Periodic Reporting for period 1 - PHOREVER (PHOtonic integrated OCT-enhanced flow cytometry for canceR and cardiovascular diagnostics enabled by Extracellular VEsicles discRimination)
Reporting period: 2023-01-01 to 2024-06-30
PHOREVER will develop a cutting-edge multi-sensing platform capable of detecting extracellular vesicles (EVs) as small as 140 nm, identifying disease-specific proteins (biomarkers) on their membrane surfaces, and determining EV concentrations in human blood samples. This innovative detection system will integrate three distinct sensing modalities:
Flow cytometry (FCM) serves as the primary sensing method for detecting and classifying particles by size in blood samples. Optical coherence tomography (OCT) functions as a secondary modality, providing micro-imaging of the sensing area and implementing a coherent gate to significantly reduce noise in FCM measurements. Fluorescence sensing is employed as the third modality, enabling the detection of target biomarkers on EV surfaces after staining with fluorescent dye. PHOREVER introduces a number of key innovations, summarized through the following 8 objectives:
Obj 1: Develop a TriPleX PIC for FCM and fluorescence sensing and use it as a dual sensing tool for detection of EVs in blood samples and detection of biomarkers on the surface of these EVs.
Obj 2: Develop a TriPleX PIC with a dual-channel spectral domain (SD-OCT) unit on-chip and use it as a coherent gate for the processing of the FCM measurement data.
Obj 3: Develop a photonic-electronic stack comprising the two sensing PICs and their companion interposers as the non-disposable part of the multi-sensing platform.
Obj 4: Develop a microfluidic unit for the pre-analytical and analytical handling of blood samples as the disposable part of the multi-sensing PHOREVER platform.
Obj 5: Develop the control electronics of the sensing platform and the algorithms for the execution of the measurements and the processing of the measurement data from the two sensing PICs.
Obj 6: Validation of the detection potential of the multi-sensing platform using reference EV materials, and development of a comprehensive data analysis tool empowered by AI algorithms for use in the medical cases of pancreatic cancer and stroke.
Obj 7: Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples about pancreatic cancer.
Obj 8: Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples in relation to stroke.
Flow cytometry (FCM) serves as the primary sensing method for detecting and classifying particles by size in blood samples. Optical coherence tomography (OCT) functions as a secondary modality, providing micro-imaging of the sensing area and implementing a coherent gate to significantly reduce noise in FCM measurements. Fluorescence sensing is employed as the third modality, enabling the detection of target biomarkers on EV surfaces after staining with fluorescent dye. PHOREVER introduces a number of key innovations, summarized through the following 8 objectives:
Obj 1: Develop a TriPleX PIC for FCM and fluorescence sensing and use it as a dual sensing tool for detection of EVs in blood samples and detection of biomarkers on the surface of these EVs.
Obj 2: Develop a TriPleX PIC with a dual-channel spectral domain (SD-OCT) unit on-chip and use it as a coherent gate for the processing of the FCM measurement data.
Obj 3: Develop a photonic-electronic stack comprising the two sensing PICs and their companion interposers as the non-disposable part of the multi-sensing platform.
Obj 4: Develop a microfluidic unit for the pre-analytical and analytical handling of blood samples as the disposable part of the multi-sensing PHOREVER platform.
Obj 5: Develop the control electronics of the sensing platform and the algorithms for the execution of the measurements and the processing of the measurement data from the two sensing PICs.
Obj 6: Validation of the detection potential of the multi-sensing platform using reference EV materials, and development of a comprehensive data analysis tool empowered by AI algorithms for use in the medical cases of pancreatic cancer and stroke.
Obj 7: Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples about pancreatic cancer.
Obj 8: Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples in relation to stroke.
WP2: The industrial use cases and application space for PHOREVER technology have been defined, along with an initial set of system requirements, module designs, component-level specifications, design rules, packaging strategies, methodologies, system-level simulation layouts, power budget analysis for intermediate modules, and AI-based models developed based on FCM retrospective data from clinical partners for cancer and stroke use cases.
WP3: The design for the FCM and OCT PIC has been completed, the swept-source laser has been replaced with spectral domain OCT, the 3D printing process for lens fabrication has been successfully implemented at PHIX, the alignment markers for lens printing has been verified and lenses have been 3D printed on the side facets of test TriPleX PICs, and the first generation of control electronics is under development.
WP4: Procedures for the collection, handling, and storage of body fluids for EV analysis were tested at AMC, including the establishment of whole blood collection and plasma preparation, the development of a process flow for blood sample handling for plasma generation and EV staining, translation of the protocol into a fluidic chip, development of a fluidic actuation instrument with pressure pumps and flow rate sensors, evaluation of blood filtration and metering concepts, integration of size exclusion chromatography in a microfluidic chip with successful dye cluster separation, the establishment of tangential flow filtration for unbound antibody filtration, hydrodynamic focusing on narrowing the sample stream to 10 μm, and the manufacture and demonstration of an early-stage fluidic cartridge.
WP5: The intermediate OCT and FCM PIC designs were successfully completed, high-level functional schematics for both OCT and FCM intermediate demonstrator systems were defined, a concept for stacked FCM+OCT assembly was established, a breadboard instrument was implemented to control the intermediate PIC and hydrodynamic focusing module, and a concept for instrument control electronics (excluding PIC control) was developed.
WP6: The generation of a biobank with blood samples from pancreatic cancer patients has begun at NKUA, and approval has been granted to access the biobank containing blood plasma samples from stroke patients at AMC.
WP7: Partners actively communicate LOLIPOP results through social media, websites, and events.
WP8: Ethics requirements have been defined and addressed. Clinical protocols, procedures, and data-sharing agreement have been established.
WP3: The design for the FCM and OCT PIC has been completed, the swept-source laser has been replaced with spectral domain OCT, the 3D printing process for lens fabrication has been successfully implemented at PHIX, the alignment markers for lens printing has been verified and lenses have been 3D printed on the side facets of test TriPleX PICs, and the first generation of control electronics is under development.
WP4: Procedures for the collection, handling, and storage of body fluids for EV analysis were tested at AMC, including the establishment of whole blood collection and plasma preparation, the development of a process flow for blood sample handling for plasma generation and EV staining, translation of the protocol into a fluidic chip, development of a fluidic actuation instrument with pressure pumps and flow rate sensors, evaluation of blood filtration and metering concepts, integration of size exclusion chromatography in a microfluidic chip with successful dye cluster separation, the establishment of tangential flow filtration for unbound antibody filtration, hydrodynamic focusing on narrowing the sample stream to 10 μm, and the manufacture and demonstration of an early-stage fluidic cartridge.
WP5: The intermediate OCT and FCM PIC designs were successfully completed, high-level functional schematics for both OCT and FCM intermediate demonstrator systems were defined, a concept for stacked FCM+OCT assembly was established, a breadboard instrument was implemented to control the intermediate PIC and hydrodynamic focusing module, and a concept for instrument control electronics (excluding PIC control) was developed.
WP6: The generation of a biobank with blood samples from pancreatic cancer patients has begun at NKUA, and approval has been granted to access the biobank containing blood plasma samples from stroke patients at AMC.
WP7: Partners actively communicate LOLIPOP results through social media, websites, and events.
WP8: Ethics requirements have been defined and addressed. Clinical protocols, procedures, and data-sharing agreement have been established.
WP2: The industrial use cases and application areas for PHOREVER technology have been identified, alongside an initial set of system requirements, module designs, component specifications, design rules, packaging strategies, methodologies, system-level simulation layouts, power budget analyses for intermediate modules, and AI-based models developed using FCM retrospective data from clinical partners for cancer and stroke applications.
WP3: The designs for the FCM and OCT PIC have been finalized, with the swept-source laser being replaced by spectral domain OCT; the 3D printing process for lens fabrication has been successfully implemented at PHIX, alignment markers for lens printing have been verified, lenses have been 3D printed on the side facets of test TriPleX PICs, and the first generation of control electronics is currently in development.
WP4: Procedures for collecting, handling, and storing body fluids for EV analysis have been tested at AMC, which includes establishing protocols for whole blood collection and plasma preparation, developing a workflow for blood sample handling to generate plasma and stain EVs, translating this protocol into a fluidic chip, creating a fluidic actuation instrument equipped with pressure pumps and flow rate sensors, evaluating concepts for blood filtration and metering, integrating size exclusion chromatography into a microfluidic chip with successful dye cluster separation, establishing tangential flow filtration for unbound antibody filtration, achieving hydrodynamic focusing to narrow the sample stream to 10 μm, and manufacturing and demonstrating an early-stage fluidic cartridge.
WP5: The designs for the intermediate OCT and FCM PICs have been successfully completed, high-level functional schematics for both the OCT and FCM intermediate demonstrator systems have been defined, a concept for a stacked FCM+OCT assembly has been established, a breadboard instrument has been implemented to control the intermediate PIC and hydrodynamic focusing module, and a concept for the instrument control electronics (excluding PIC control) has been developed.
WP6: The creation of a biobank with blood samples from pancreatic cancer patients has commenced at NKUA, and approval has been received to access the biobank containing blood plasma samples from stroke patients at AMC.
WP7: Partners are actively disseminating LOLIPOP results through social media, websites, and various events.
WP8: Ethics requirements have been defined and addressed, with clinical protocols, procedures, and a data-sharing agreement established.
WP3: The designs for the FCM and OCT PIC have been finalized, with the swept-source laser being replaced by spectral domain OCT; the 3D printing process for lens fabrication has been successfully implemented at PHIX, alignment markers for lens printing have been verified, lenses have been 3D printed on the side facets of test TriPleX PICs, and the first generation of control electronics is currently in development.
WP4: Procedures for collecting, handling, and storing body fluids for EV analysis have been tested at AMC, which includes establishing protocols for whole blood collection and plasma preparation, developing a workflow for blood sample handling to generate plasma and stain EVs, translating this protocol into a fluidic chip, creating a fluidic actuation instrument equipped with pressure pumps and flow rate sensors, evaluating concepts for blood filtration and metering, integrating size exclusion chromatography into a microfluidic chip with successful dye cluster separation, establishing tangential flow filtration for unbound antibody filtration, achieving hydrodynamic focusing to narrow the sample stream to 10 μm, and manufacturing and demonstrating an early-stage fluidic cartridge.
WP5: The designs for the intermediate OCT and FCM PICs have been successfully completed, high-level functional schematics for both the OCT and FCM intermediate demonstrator systems have been defined, a concept for a stacked FCM+OCT assembly has been established, a breadboard instrument has been implemented to control the intermediate PIC and hydrodynamic focusing module, and a concept for the instrument control electronics (excluding PIC control) has been developed.
WP6: The creation of a biobank with blood samples from pancreatic cancer patients has commenced at NKUA, and approval has been received to access the biobank containing blood plasma samples from stroke patients at AMC.
WP7: Partners are actively disseminating LOLIPOP results through social media, websites, and various events.
WP8: Ethics requirements have been defined and addressed, with clinical protocols, procedures, and a data-sharing agreement established.