range of applications, such as ability to quantify down to few bacteria, to acess antibioresistance and bacterial specie identification.
In the project "Smartpolymer" we develop a new method of analysis that uniquely combines PCR and Petri dish technologies in so called “Molecular Petri dish” technology. This is achieved by changing the PCR media from water to a specially designed polymer matrix. This matrix is able to capture every bacteria or virus in the sample separately from each other. After the PCR the matrix also captures generated multiple copies of bacterial or viruses’ genetic material nearby their “parent locations”. The principle is the same as in traditional Petri dish, however our technology allows way more fast and sensitive detection of pathogens in less than 1 hour. What is more, in combination with BforCure Chronos machine the analysis can be performed outside the big laboratories without the need of trained personnel and special equipment.
So, the main objective of the project "Smartpolymer" is to design, synthesize and optimize smart polymer compositions that would allow fast and precise detection of multiple pathogens.
Nowadays being confronted to a huge world sanitary crisis caused by COVID-19 virus this is clear for everybody that we need further and further to develop fast, sensitive and easy-to-perform tests for detection of different pathogens. These are all kind of viruses like Coronaviruses, Influenza viruses, plurality of bacteria responsible for water and food contamination, causing such dangerous diseases as Pneumonia, bacterial meningitis, a lot of sexually transmitted diseases and so on. Thefore, the important mission is to develop ultrafast PCR machines and tests, able to detect all of these pathogens in a fast, user-friendly and highly sensitive way.
PCR, polymerase chain reaction, is a molecular diagnostic method based on rapid production of millions to billions of copies of DNA or RNA of bacteria or virus, allowing to detect ultra-small quantities of pathogens in a sample. PCR technique greatly overtops traditional petri-dish culture methods due to its superior fastness (the analysis takes several hours instead of several days), ability to detect viruses and non-growable bacteria. However, culturing methods still remain in use because of their reliability, efficiency, sensitivity, and range of applications, such as ability to quantify down to few bacteria, to acess antibioresistance and bacterial specie identification.
In the project "Smartpolymer" we develop a new method of analysis that uniquely combines PCR and Petri dish technologies in so called “Molecular Petri dish” technology. This is achieved by changing the PCR media from water to a specially designed polymer matrix. This matrix is able to capture every bacteria or virus in the sample separately from each other. After the PCR the matrix also captures generated multiple copies of bacterial or viruses’ genetic material nearby their “parent locations”. The principle is the same as in traditional Petri dish, however our technology allows way more fast and sensitive detection of pathogens in less than 1 hour. What is more, in combination with BforCure Chronos machine the analysis can be performed outside the big laboratories without the need of trained personnel and special equipment.
So, the main objective of the project "Smartpolymer" is to design, synthesize and optimize smart polymer compositions that would allow fast and precise detection of multiple pathogens.
In general, the project has achieved most of its objectives and milestones for the period, with relatively minor deviations.
(1) Scientific objectives:
• We designed and synthetised several bio-compatible polymer compositions which display a low viscosity at room temperature (r.t.) allowing mixing target molecules/cells and amplification reagents at that temperature and subsequent user manipulations required for production, but rapidly form a homogeneous hydrogel network during PCR.
• The gel components and the gelation procedure are not damaging to PCR reagents, target and amplification process.
• The design of gel matrices was done in a way that they will be able to withstand the rapid changes of temperature and retain structural integrity under stress during the nucleic acid amplification in a wide range of temperatures. The gels designed are characterized by homogeneous structure and mechanical stability. We showed their usability in different kinds of PCR tests on different PCR thermal cyclers including ultra-fast PCR thermocycler Chronos, that is designed and produced by BforCure.
• The obtained gels possess homogeneous structure, the formed molecular colonies are uniform and their quantity reflects the actual quantity of the target. The gels' percentage and composition, and, consequently, the mesh size can be modified depending on the target and PCR reagents used.
(2) Training objectives:
• Marketing and communication strategy. The ER participated in the International conference Analytica 2020 (19-22 October 2020, online) where she communicated to a lot of researchers, representatives of laboratories, private companies, industry in order to establish partnership, advertise the technology and attract the future clients.
• Product- and market-oriented R&D work, entrepreneurship evolution. In the course of the project the ER communicated to all of the BforCure team: with the CEO who helped in identification of her career plans and goals, in studying entrepreneurship; with the marketing team that taught her the basics of communication and advertising. The ER participated in CE Marking Training session (04/2020) hold by the leading specialist in the field, Dr Gruszka (linkedin.com/in/fatima-gruszka-4621b41).
• IP. In the beginning of the project, the ER was trained to do a Freedom-To-Operate (FTO) analysis (by participating in the workshop on intellectual property (01/2020) hold by the leading specialist in the field, Mr. Jacques Vesin (linkedin.com/in/jacques-vesin-4785b417)). ER studied >100 patents and summarized which technology will be an obstacle to develop the product and how to cross over the protected property right to continue conducting the R&D. As a result 2 patent drafts were sent to the patent office.
• Training in the complementary to ER expertise areas. The ER got a full training in PCR techniques and equipment, training in the basics of routine R&D work in the field of molecular and microbiology (design of PCR kits and performing a variety types of PCR – qPCR, reverse transcription PCR, dPCR; sample preparation techniques; growing and analysis of different bacterial cultures, DNA and RNA extraction techniques etc.) ER attended several webinars, e.g. “Lyophilization-Changing the Game for Molecular Diagnostics” (27/10/2020). Based on received training ER performed a big panel of abovementioned experiments on a high scientific level and mastered the technique.
(1) Scientific objectives:
• We designed and synthetised several bio-compatible polymer compositions which display a low viscosity at room temperature (r.t.) allowing mixing target molecules/cells and amplification reagents at that temperature and subsequent user manipulations required for production, but rapidly form a homogeneous hydrogel network during PCR.
• The gel components and the gelation procedure are not damaging to PCR reagents, target and amplification process.
• The design of gel matrices was done in a way that they will be able to withstand the rapid changes of temperature and retain structural integrity under stress during the nucleic acid amplification in a wide range of temperatures. The gels designed are characterized by homogeneous structure and mechanical stability. We showed their usability in different kinds of PCR tests on different PCR thermal cyclers including ultra-fast PCR thermocycler Chronos, that is designed and produced by BforCure.
• The obtained gels possess homogeneous structure, the formed molecular colonies are uniform and their quantity reflects the actual quantity of the target. The gels' percentage and composition, and, consequently, the mesh size can be modified depending on the target and PCR reagents used.
(2) Training objectives:
• Marketing and communication strategy. The ER participated in the International conference Analytica 2020 (19-22 October 2020, online) where she communicated to a lot of researchers, representatives of laboratories, private companies, industry in order to establish partnership, advertise the technology and attract the future clients.
• Product- and market-oriented R&D work, entrepreneurship evolution. In the course of the project the ER communicated to all of the BforCure team: with the CEO who helped in identification of her career plans and goals, in studying entrepreneurship; with the marketing team that taught her the basics of communication and advertising. The ER participated in CE Marking Training session (04/2020) hold by the leading specialist in the field, Dr Gruszka (linkedin.com/in/fatima-gruszka-4621b41).
• IP. In the beginning of the project, the ER was trained to do a Freedom-To-Operate (FTO) analysis (by participating in the workshop on intellectual property (01/2020) hold by the leading specialist in the field, Mr. Jacques Vesin (linkedin.com/in/jacques-vesin-4785b417)). ER studied >100 patents and summarized which technology will be an obstacle to develop the product and how to cross over the protected property right to continue conducting the R&D. As a result 2 patent drafts were sent to the patent office.
• Training in the complementary to ER expertise areas. The ER got a full training in PCR techniques and equipment, training in the basics of routine R&D work in the field of molecular and microbiology (design of PCR kits and performing a variety types of PCR – qPCR, reverse transcription PCR, dPCR; sample preparation techniques; growing and analysis of different bacterial cultures, DNA and RNA extraction techniques etc.) ER attended several webinars, e.g. “Lyophilization-Changing the Game for Molecular Diagnostics” (27/10/2020). Based on received training ER performed a big panel of abovementioned experiments on a high scientific level and mastered the technique.
To the best of our knowledge, there is no available PCR assay using thermally- of light-triggered polymers. Such new polymer compositions were designed and applied for the first time within the PCR assay allowing fast, sensitive and selective quantification of different targets within the single reaction.
The PCR assay combined with the use of smart polymer matrix greatly contributes to digitalizing assays and making multiplex target quantification straightforward. PCR techniques are extremely useful in such fundamental research areas as Genetics, Medicine, Forensic Science, and Environmental Microbiology. The advanced variant, multiplex PCR, allows simultaneous amplification of several DNA fragments within one reaction. This results, on the one side, in saving time and money and, on the other side, in further improvement of this technology and its application to increasingly higher multiplex systems. The new designed polymer compositions will impact on digitalizing assays and making multiplex target quantification straightforward and highly sensitive. The ability of easy loading and assembling homogeneously all PCR reagents and sample at room temperature into the PCR chip within the method proposed will assure its using outside of the laboratory such as fast Point of Care diagnosis.
The PCR assay combined with the use of smart polymer matrix greatly contributes to digitalizing assays and making multiplex target quantification straightforward. PCR techniques are extremely useful in such fundamental research areas as Genetics, Medicine, Forensic Science, and Environmental Microbiology. The advanced variant, multiplex PCR, allows simultaneous amplification of several DNA fragments within one reaction. This results, on the one side, in saving time and money and, on the other side, in further improvement of this technology and its application to increasingly higher multiplex systems. The new designed polymer compositions will impact on digitalizing assays and making multiplex target quantification straightforward and highly sensitive. The ability of easy loading and assembling homogeneously all PCR reagents and sample at room temperature into the PCR chip within the method proposed will assure its using outside of the laboratory such as fast Point of Care diagnosis.