Periodic Reporting for period 2 - PoC-ID (Platform for ultra-sensitive Point-of-Care diagnostics for Infectious Diseases) Periodo di rendicontazione: 2016-11-01 al 2018-11-30 Sintesi del contesto e degli obiettivi generali del progetto The aim of the PoC-ID project was to develop new micro- and nanoelectronic-based sensing and integra-tion concepts for advanced miniaturised in-vitro diagnostic devices. The project addressed the increasing demand for rapid and ultra-sensitive point-of-care diagnostics to reduce healthcare costs and increase the quality of life with a focus on infectious diseases, one of the world’s leading causes of morbidity and death. Interdisciplinary collaboration using the technology and expertise of the consortium members was applied to develop and test a breakthrough PoC prototype for the diagnosis of respiratory syncytial virus infections and host responses in the paediatric context. PoC-ID has enabled new types of point-of-care diagnostics for virtually any type of complex liquid sample.Applications of PoC-ID are disease diagnosis, monitoring of therapeutic responses, clinical research of path-ogen-host interaction and personalised medicine. PoC-ID aimed to combine the detection of both patho-gens and host responses leading to more accurate diagnosis as compared to the current standard, which is focused on detection of pathogens only. This novel approach will support prevention and control of patho-gen spread and enable faster and more personalised patient treatment. Furthermore, the platform technol-ogy can easily be adapted to a variety of diagnostic or bio-sensing purposes, such as in health/environmental monitoring or food quality testing.Improved performance in terms of robustness, sensitivity and selectivity has been reached by a combination of innovative nano-membrane technology, molecular-engineered capture molecules and two novel sensing concepts (BioMEMS and BioGrFET), increasing device-flexibility and reducing risk. Both concepts work label-free (no expensive biological active buffers or enzymes required for detection) and allow rapid and multi-plexed detection due to parallel electric readout. Further advances have been realised in terms of usability and speed of data analysis arising from the integration of sensors, read-out electronics and microfluidics into one user-friendly point-of-care (PoC) platform. Costs of the new disposable sensors will be ultra-low if pro-duced at high volumes, thanks to designing into microelectronics production flows. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti All of the PoC-ID goals were achieved during the project duration. Based on a specification document com-prising the overall system requirements from the user’s perspective, technological demands were defined. Sequences of host biomarkers and viral proteins were selected for in vitro screening of new capture mole-cules. The focus for the first phase was on device design and process development aiming for proof-of-concept prototypes for BioMEMS and BioGrFET and flexible technology toolboxes (functionalisation schemes, packaging and device design). During this first phase, the sensors showed good results in artificial environments and buffer solutions. Early definition of the protocols for sample collection and storage en-sured sample availability for the sensor evaluation phase. Also, early development of chemistry for immobi-lisation of capture molecules to the sensors paved the way for Biosensor development. In the second half of the project, the performance of the BioGrFET to detect the PoC-ID targets was optimised using theoretical modelling. By fabricating 5 batches of the device, the optimum parameters were found, experimentally realised and linked to modelling results. Specific biorecognition molecules (Spiegelmers) against three iden-tified targets (CXCL8, CCL5, and CXCL10) have been successfully immobilised to surfaces via a Carbon Nanomembrane (CNM) nanointerposer combined with the BioGrFET sensor. Such functionalised sensors showed good results on exposure to CXCL8 target in a laboratory setup and even in a clinical setting. The sensor thus covers the clinically most relevant range of CXCL8 for the diagnosis of RSV [Respiratory Syncyti-al Virus]. Furthermore, the clinical value of inflammatory markers present in the NPS [nasopharyngeal swab] during RSV infection have been further explored and CXCL8 was confirmed as a biomarker able to differentiate children with moderate and severe disease and to determine who needed supportive care in a hospital. Testing of the PoC-ID device was done under lab conditions and in a clinical environment, proving the applicability of PoC-ID results for the target applications.Overall, an integrated PoC-ID benchtop device has been designed and developed which integrates the fluidics system, the driving electronics and sensor module. The device is able to acquire measurements from both the BioGrFET and BioMEMS sensors and provides accurate analysis results in only approx. 6 minutes. Hence, it was demonstrated, that PoC-ID results are a large step towards low cost and fast detection of specific biomarkers in the clinically relevant range. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The immobilisation chemistry, sensor concepts and first biosensor-packaging concepts have been developed beyond the previous state of the art. The BioGrFET concept will have a strong impact on bioanalytics and sensor technology. The combination of two-dimensional graphene with carbon nanomembrane demon-strated a sensitivity in the pM range and will extend the applicability of all-electrical sensing for medical and other applications. Two international patent applications have been filed on the immobilisation of oli-gonucleotides on CNM, which enables the use of all of the L-aptamers that were newly selected in the project. Furthermore, some novel chemokine-binding L-aptamers have been discovered and a patent appli-cation on a novel inflammatory marker-binding L-aptamer-based capture molecule was filed. Using this molecule, the proof-of-concept for the possibility to detect a biomolecule with the BioGrFET sensor that has been covered with a CNM as a nanointerposer has been reached. This was not only possible from laborato-ry samples but also from patient material (nasal swab). When commercialised the CXCL8 measurement may add to the information of a Procalcitonin or CRP measurement that is obtained using other rapid tests to facilitate the diagnostics (viral/non-viral pneumonia) and aid in the treatment decision (anti-viral experi-mental drugs vs. antibiotics). However, for commercialisation of the PoC-ID device more work on automa-tion, calibration and reproducibility is necessary. Once this, and ideally the selective detection of the re-maining biomarkers, has been reached, the impact will remain as relevant as proposed at the beginning of the project: The high selectivity of biomarker detection facilitates application for personalised medicine and the rapid measurement provides the opportunity of bedside patient monitoring. Increased sensitivity and reliability as well as rapid detection will enable earlier disease diagnosis, which will reduce the length of stay in a hospital. Once successfully implemented, the PoC-ID platform will lead to (1) strong improvements in patient care, (2) earlier recovery of patients, (3) significant cost reduction for the healthcare system, (4) accelerated development towards personalised medicine and (5) new market opportunities for SMEs, which will benefit the EU economy. Bio-GrFET sensor principle and fan-out wafer level package Assay Geometry Bio-GrFET sensor assembled in microfluidics PoC-ID System Principle
