Periodic Reporting for period 2 - PHOCNOSIS (Advanced nanophotonic point-of-care analysis device for fast and early diagnosis of cardiovascular diseases)
Reporting period: 2017-03-01 to 2019-02-28
There is an ever-increasing demand for novel and more efficient point-of-care testing (POCT) devices for the early diagnosis of diseases, with a special interest in cardiovascular diseases (CVD). The most recent statistics from the European Heart Network (EHN) and the European Society of Cardiology (ESC) show that CVD remain as the leading cause of death in the European Union (EU) accounting for 1.9 million deaths each year (40% of all deaths in the EU) with an estimated associated cost of almost €196 billion per year. Despite these numbers, CVD can be successfully treated when detected early and managed according to best practices. Therefore, having efficient detection tools for the implementation of preventive testing and early diagnosis is a key factor for reducing CVD mortality rates.
The PHOCNOSIS project aims at developing an ultra-sensitive, compact and low-cost POCT device based on nanophotonic and micro-/nanofluidic technology for its application to minimally-invasive early diagnosis of CVD. This POCT device will significantly help to reduce the actual costs designated for early diagnosis and to implement mass screening programs, allowing a significant contribution to the improvement of the citizens’ health status and to the sustainability of healthcare systems.
The PHOCNOSIS project aims at developing an ultra-sensitive, compact and low-cost POCT device based on nanophotonic and micro-/nanofluidic technology for its application to minimally-invasive early diagnosis of CVD. This POCT device will significantly help to reduce the actual costs designated for early diagnosis and to implement mass screening programs, allowing a significant contribution to the improvement of the citizens’ health status and to the sustainability of healthcare systems.
In summary, the main outcomes from the work carried out during Period 2 have been:
- The panel of potential biomarkers for CVD has been completed, including C-reactive protein (CRP) and myoglobin (Myo) to those already selected in Period 1 (cardiac troponin I (cTnI), cardiac troponin T (cTnT) and Creatine Kinase MB isoenzyme (CK-MB)). From them, cTnI is the most relevant for the development of the type of fast and early diagnosis device proposed in PHOCNOSIS.
- Consideration of a novel CVD biomarker (cardiac myosin-binding protein C (cMyC)) to be potentially included in the PHOCNOSIS system.
- To continue with the optimization of the defined protocol using ELISA technique for the highly sensitive and specific detection of the rest of CVD biomarkers (cTnT, CK-MB and Myo).
- A microfluidic concentration system was developed and its functionality was proven in chips made out of PDMS. Initial (but promising) results were obtained with the mass fabricated chips made out of Polycarbonate (PC).
- The interaction between the concentrated biomarkers (via the microfluidic system) and the immobilized antibodies has been investigated.
- The manufacturing process suitable for series production for mastering and replication of the microfluidic concentrator chip on wafer level was finally developed and optimized concerning channel depth and surface quality for bonding. The design of the microfluidic chip was adapted in order to allow that mass production approach.
- The approaches of using direct laser writing (DLW) into photoresist for mastering and injection compression molding (ICM) for replication could be approved.
- The transference of the fabrication process of the photonic chips to NIL (nanoimprint lithography) has been successfully completed.
- An approach for the fabrication of the photonic chips at wafer level using e-beam lithography (EBL) has also been established.
- Proposal of a novel configuration of photonic sensing structure based on the use of subwavelength grating (SWG) structures for the creation of a single channel interferometric structure. This configuration, as it is based in the combination of these two concepts able to increase the sensitivity, might mean a significant breakthrough in the field of the development of photonic biosensors.
- Additional optimization of the TEC and LAMI biofunctionalization approaches has been carried out, aiming at determining their multiplexing capability and their detection limit.
- CVD biomarkers detection has been demonstrated using the TEC biofunctionalization approach. However, a lack of repetitivity and a lower sensitivity than required have been determined. For the case of the LAMI biofunctionalization approach, photonic detection has been only observed for CRP using high concentrations.
- Alternative approaches for the biofunctionalization of the photonic chips have been considered. However, the sensitivity achieved was not enough to reach the limits initially targeted.
- A room-temperature assembly concept for PHOCNOSIS chips has been defined and demonstrated using double-sided adhesive tapes with laser-patterned structures.
- The integration of initial optofluidic chip samples has been successfully achieved.
- The integrated optofluidic chip comprising the microfluidic concentrator and the photonic chips has been tested. However, it was not possible to perform biosensing as the optical losses of the photonic chip significantly increased when applying the electrical current required for the fluidic concentration of the target CVD biomarkers.
- All the systems required for the operation and interrogation of the optofluidic chips in the final “product-like” version of the readout platform have been developed and tested.
- A packaged readout platform has been developed, including an adequate loading mechanism for the packaged biochip.
- The sensing capabilities of the final readout platform have been demonstrated by means of refractive index sensing experiments.
- Two panels of experts have been carried out in which relevant players have been involved in the analysis of the results achieved as well as in the determination of the best solution for the end users.
- In terms of standardization, we have collaborated with the UNE (Spanish National Standardization Body) for the revision of the current standards and the presentation of the PHOCNOSIS concept to the members of the European Technical Committee.
- Consensus has been reached at the consortium level to ensure the continuity of the project and the arrival to the final market, including the elaboration of an Exploitation Agreement.
- At the macro level, an in-depth study of the market and its growth potential has been made, including analysis of competitors and other relevant players. At the micro level we have studied the different applicable business models, and a business plan has been developed including market and financial strategy.
- Dissemination/communication activities targeting the promotion of the PHOCNOSIS project have been carried out.
- The panel of potential biomarkers for CVD has been completed, including C-reactive protein (CRP) and myoglobin (Myo) to those already selected in Period 1 (cardiac troponin I (cTnI), cardiac troponin T (cTnT) and Creatine Kinase MB isoenzyme (CK-MB)). From them, cTnI is the most relevant for the development of the type of fast and early diagnosis device proposed in PHOCNOSIS.
- Consideration of a novel CVD biomarker (cardiac myosin-binding protein C (cMyC)) to be potentially included in the PHOCNOSIS system.
- To continue with the optimization of the defined protocol using ELISA technique for the highly sensitive and specific detection of the rest of CVD biomarkers (cTnT, CK-MB and Myo).
- A microfluidic concentration system was developed and its functionality was proven in chips made out of PDMS. Initial (but promising) results were obtained with the mass fabricated chips made out of Polycarbonate (PC).
- The interaction between the concentrated biomarkers (via the microfluidic system) and the immobilized antibodies has been investigated.
- The manufacturing process suitable for series production for mastering and replication of the microfluidic concentrator chip on wafer level was finally developed and optimized concerning channel depth and surface quality for bonding. The design of the microfluidic chip was adapted in order to allow that mass production approach.
- The approaches of using direct laser writing (DLW) into photoresist for mastering and injection compression molding (ICM) for replication could be approved.
- The transference of the fabrication process of the photonic chips to NIL (nanoimprint lithography) has been successfully completed.
- An approach for the fabrication of the photonic chips at wafer level using e-beam lithography (EBL) has also been established.
- Proposal of a novel configuration of photonic sensing structure based on the use of subwavelength grating (SWG) structures for the creation of a single channel interferometric structure. This configuration, as it is based in the combination of these two concepts able to increase the sensitivity, might mean a significant breakthrough in the field of the development of photonic biosensors.
- Additional optimization of the TEC and LAMI biofunctionalization approaches has been carried out, aiming at determining their multiplexing capability and their detection limit.
- CVD biomarkers detection has been demonstrated using the TEC biofunctionalization approach. However, a lack of repetitivity and a lower sensitivity than required have been determined. For the case of the LAMI biofunctionalization approach, photonic detection has been only observed for CRP using high concentrations.
- Alternative approaches for the biofunctionalization of the photonic chips have been considered. However, the sensitivity achieved was not enough to reach the limits initially targeted.
- A room-temperature assembly concept for PHOCNOSIS chips has been defined and demonstrated using double-sided adhesive tapes with laser-patterned structures.
- The integration of initial optofluidic chip samples has been successfully achieved.
- The integrated optofluidic chip comprising the microfluidic concentrator and the photonic chips has been tested. However, it was not possible to perform biosensing as the optical losses of the photonic chip significantly increased when applying the electrical current required for the fluidic concentration of the target CVD biomarkers.
- All the systems required for the operation and interrogation of the optofluidic chips in the final “product-like” version of the readout platform have been developed and tested.
- A packaged readout platform has been developed, including an adequate loading mechanism for the packaged biochip.
- The sensing capabilities of the final readout platform have been demonstrated by means of refractive index sensing experiments.
- Two panels of experts have been carried out in which relevant players have been involved in the analysis of the results achieved as well as in the determination of the best solution for the end users.
- In terms of standardization, we have collaborated with the UNE (Spanish National Standardization Body) for the revision of the current standards and the presentation of the PHOCNOSIS concept to the members of the European Technical Committee.
- Consensus has been reached at the consortium level to ensure the continuity of the project and the arrival to the final market, including the elaboration of an Exploitation Agreement.
- At the macro level, an in-depth study of the market and its growth potential has been made, including analysis of competitors and other relevant players. At the micro level we have studied the different applicable business models, and a business plan has been developed including market and financial strategy.
- Dissemination/communication activities targeting the promotion of the PHOCNOSIS project have been carried out.
The use of the microfluidic and photonic technologies proposed in the PHOCNOSIS project would allow reaching sensitivity levels not obtained before using low footprint nanophotonic sensing technology. This sensitivity level would allow us using the developed technology for the detection of the extremely low concentration levels of cardiac biomarkers present on human samples. Additionally, the analysis concept proposed in PHOCNOSIS would allow having a final device being fast, compact and low-cost, what will open the door to its practical use by the healthcare systems around Europe. The performance of the PHOCNOSIS device would allow analysing several relevant CVD biomarkers in only a few minutes, thus providing very relevant information in order to immediately apply the proper treatment to patients potentially having a cardiac problem.