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.
