Project Objectives

Objective 1:
Develop a TriPleX® PIC for flow-cytometry (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

PHOREVER will develop a disruptive sensing PIC on TriPleX® platform that will support the simultaneous execution of flow-cytometry (FCM) measurements and fluorescence sensing on blood samples. The PIC will have a through-hole with 800 µm diameter that will accommodate the use of a flow-channel normal to the PIC. Pretreated blood samples (plasma) will flow in this channel serving as the subject of investigation for both sensing modalities. In the first part of the overall process, the FCM modality will serve as a quality control to confirm the absence of blood cells and hemolysis byproducts from the sample, and to estimate the total number of particles inside the plasma sample. In the second part that involves the actual measurements, the FCM modality will be responsible for the detection and size classification of all the EVs with diameter size down to 80 nm, whereas the fluorescence sensing modality will be responsible for the detection of EVs that carry the biomarkers of interest on their membrane. A step for staining the biomarkers with fluorescent antibodies will take place between the two parts.

The FCM subcircuit in the PIC will comprise 4 laser diodes (LDs) at 405, 488, 633 and 785 nm emitting up to 100 mW each. The 4 wavelengths will be brought to the sensing area by a common illumination waveguide, in which the wavelengths will be combined using a cascade of wide-band couplers. A time-division multiplexing (TDM) scheme will be applied for the activation and deactivation of each LD in order to ensure the illumination of the sample by a single wavelength at each point of time. The scattered photons will be collected by a group of 12 waveguides, supporting the detection of both forward and side-scattered photons. The exact position of the collection waveguides will maximize their spatial density in the parts of the flow-channel circumference that can offer most useful information about the size and the refractive index of the scatterers. At the other end of each collection waveguide, the photons will be detected by an avalanche photodiode (APD) on-chip. In front of each APD, a narrow-band filter based on a Mach-Zehnder interferometer (MZI) and a micro-ring resonator (MRR) will be periodically tuned over the 4 wavelengths in a synchronous way with the TDM scheme in order to select the active wavelength at each point of time, blocking any background noise from fluorescence emission. Finally, a waveguide at the opposite side of the through-hole with respect to the illumination waveguide will act as a beam sink, collecting the un-scattered part of the beam, and minimizing the stray light. The fluorescence subcircuit in the same PIC will comprise the LD at 488 nm, 2 collection waveguides in the circumference of the flow-channel, and 2 photo-multiplier tubes (PMTs), hybridly integrated on-chip, for ultra-sensitive detection of the fluorescence light at 500-600 nm. In front of each PMT, a monolithic notch filter based on an MZI and an MRR will block the excitation light at 488 nm. Last but not least, both the illumination and the collection waveguides will be equipped at the edge of the through-hole with monolithic spot-size converters and hybrid (polymer) micro-lenses that will adjust their illumination or light collection parameters as summarized in Table 1. The total size of this first sensing PIC will be 10 mm × 20 mm.

Objective 2:
Develop a TriPleX® PIC with a dual-channel swept-source optical coherence tomography (SS-OCT) unit on-chip and use it as a coherent gate for the processing of the FCM measurement data

PHOREVER will develop the first ever PIC that will support the entire process of the swept-source optical coherence tomography (SS-OCT) on-chip. The PIC will be developed on the TriPleX® platform, and will have a through-hole for the accommodation of the same flow-channel as the first sensing PIC. The OCT sensing modality will make an image of the sensing area inside the flow-channel offering information about the number and the position of the plasma particles that will be illuminated inside the flow-channel at each point of time. In this respect, it will offer a coherent gate or in other words, a means for the validation of the scattering patterns from the FCM measurements that will be performed with the first sensing PIC on the same volume of sample.

The key element of the SS-OCT PIC will be a world’s first photonic integrated swept laser source with sub-micron operation, which will offer 100 nm sweeping span around 750 nm. This source will be built as an external cavity laser (ECL) on the TriPleX® platform. Two GaAs active elements with small overlap in their gain spectra will be hybridly integrated in the TriPleX® PIC to serve as the gain media of the ECL. The cavity will extend to the TriPleX® motherboard, where a set of MRRs will form a semi-transparent mirror with wide tunability based on the Vernier effect. Wavelength sweeping will be based on the reconfiguration of the MRRs and the phase tuning of the laser cavity, both by means of lead zirconate titanate (PZT) phase shifters with reconfiguration speeds on the order of 10 MHz. Using an approximate number of 100 wavelength steps of 1 nm each for the coverage of the total 700-800 nm span, the resultant sweeping rate is expected to be close to 100 kHz. The mode-hops that will be present in this wavelength sweeping process due to the Vernier effect will be deterministic, and thus well controlled, resulting in stable and low-noise sweeping iterations. The light at the ECL output will be split in two parts to support the operation of two SS-OCT channels. Each one of these parts will be further split to form the sensing and the reference arm of the corresponding channel. The two sensing arms will illuminate the flow-channel from two points allowing for the creation of two perpendicular tomographic slices of it. A monolithic spot-size converter and a hybrid micro-lens will be integrated again at the edge of each illumination waveguide to form a collimated beam with the same cross-section as in first sensing PIC (10 µm × 20 µm). Finally, the back-reflected light from each sensing part will be combined with the corresponding reference light before their detection by a photodiode hybridly integrated on-chip. Given the target parameters of the swept source, the axial resolution and the imaging depth of each OCT channel will be 2.5 µm and 1.4 mm, respectively. The total size of this second PIC will be similar to the first one (i.e. 10 mm × 20 mm).

Objective 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

PHOREVER will develop two printed circuit boards (PCBs) that will serve as interposers for the mounting of each sensing PIC and the routing of the electrical lines that will activate their active and detection elements, and their phase shifters. At the first stage of this development process, each PIC will be mounted onto the upper side of its companion interposer. A through-hole in each interposer will be aligned with the through-hole in the corresponding PIC. The electrical connections between the pads in the two PICs and the pads on their interposers will be realized by means of wire-bonds. At the second stage, one the two subassemblies will be flipped and mounted onto the other one (back-to-back) forming the final 4-layer photonic-electronic stack. The through-holes at the 4 layers will remain aligned accommodating the use of the flow-channel. Within this stack, the vertical distance between the two PICs along the flow-channel will be known with high precision, enabling the synchronization between the FCM and the OCT modalities. This 4-layer stack will be used as the non-disposable core of the multi-sensing platform. A single electrical connector will enable the electrical connection of the entire stack to the control electronics of the platform.

Objective 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

PHOREVER will develop a microfluidic unit that will be responsible for the pre-analytical and analytical handling of blood samples. In the pre-analytical stage, the blood sample will be loaded through a funnel. The funnel will then be closed, and the sample pushed through a blood filter driven by over pressure. The generated plasma is in a next step immunolabeled before it is transferred into the analytical stage. In the analytical stage the sample is first passively aligned with inertial forces within a meander structure. In a second step the sample is finally actively aligned and separated by the introduction of a concentric sheath flow. When the particles are well centered and spaced in the flow focusing channel, they pass the detector unit. In the first round of measurement, the FCM signal of the particles will be used to estimate their total concentration in order to determine the required dilution, as a preparation for the second round of the measurement. The sample will pass through an intermediate tank, for proper dilution in Phosphate Buffered Saline (PBS) solution. Then, passing through a second tank, the sample will be stained with specific reagents. A final dilution step follows before the final pass through the flow channel, where being actively aligned, finally be measured by the FCM and OCT sensing modules.

Objective 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

HOREVER will develop the control electronics of the multi-sensing platform, supporting the operation of the microfluidic unit for the pre-analytical and analytical treatment of the blood samples, and the operation of the two PICs with the three sensing modalities (FCM, fluorescence sensing and SS-OCT). The control electronics will be connected to the PCB of the microfluidic unit and the interposer of the two sensing PICs in the 4-layer stack. Apart from the digital and analog parts of the control electronics at the hardware level, PHOREVER will also develop the smart algorithms for the execution of the measurements, the data acquisition and the data processing associated with:

The control of the microfluidic elements that will be responsible for: a) the pre-analytical treatment of the blood sample and the extraction of plasma, b) the flow of the plasma through the flow-channel for the execution of the quality control process, c) the dilution and the staining of the sample, d) the flow of the plasma through the same flow-channel for the performance of the actual EV measurements, and e) the management of the measurement waste.
The operation of the PMTs in the first sensing PIC for the sensing of fluorescence emission from the stained EVs.
The implementation of the TDM scheme for the operation of the 4 LDs in the first sensing PIC, and the execution of the FCM measurements for the extraction of the scattering patterns with the forward- and side-scattered photons.
The tuning of the MRRs and the phase sections inside the cavity of the ECL in the second sensing PIC, and the execution of the wavelength sweeping process without random mode-hopes and instability in the sweeping iterations.
The use of the SS-OCT in the second sensing PIC for the imaging of the sensing area, and the use of AI algorithms (convolutional neural networks) for the identification of the number and the positions of the particles inside this area.
The fusion of the FCM and the OCT data sets for the interpretation of the FCM data (i.e. the extracted scattering patterns) into credible information about the size and the refractive index of the flowing particles inside the samples.

Objective 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 the pancreatic cancer and the stroke

PHOREVER will use reference materials that are clinically relevant and are developed within the METVES project. These materials will contain healthy EVs from human plasma that are further pre-stained with fluorescent antibodies. They will resemble the natural heterogeneity of the EVs in the blood, in terms of size distribution, presence of multiple EV types (exosomes, micro-vesicles), and multiple cellular origin. From a physical (scattering) point of view, the EV reference materials will be spherical particles with a pre-defined concentration of less than 1012 particles per mL, size range from 50 to 1000 nm, homogeneous refractive index from 1.38 to 1.63, and quantified number of fluorophores per particle. Since these physical properties of the reference materials are traceable, they will enable the simulation of their scattering and fluorescence responses during and after the consolidation of the multi-sensing platform design. The signals from the EV reference materials will be measured by the multi-sensing platform, and will be related to the size and the refractive index of the particles, as well as to the number of the fluorescent labels that will be attached. Based on these sets of measurement and validation data, PHOREVER will develop its artificial intelligence-empowered tool for the medical translation of the EV detection process, and its correlation to the two main medical use cases of the project: the pancreatic cancer and the arterial thrombi present in stroke patients.

Objective 7:
Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples in relation to pancreatic cancer

Whole blood samples from healthy volunteers will be used to experimentally define and validate the process flow for the collection, handling and storage of blood plasma for downstream EV analysis. With this as a starting point, PHOREVER will investigate and demonstrate the use of the multi-sensing platform for the detection of EVs in blood samples from patients with pancreatic adenocarcinoma. The patients will need to have a confirmed tissue biopsy diagnosis for a locally advanced (LAPC) or metastatic (MPC) pancreatic cancer. Liquid biopsy by means of our multi-sensing platform will be performed before any treatment, either surgical or medical chemotherapy. Patients with benign pancreatic disease and healthy volunteers will serve as the control group. A second set of blood samples will be drawn after the end of the treatment for association of the clinical findings with the EV detection and quantification. Clinical findings that will constitute the basis for this association include the radiological response of the pancreatic tumor to chemotherapy, and the surgical tumor resection. The detected EVs will be evaluated as potential circulating biomarkers for diagnosis of pancreatic cancer via the comparison of the malignant group with the control groups, and as potential biomarkers for the assessment of the pancreatic tumor response to treatment.

Objective 8:
Demonstrate the use of the multi-sensing platform (PHOREVER platform) for the clinical analysis of EVs in blood samples in relation to stroke

PHOREVER will also investigate as a second use case the presence of EVs in stroke patients. The two main types of stroke, the ischemic and the hemorrhagic stroke, are known to need quite different medical treatment. To limit the stroke-induced brain damage, a reliable and blood-borne biomarker that can distinguish between the two types is needed. To identify EV-associated biomarkers for ischaemic stroke, arterial thrombi is collected during aspiration thrombectomy. Arterial thrombi release EVs in vitro, and up to now two potential EV-associated ischemic stroke biomarkers have been identified. Both biomarkers can be fluorescently labelled. PHOREVER will utilize EVs derived from human arterial thrombus to spike blood or plasma from healthy human individuals. Furthermore, an available biorepository will be used, containing plasma from haemorrhage stroke patients (20 individuals), ischemic stroke patients (20 individuals), and stroke mimics (20 individuals), to measure the level of arterial thrombus derived EVs with our multi-sensing platform, and as a reference also with a calibrated commercial flow cytometer instrument.

Project Objectives

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PHOREVER Project