The scientific and technological objectives
The project involves a multidisciplinary consortium of technology developers, three leading biomedical groups in clinical neuroscience for definition of specifications and end-user pre-clinical validation, and three research-oriented SMEs in biotechnology, nanosensing and microfluidics.
The project is divided into eight workpackages: four of these concern themselves with basic technological brick and the development and improvement of these technologies. Two further workpackages look into aspects of combining all or selected technologies and testing them for suitability to be incorporated, and then proceed to prepare a demonstrator unit for the final testing and evaluation phase. This final phase is governed in another workpackage preparing and coordinating both pre-clinical and clinical tests and evaluations. The final two workpackages deal with dissemination and exploitation activities, and with the overall management of the project (including risk management), respectively.
WP1 Innovative nanoparticles
Preparation of superparamagnetic particles with requested parameters, such as no aggregation in liquid medium, high monodispersity, hydrophilic layer for minimal non-specific sorption, convenient surface active groups for subsequent immobilization of antibody.
- Development of antibody modified nanoparticles (NPs) with controlled electrochemical properties based on gold nanoparticles (AuNP) and quantum dots (QDs, i.e. CdS, ZnS, PbS etc.).
Development of an advanced immobilization protocol for covalent immobilization of IgG molecules to superparamagnetic particles. Optimization and verification of antigen immunocapturing conditions unavoidable for the subsequent routine isolation of AD biomarkers from biological samples. Providing of efficient immunosorbents to other partners.
Identification and characterization of relevant hazards associated with medical application of nanoparticles.
In workpackage 1 (WP 1) innovative micro- and nanoparticles for use in sample preparation and sensing were developed. A range of new surface-modified colloidally stable iron oxide (magnetic) nanoparticles and monodisperse magnetic microspheres was synthesized. The particles can be provided with different coatings to, e.g., prevent non-specific adsorption. Several immunosorbents and protocols were prepared for other partners and tested with these partners on real samples.
WP2 Micro fluidics and micro fabrication
Development and availability of precise fluidic control over nine orders of magnitude in flow rate range using pressure feedback control.
Development and availability of low-cost polymer microfabrication approaches, with rapid prototyping, integration and possible mass-production in mind.
- Development and availability of novel protocols for surface treatment on a number of substrates and for various application. requirements.
- Development of an alternative protein enrichment approach using magnetic beads and electrochemical detection.
- Identification and characterization of relevant hazards associated with medical application of nanoparticles.
WP 2 covered the fundamental microfluidic and microfabrication needs for the consortium. To this end, a feedback-controlled pressure source for generating flow has been further optimized. A number of potential chip materials and their microfabrication has been investigated, mostly focusing on polymers. Disposable systems were tested using nanoparticles for detection, implementing a magneto-immunoassay followed by a QD mediated detection.
WP3 Ultrasensitive detection
- To develop an ultra-sensitive electrochemical detection system for nano-ELISA.
- To develop waveguides, integrated with separation chips for sensitive on-line detection.
- To develop chip-integrated evanescent-wave detection, using the new waveguide technology.
- To develop nanotargets for MALDI-TOF mass spectrometry, aiming for an improved sensitivity.
- To fabricate nanoparticles for ultrasensitive electrochemical detection.
- To develop a new detection system for microarrays based on a droplet-confined chemistry.
WP 3 provided several avenues for ultra-sensitive detection. One approach builds on a platform originally developed by one of the SME partners. Improvements allowed electrochemical detection of A-beta1-42 within about two hours.
Mass spectrometric detection nanotargets for MALDI-TOF were made and tested for different test substances. This was also coupled on-line to a magnetic immunoprecipitation. Excellent results were achieved in patient samples (both CSF and blood) and even in very limited sample volumes the A-beta spectrum was detected. The technology is further developed to be quantitative.
Droplet arrays for digital ELISA were tested. The result using ApoE3 model system, showed that the digital ELISA excellent sensitivity and linear quantitative range which enables detection of biomarkers in blood.
WP4 Lab-on-chip technologies
- To develop different microfluidic components or « technological bricks », to be combined and integrated in WP6
WP 4 developed more specific microfluidic technologies making use of input from WPs 1-3. A special way to perform magnetic immunocapture to get proteins out of a biological liquid based on using the fluidized bed approach was developed. Very good enhancement factors with A-beta were obtained and will be used in the final demonstrator.
The spotted peptide and protein microarray experiments were performed with Si chips. An antibody microarray of biomarkers are ready to be tested on clinical material.
WP5 Optimization and comparison of technologies
- To provide the biological material and expertise, necessary to ensure that the technological bricks developed in WP1 to 4 are from the start guided by the most realistic biological and medical issues, and be faced early enough with the problems raised by real biological and clinical samples.
- To provide a panel of samples and criteria, upon which the different outcomes of WP1 to WP4 will be compared, and thus to prepare technologies selection and integration, to be done in WP6.
- To provide innovative data analysis and signature elaboration strategies, in order to support the above objectives, and to prepare the software development planned in WP6.
Within WP3, the project we defined a group of biomarker which either serve currently as direct diagnostic marker (e.g. tau-proteins and abeta-peptides), which are candidates for furher biomarker studies (e.g. neurofilaments and ubiquitin) and markers which serve as proof-of-principle for the techniques (e.g. ApoE-proteins). We were able to set-up a new proteomic method for direct quantification of ubiquitin, which was significantly elevated in patients with AD and CJD. We were also able to adopt a 2D-detection method for post-translational modified SerpinA1 on a nanoscale method. By this we are now able to evaluate the risk for patients for Parkinsons´s disease to develop a Parkinson´s disease dementia on large scale numbers. We have also produced a library of antibodies to be used for biomarker discovery.
WP6 Integration and prototyping
To realize a microfluidic automated system by integrating the best techniques developed during WP1-WP5 regarding sensitivity and selectivity, compliance with the end users demands and microfluidic integration.
Consider and integrate in the global optimization parameters regarding production cost, economic viability, and regulatory issues.
In WP6 we have successfully automated sample pre-concentration and microarray analysis but we still need to combine these two methods into one demonstrator and test the combination for possibilities to diagnose patients. Characterization of a commercial platform from a SME partner suggests robust automated analysis for biomarkers relevant for neurological diseases.
WP7 Pre-clinical and clinical testing
- To provide a high quality collection of blood plasma and CSF samples from well characterized patient- and control groups.
- To evaluate the performance of the fully integrated prototype instrument from WP6 by model samples.
- To evaluate the prototype instrument under clinical conditions and analyze the clinical relevance of the elaborated biomarker signature.
In WP7 the clinical partners have been gathering patient material and produced a biobank with samples from >1200 individuals, which can be accessed by other partners in the consortium for testing technology platforms and instruments. Commercial gold standard assays have also been established during the project. The biobank and range of commercial assays will for the basis for a large comparison study of the newly developed micro- and nanotechnology systems.
WP8 Dissemination and exploitation
- Dissemination of results in website, publications and conferences.
- Training of health care professionals in clinical use of the prototype instrument.
- Preparing a strategy for exploitation.
- Investigation of other applications of microfluidic systems.