Periodic Reporting for period 2 - SiMBiT (Single molecule bio-electronic smart system array for clinical testing) Reporting period: 2020-01-01 to 2021-06-30 Summary of the context and overall objectives of the project The general objective of the SiMBiT project is to develop a bio-electronic smart system that can perform single-molecule detection of both proteins and DNA bio-markers. Specifically, the SiMBiT activities will develop a lab-based device into a cost-effective portable multiplexing array prototype with extremely fast time-to-results. During the second and half year, the programme of activities, described in the Annex 1 of the Grant Agreement, has been successfully carried out, meeting what foreseen besides few minor deviations (some foreseen and some not foreseen) that have been successfully tackled. An amendment has been submitted to the commission to include a small delay due to the COVID-19 situation. However, the deliverables and the milestones have been completed and submitted on time. Only in one case a small delay was incurred and justified. Overall, the SiMBiT Consortium has successfully reached the end of the second period via a systematic cooperative work involving an intensive inter-sectorial research effort from academia and industrial partners, accomplishing all planned steps towards the development of the bio-electronic smart system array. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far W1: After the conclusion of first and second Task of the WP1 (T1.1 on technical specifications of the SiMBiT bio-electronic smart system and T1.2 on the standard operating procedures) with the preparation of two deliverables: D1.1 (M3) and D1.2 (M4), the work package activity has continued with the task T1.3 on the quality control data reporting and the preparation of the first (intermediate) version of the Exploitation Plan. The conclusion of the quality control data reporting is due at the conclusion of the project (D1.3) while the first version of the exploitation plan was delivered at M24 (June 2020) and it is available as document with version 1.3. A final version of the Exploitation Plan will be delivered at the conclusion of the project.W2: WP2 is dedicated to the fabrication, characterization and optimization of the single sensing device. During the second reporting period of the project, the efforts were focused on the optimization of the biofunctionalization protocol and on the design and validation of the 3D printed sensing gate. The activities of WP2 have been focused on the study and optimization of the bio-functionalization protocol of the gate surface based on a systematic molecular characterization. The outputs on this activity are detailed in D2.3 (M18) and D2.4 (M24). As far as the gate is concerned, the activity carried out aims at the demonstration and validation of a 3D printed gate structure suitable for the development of the final gate cover plate that will fit the standard ELISA plate geometries. The outputs on this activity are given in D4.3 (M24). Moreover, the validation and the assessment of the analytical figures of merit of the final single SiMBiT sensor is reported in D2.5 (M24) and D2.6 (M30), using KRAS and MUC1 markers are detected in phosphate buffer saline solution and in whole blood serum at the single molecule detection limit.W3: In M13 – M30 WP3 focused on the characterization, simulation, modelling and design optimization of EG-TFTs with bare and biofunctionalized gate. Both single biosensors and 4x4 biosensor matrixes were addressed. More in detail, Task 3.1 was devoted to the electrical and spectroscopic characterization. Task 3.2 developed a numerical framework for the two-dimensional (2D) physical-based simulation of electrolyte-gated transistors with bare and biofunctionalized gate. The framework accounted for the relevant physics, the meaningful geometries and the key device parameters. The combination f the electrical characterizations (Task 3.1) and 2D numerical simulations (Task 3.2) provided insight on the biosensor operation, and supported the development of physically based numerical and analytical models (Task 3.3). The analytical model is currently used for (i) the quantitative assessment of the SiMBiT technology, (ii) technology and system optimizations, and (iii) for the automated analysis of the SiMBiT bioelectronic cartridge.W4: The process flow for the fabrication of single EG-TFTs, 4x4 and 8x12 arrays has been optimized and validated, by producing these devices and characterizing them in terms of performance, reproducibility and stability. Tridimensional gate arrays have also been realized according to an optimized 3D printing process, matching the same geometry of the commercial ELISA plates used as fluidics for the EGTFTs.W5: During the second reporting period the module design of the first prototype was completed and reported in D5.2. The components of the first prototype were fabricated and then the electrical verification of the OTFT addressing electronics, Si-IC and PCB were presented in D5.4. Based on learnings from the first prototype design, fabrication and testing, the system and module design for the final prototype was reported in D5.3.W6:Work Package 6 is dedicated to sample collection and analysis. Results of Next generation sequencing (NGS) for genomic markers and of ELISA and SIMOA for protein markers are then compared to results obtained by the SiMBiT device. A further objective is the screening for possible additional biomarkers in pancreatic cyst fluids, which could be analyzed by SiMBiT in the future. In this period, sample collection has proceeded according to the logistic established during the first period, facing the difficulties and delays in patients’ recruitment due to the COVID-19 pandemics. Additionally, a cyber-attack to the University Hospital of Dusseldorf, with following shutdown of all clinical activities as well as of clinical and research servers in September/October 2020 has further dramatically impaired sample collection and analysis. Nevertheless, sample collection has slowly proceeded and partially recovered during the last six months of this second period; all NGS and ELISA analyses have been performed and the screening for additional markers has delivered important results.W7: WP7 is dedicated to communication, dissemination and outreach activities. One of SiMBiT communication and dissemination objective is creating awareness, raising interest, fostering engagement and accelerating the market uptake. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The information on section 2.1 of the Annex 1 is still relevant for CSGI, UDUS, UNIBS, ABO, IIT, TU/e, FE, MASMEC and Efficient Innovation. Leaflets
