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Super-sensitive detection of Alzheimer’s disease biomarkers in plasma by an innovative droplet split-and-stack approach

Periodic Reporting for period 2 - SensApp (Super-sensitive detection of Alzheimer’s disease biomarkers in plasma by an innovative droplet split-and-stack approach)

Reporting period: 2020-01-01 to 2022-06-30

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder, which leads to death. It represents the major cause of dementia in the elderly population, with a great socio-economic impact on the worldwide community. The current guidelines for clinical diagnosis of AD establish the determination of specific protein biomarkers (Amyloid-beta, tau, P-tau) in cerebrospinal fluid (CSF) through ELISA kit and positron emission tomography (PET) of the brain with amyloid tracer. However, PET is highly expensive and not always available in clinics and lumbar puncture for CSF collection is an extremely invasive intervention that requires hospitalization and hinders follow-up programs during therapies. Nowadays the traditional ELISA kits cannot determine such biomarkers in peripheral blood due to their abundance well below the standard sensitivity that is of 50-100 pg/mL. Therefore, by the time it is recognized, the disease has been progressing for many years. In this framework an early and non-invasive diagnosis of AD is crucial for saving lives. SensApp aims at developing a device prototype that we call “super-sensor” for determining low abundant AD biomarkers in human plasma for non-invasive AD diagnosis in routine clinical practice. An innovative integrated optical system detects the fluorescence signal directly on the reaction support. The super-sensor makes use of a new technology that we call ‘droplet-split-and-stack’ (DSS), which key innovation relies on the capability of drawing tiny and repeatable droplets of a sample through a pyroelectric effect and stack them on a restricted area of the reaction support. As a consequence, the number of fluorescent biomolecules per unit area enhances significantly after the successive immunoreaction steps pushing the sensitivity down to sub-picogram level.
The work plan was divided into 5 workpackages: (WP1) management and dissemination; (WP2) identification of the very first features of the super-sensor; (WP3) implementation and characterization of each building block; (WP4) integration and comparative study; (WP5) first tests on body fluids. The WP1 ran during the entire duration of the project. The WP2 and WP3 developed mainly in the first two years of the project, while the activities in WP4 and WP5 were carried out in the last two years. The impact of covid-related restrictions was detrimental from different points of view. The lack of continuity in lab activity, especially during the 2020 year, delayed significantly the first tests on the implementation of the immunoreaction protocol, thus transferring to WP4 and WP5 the further optimization of the procedure. The two partners closer to the clinical field (Ginolis and Pulejo) suffered from the covid-related emergency with a consequent lack of personnel focusing on SensApp, thus undermining the achievement of the final tests in a clinical setting. Conversely, the more technological objectives of the project were achieved successfully with additional efforts through teleconference meetings to overcome the lack of face-to-face joint-measurement sessions. We realized the prototypes of the two expected modules of the super-sensor: the DSS module and the RO (read-out) module. Both prototypes were characterized and demonstrated successful to detect fluorescent biomolecules down to the sub-picogram level in pure samples and even in synthetic body fluids such as urine. In summary, the overall result is the demonstration of the DSS technology’s ability to concentrate biomolecules through highly reproducible spots and its compatibility with immunoreaction steps on reaction slides. In this way the clinical validation of the super-sensor will be a matter of robust immunoreaction protocol post-DSS, able to minimize the background level especially when working with low abundant biomarkers. We believe that further research in a new collaborative project (e.g. EIC Transition) will allow us to continue developing the SensApp super-sensor by validating the technology in a real clinical setting for early diagnosis applications. Moreover, the comparative study of the DSS technology performed with a piezo-driven micro-spotter encourages us to develop a new collaborative project for testing the DSS technology in the field of microarray printing.
Regarding the dissemination of the results, we achieved the following results:
• Visual identity of the project with own logo, website, document templates, brochures, social accounts (Facebook, Twitter, Instagram, YouTube) and community on the repository Zenodo
• IP Agreement & plan for data management
• Three promotional movies
• Manuals for the prototypes
• International symposium
• More than 10 scientific papers published in peer-reviewed journals with others under preparation
• More than 20 presentations at international conferences
• Presentations at public and local events (e.g. European Researchers’ Night, etc.)
• Various master theses
• Joint press release

Our plan for exploitation of the results includes:
• Use results in further research projects, internally or as background in new collaborative projects
• License the patents filed after the end of the project (both individually or jointly among partners)
• Evaluate joint ventures with companies working in the field of microarray technologies
• Continue promoting SensApp topics in PhD and master theses
• Evaluating the opportunity to develop a spin-off
The scientific results obtained during the SensApp project are expected to open up several new perspectives both from a fundamental and application side. A novel perspective is the introduction of pyroelectric-based printing in the field of microarray technology with new advantages in terms of spot quality and reproducibility. The demonstration of the DSS technology to overcome the typical limits of detection in immunodetection procedures will allow us to continue optimizing the biochemical aspects of the process in a new collaborative project (e.g. EIC Transition) and to launch the DSS technology in the market of diagnostic tools with a new challenging sensitivity. In particular, we envisage the application of DSS in the case of cancer-oriented applications where the detection and monitoring of low abundant biomarkers in peripheral body fluids would help clinicians in optimizing the follow-up therapy. Therefore, clinical validation will be of fundamental importance, in such a new project, in order to introduce DSS in routine diagnostic practice and let clinicians benefit from sub-picogram levels of detection.
Logo of the project SensApp
last consortium meeting at VUB