Periodic Reporting for period 1 - 2D-BioPAD (Supple Graphene Bio-Platform for point-of-care early detection and monitoring of Alzheimer’s Disease)
Periodo di rendicontazione: 2023-10-01 al 2025-03-31
2D-BioPAD aims to introduce a fast and cost-effective, minimally invasive, reliable, digitally and graphene-enabled Point-of-Care (PoC) in-vitro diagnostic (IVD) system for supporting the early diagnosis and progression monitoring of AD directly at primary healthcare settings.
To achieve this, and tackle the scientific challenge, the technological and market gap of PoC IVD for AD, 2D-BioPAD leverages the unique properties of 2D materials, such as graphene and its derivatives.
Towards that direction, 2D-BioPAD goes beyond the state-of-the-art of its 2D Materials’ pioneer consortium to deliver a graphene-based PoC IVD system that will (i) introduce a versatile surface chemistry that combines nano and DNA technologies towards improved biocompatibility, stability, as well as high sensitivity and specificity for enhanced (bio-) sensing; (ii) be able to reliably identify and quantify in real-time and simultaneously up to 5 AD biomarkers in blood samples effectively supporting healthcare professionals in early diagnosis; (iii) offer an easy to use and understand digital interface with key metrics and insights regarding the measured results; and (iv) employ Artificial Intelligence (AI) to improve the overall system implementation.
The 2D-BioPAD system and its impact will be demonstrated in 3 clinical centres in Europe (UEF-Finland, GAADRD-Greece, and ZI-Germany) under two clinical pilot studies.
To achieve this, and tackle the scientific challenge, the technological and market gap of PoC IVD for AD, 2D-BioPAD leverages the unique properties of 2D materials, such as graphene and its derivatives.
Towards that direction, 2D-BioPAD goes beyond the state-of-the-art of its 2D Materials’ pioneer consortium to deliver a graphene-based PoC IVD system that will (i) introduce a versatile surface chemistry that combines nano and DNA technologies towards improved biocompatibility, stability, as well as high sensitivity and specificity for enhanced (bio-) sensing; (ii) be able to reliably identify and quantify in real-time and simultaneously up to 5 AD biomarkers in blood samples effectively supporting healthcare professionals in early diagnosis; (iii) offer an easy to use and understand digital interface with key metrics and insights regarding the measured results; and (iv) employ Artificial Intelligence (AI) to improve the overall system implementation.
The 2D-BioPAD system and its impact will be demonstrated in 3 clinical centres in Europe (UEF-Finland, GAADRD-Greece, and ZI-Germany) under two clinical pilot studies.
During the first period, 2D-BioPAD has made significant progress in terms of developing the envisioned biosensors, delivering several prototypes on individual components (aptamers, MNPs, graphene derivatives, electrochemical and GFET biosensing technologies). To better define our implementation, a landscape analysis was performed, covering a desk research, 26 semi-structured interviews and an online survey engaging 196 participants. Through this PPIE-driven methodology clear guidelines and requirements for the 2D-BioPAD system have been identified regarding: (i) Users’ Needs and Challenges, (ii) Clinical Practice, (iii) Biomarkers, (iv) PoC IVD requirements, and (v) Ethics and Safety. For the latter, an Ethical Consideration Roadmap was developed to guide ethical principles and guidelines for the 2D-BioPAD project. Based on these insights, the first version of the 2D-BioPAD system architecture was co-designed, supporting the subsequent implementation steps.
These steps include: The successful selection and optimization of several DNA aptamers for specific AD protein biomarkers (Aβ40, Aβ42, p-tau 217, GFAP and NfL). Aptamer selection was successful for GFAP and NfL, it is ongoing for pTau-217 and a revised protocol has been agreed for Aβ1-40 and Aβ1-42. Three distinct magnetic nanoparticles (MNPs) systems successfully synthesized and characterised. Core-shell Fe3O4/Au (~50 nm) MNPs have been selected as the best performing actor with respect to feasibility, binding efficiency and magnetic features. A MNPs/Aptamer conjugation protocol on thrombin (TBA) has been validated, while Aβ40 and Aβ42 are effectively conjugated with synthesized MNPs.
In terms of the 2D-materials, seven covalent graphene derivatives have been synthesized (two doubly-functionalised) with a functionalisation degree up to 23%. A first prototype for a lateral flow electrochemical sensor has been developed, with qualitative, selective and sensitive read-out, reproducible from sensor to sensor, with a LoD below 10 nM (for Thrombin as a proof-of-principle). A first prototype for a GFET sensor has been developed, supporting two channels and with a LoD as low as 4pΜ (for GFAP). These prototypes were also accompanied by the realization of a microfluidic passive and compact sample preparation kit that is filtering red blood cells to bring in contact to the biosensors a serum-like fluid for analysis and an initial design of the hardware and software for the digitalization of the 2D-BioPAD system.
Finally, 2D-BioPAD has prepared the protocol for the Clinical Studies and submitted the protocol to the ethical committees in Finland, Greece and Germany for an Ethics Check. The retrospective pilot study was approved by ethics committees at all sites - Finland, Greece and Germany, whereas for the prospective pilot study protocol the consortium has initiated the procedures for both ethical and regulatory approval.
At the same time 2D-BioPAD has a strong collaboration with the Graphene Flagship and its biomed projects.
These steps include: The successful selection and optimization of several DNA aptamers for specific AD protein biomarkers (Aβ40, Aβ42, p-tau 217, GFAP and NfL). Aptamer selection was successful for GFAP and NfL, it is ongoing for pTau-217 and a revised protocol has been agreed for Aβ1-40 and Aβ1-42. Three distinct magnetic nanoparticles (MNPs) systems successfully synthesized and characterised. Core-shell Fe3O4/Au (~50 nm) MNPs have been selected as the best performing actor with respect to feasibility, binding efficiency and magnetic features. A MNPs/Aptamer conjugation protocol on thrombin (TBA) has been validated, while Aβ40 and Aβ42 are effectively conjugated with synthesized MNPs.
In terms of the 2D-materials, seven covalent graphene derivatives have been synthesized (two doubly-functionalised) with a functionalisation degree up to 23%. A first prototype for a lateral flow electrochemical sensor has been developed, with qualitative, selective and sensitive read-out, reproducible from sensor to sensor, with a LoD below 10 nM (for Thrombin as a proof-of-principle). A first prototype for a GFET sensor has been developed, supporting two channels and with a LoD as low as 4pΜ (for GFAP). These prototypes were also accompanied by the realization of a microfluidic passive and compact sample preparation kit that is filtering red blood cells to bring in contact to the biosensors a serum-like fluid for analysis and an initial design of the hardware and software for the digitalization of the 2D-BioPAD system.
Finally, 2D-BioPAD has prepared the protocol for the Clinical Studies and submitted the protocol to the ethical committees in Finland, Greece and Germany for an Ethics Check. The retrospective pilot study was approved by ethics committees at all sites - Finland, Greece and Germany, whereas for the prospective pilot study protocol the consortium has initiated the procedures for both ethical and regulatory approval.
At the same time 2D-BioPAD has a strong collaboration with the Graphene Flagship and its biomed projects.
During the first period, we have introduced new aptamer sequences for GFAP and NfL protein biomarkers, three distinct MNPs systems (Au/Fe3O4, Fe3O4@Au, and Janus Au-Fe3O4 NPs) and a structured, reproducible conjugation protocol for the robust conjugation of aptamers on the MNPs
In addition, extended research has been performed on the synthesis of new graphene derivatives and their functionalisation (including dual functionalization), introducing new synthesis steps for delivering densely, selectively functionalised and electrochemically sensitive covalent graphene derivatives. On the same direction, two detection strategies have been designed, developed and validated (proof-of-principle with thrombin) for the detection of AD biomarkers employing electrochemical three-electrode cells produced using an environmentally friendly, low-cost printing/stamping technology in a single step. In parallel, the immobilisation of new aptamers on GFET biosensors, supporting currently two simultaneous channels, has been achieved, offering a first of its kind PoC multianalyte biosensing prototype (proof-of-principle for GFAP and NfL). Finally, scientific progress has already been made on the clinical aspects, with the two clinical pilot studies’ protocols paving the way for both the technical and clinical validation of the biosensors.
The project has produced several scientific papers during the first reporting period to both international conferences and high impact journals with reviewers, showcasing significant progress beyond the state of the art in 2d materials – specifically graphene synthesis and functionalisation – aptamer selection and optimisation (including advanced computational models), nanomaterials’ synthesis and functionalisation, as well as electrochemical and GFET biosensors (both immunosensors and aptasensors).
In addition, extended research has been performed on the synthesis of new graphene derivatives and their functionalisation (including dual functionalization), introducing new synthesis steps for delivering densely, selectively functionalised and electrochemically sensitive covalent graphene derivatives. On the same direction, two detection strategies have been designed, developed and validated (proof-of-principle with thrombin) for the detection of AD biomarkers employing electrochemical three-electrode cells produced using an environmentally friendly, low-cost printing/stamping technology in a single step. In parallel, the immobilisation of new aptamers on GFET biosensors, supporting currently two simultaneous channels, has been achieved, offering a first of its kind PoC multianalyte biosensing prototype (proof-of-principle for GFAP and NfL). Finally, scientific progress has already been made on the clinical aspects, with the two clinical pilot studies’ protocols paving the way for both the technical and clinical validation of the biosensors.
The project has produced several scientific papers during the first reporting period to both international conferences and high impact journals with reviewers, showcasing significant progress beyond the state of the art in 2d materials – specifically graphene synthesis and functionalisation – aptamer selection and optimisation (including advanced computational models), nanomaterials’ synthesis and functionalisation, as well as electrochemical and GFET biosensors (both immunosensors and aptasensors).