The project aims to develop a prototype of a miniaturized system for diagnostics in the early stage of Alzheimer disease and other neurodegenerative diseases, or as a point-of-care instrument for patient follow-up. The system to be developed belongs to the emerging field of “lab-on-chip” systems. It incorporates several innovative enabling technologies, including microfluidic flow control, highly sophisticated nanobiodevices with integrated detection, and novel magnetic nanoparticles. These approaches will lead to unprecedented integration and automation, and allow routine implementation of tests that can presently only be performed in a small number of specialized research laboratories. The system will use biomarkers present in blood, such as differently cleaved b amyloid peptides and post-translational modifications of the Tau protein. The miniaturization and integration of innovative detection technologies are intended to extend the sensitivity of biomarker detection, and thus improve the precocity of diagnosis. This is of paramount importance for the treatment of neurodegenerative diseases, since therapeutic approaches able to retard the evolution of the diseases are progressing and promising, but little hope exists for the repair of existing brain damage. The method also aims at allowing the simultaneous study of a wide range of markers, improving the early discrimination between different neurodegenerative diseases, and thus the choice of treatment. Indeed, the NeuroTAS system will have a modular and evolutive structure, and it will be able to progressively test and integrate into its diagnostic scheme new biomarkers that may be discovered during the prototype development. The consortium is a combination of 4 academic, methodology-oriented laboratories with complementary competences in biochemistry, analytical chemistry, biophysics and microfabrication, two SMEs in the field of microfluidics, and two “end-users” directly involved in patient diagnosis and treatment.
The envisioned device will present the following advantages:
- Full automation (from plasma to result) for low cost, reproducibility and usability in routine diagnosis or patient follow-up.
- Fast analysis (the ultimate objective is one hour, and the criterion for success will be set at 3 hours total time, to be compared to present protocols that last several days)
- High sensitivity
- Multi-parameter, multiplexed analysis, to allow for differentiation between different types of neurological diseases that cannot presently be distinguished at an early stage, and to permit a detail investigation of the evolution of biomarkers in patients; this will hopefully help both on the research side, to better understand the disease, and in routine for patients follow-up.
The prototype to be developed in the project is an automated system capable of performing sequential capture, analysis and detection of protein and peptide biomarkers. For both, Ab peptides and tau proteins, two strategies will be considered:
a: “direct track”: this strategy consists of multiplexed immunocapture followed by direct detection by miniaturized ELISA. This strategy will be relevant if antibodies specific enough for a complete medically relevant profiling are available.
b: “electrophoretic profiling track”: This strategy, more powerful but also more demanding on the instrumental side, involves immunocapture followed by electrokinetic separation and post-separation detection.
The lab-on-chip instruments will be designed in a modular format incorporating the following principal modules:
(1) The multiplexed immunocapture module (MI) is common to both tracks: The protein capture will be performed by circulating plasma in a sequential series of immunoaffinity microcolumns dedicated to the capture of specific peptides (one Ab species) or one specific tau form per chamber. The main strategy will rely on an innovative approach based on magnetic nanoparticles. This system uses self-organization of the nanoparticles to create a compact microcolumn with auto-calibrated micron-sized pores. It combines a high loading capacity and fast affinity reaction, thanks to a high surface to volume ratio and extremely thin diffusion layer.
(2) Electrochemical detection module (ECD). In the direct track option, an electrochemical detection will be performed directly after immunocapture. The system to be developed will have the capability of detecting in parallel biomarkers from several capture chambers, using a secondary antibody labelled with an enzyme able to transform a substrate into an electrochemically active compound. For Ab peptides, specific antibodies against several b amyloid species exist, so that a direct capture and electrochemical detection strategy of the ELISA type can be contemplated.
(3) Microchannel Electrophoresis-Laser Induced Fluorescence profiling module (ME-LIF). For some other biomarkers a more elaborate profiling strategy (electrophoretic profiling track) should be employed. In this approach, the preconcentrated proteins/peptides will be transferred to an electrokinetic profiling module, in which they will be profiled by high resolution capillary electrophoresis (CE) on chip. Two different detection strategies will be developed. A first one will be based on fluorescent labeling and direct on-chip detection using an innovative technology based on the incorporation of planar waveguides integrated into the chip during fabrication.. It simplifies dramatically the detection scheme, avoiding complicated and expensive optics, and is thus particularly well suited to the development of an integrated lab-on-chip. As part of the project, this approach will be implemented in an “all-polymer” scheme, to keep full compatibility with the other modules of the project.
(4) Hyphenated Microchannel Electrophoresis-Immunodetection module (”Micro-Western”). As an alternative to the ME-LIF module above, a second strategy will combine CE separation followed by translation into a secondary multiplexed immuno-immobilization element. This new approach, which will be a major foreseeing and innovative aspect of the project, will combine the resolution of CE with the sensitivity and specificity of ELISA-type immunoassays. Essentially, this development will introduce the power of Western blotting to the “lab-on-chip” world.
This option will be selected if either the online fluorescent labelling strategy (ME-LIF) does not provide sufficient resolution (this may be the case in particular for tau proteins, since differences in phosphorylation levels only lead to small differences in mobilities that may be hidden by the labelling step), or to increase the sensitivity and the specificity of the detection. Sensitivity enhancement will be made possible, in particular because this approach allows for an ELISA type amplification step.
Final system
The final system will comprise two sequential blocks, one dedicated to Ab peptides, and one dedicated to tau proteins. Depending on the options chosen for each family of biomarkers, the corresponding block will itself comprise several “modules” selected among the ones described above, depending on the option chosen for profiling: the “direct track” involves an immunoaffinity module and an electrochemical detection module, whereas the “electrokinetic profiling” will involve an immunoaffinity module, followed by an electrokinetic profiling module involving either the MELIF strategy or the MicroWestern strategy. The modular strategy will allow for parallel development and optimization, but a requirement of compatibility between the different modules will be included from the start, to allow the integration of all elements into a single microfluidic system.