Projet ANR-07-PCVI-0036 – D-AminoChip : New Lab-on-Chip for the enantiospecific analysis of free D-amino acids in biological fluids
Programme : Physique et chimie du vivant (PCV) 2007
Année de financement : 2007
Coordinateur du projet : (UNIVERSITE CLAUDE BERNARD - LYON I)
Partenaires : Centre national de la recherche scientifique; Institut polytechnique de Bordeaux; Université Claude Bernard - Lyon 1; Université de Grenoble Alpes
Montant : 700000.0 euros
Durée : 48 mois
Résumé : 1-Scientific background and objectives : -  D-enantiomers of amino acids play a preponderant role in biochemistry and especially in -  neurotransmission. For example, D-serin is involved in neurological diseases evolution, such as -  schizophrenia or epilepsy. The presence of D-aspartate has been detected in the case of Alzheimer -  disease or cataract disease. Understanding the role of D-amino acids in the neurological function -  would represent a considerable step towards the understanding of the resulting disease mechanisms. -  However, these compounds of interest are found at trace levels in particularly complex matrices, with -  extremely high content of L-enantiomers, and very small volumes available. A disposable Lab-on- -  Chip constituted of a preconcentration step, specific to the D-enantiomer, followed by an -  enantioselective separation stage appears as the most adapted tool. It would be of great interest -  during the study of neurodegenerative diseases mechanisms as it is likely that a large number of -  samples are required to study the role of amino acids action mechanisms and kinetics...Moreover, if it -  is acknowledged that the determination of amino acids may contribute to the diagnosis or the -  monitoring of these diseases, the availability of an integrated automatized analytical tool would be -  highly relevant. -  This project proposes the extraction and selective preconcentration of D-enantiomers of amino acids, -  followed by chiral electrochromatography separation and subsequent fluorescence detection, allowing -  high sensitivity for the determination of traces, all these steps being integrated online in a -  microsystem. -  2-Description of the project, methodology : -  In order to obtain sufficient sensitivity for the analysis of D-enantiomers of amino acid, a derivatization -  step allowing laser-induced fluorescence (chemical reaction with a fluorophore) is required. In this -  project, it will be realized off-line prior to the introduction of the sample in the microsystem, as the offline -  derivatization does not affect the quality of analytical results. -  An innovative microsystem will be developed, with two integrated analytical steps: -  a) An immunopreconcentration step, highly specific towards the molecules of interest, based on the -  immobilization of D-specific antibodies on a monolithic support. The key of this strategy lies in the -  production of specific antibodies exhibiting high affinity for the derivatized D-amino acids, while -  offering no recognition to other derivatized compounds or to the derivatization by-products. -  b) An enantiospecific separation based on the motivated choice of an adequate chiral selector, -  which will be dynamically adsorbed on a stationary phase allowing electrochromatography. This -  chiral separation is vital. Indeed, despite the high recognition ability of antibodies, it is necessary -  to make sure that the presence of L-enantiomers in high concentrations does not create any -  interference during the antigen-antibody recognition. The coupling of both recognition -  dimensions, by antibodies and by enantiospecific determination, guarantees the accuracy of the -  results. -  3-Expected results : -  In a first stage, this Lab-on-Chip will be dedicated to the determination of one D-amino acid in order to -  optimize the analytical steps as well as their integration within the microsystem. -  In a second time, it will be interesting to immobilize a mixture of antibodies in order to simultaneously -  determine the D-enantiomers of many amino acids, especially serine and aspartic acid, in biological -  fluids. This novel analytical tool could then be available to study the mecanisms of evolution of -  neurodegenerative diseases.                                                                                                                                                         ...