/ "Chimie, Electrochimie Moléculaires et
Chimie Analytique"
CEMCA
UMR CNRS 6521 /
OFFRE DE THESE FINANCEE REGION BRETAGNE - INDUSTRIE
Pr Raphaël TRIPIER
Dr Maryline BEYLER
Université de Bretagne Occidentale
UMR-CNRS 6521
: 02 98 01 61 38; :
: 02 98 01 79 33; :
Cell analysis (healthy or tumor), although of great importance, is still very limited and difficult to implement. The later must be very sensitive in order to offer a maximum of data to properly assess the structure and the nature of the cell. Cellular imaging appears to be very promising if progress can be made concerning its sensitivity. Mass cytometry, a cell imaging method based on the biological analysis by coupled spectroscopic and spectrometric techniques can provide the sensitivity required for performant analysis. The technique however requires the use of specific markers which permit a targeted detection of high sensitivity.
The Fluidigm Company (Toronto, Canada), industrial partner of this thesis, recently invented, produced and marketed an instrument for immunoassays on isolated cells by broadband and multi-parametric mass cytometry. The methodology developed is based on the use of polymers functionalized by metallic cation chelators(MCPs: metal-chelating polymers) covalently link to antibodies (Abs). These biomarkers can then offer a highly sensitive study. Each Ab-MCP immunoconjugate can indeed be marked by multiple stable isotopes of natural origin of the same metal (typically an isotopically pure lanthanide ion). Multi-parametric capabilities of mass cytometry rely on the ability of detection of torch spectrometry combined to mass spectrometry (ICP-MS) which has a resolution within one mass unit thanks to a wide variety of stable isotopes.
Many recent reviews in literature report the importance of this multi-parametric analysis technique in cell biology (including isolated cells), but underline that the limitations of mass cytometry lie in its multiplicity and sensitivity.
If specific antibodies or molecules are now readily available and varied enough to consider the analysis of a large number of biological targets, limitations concern the range of vectorizable isotopes which restrict the amount of exploitable information. The type of instrumentation currently available is in fact limited to around forty analyzable parameters per cell. However, it is estimated that with the use of additional isotopes, this technology could allow measurements of 50 to 60 parameters per cell.
In the large family of macrocyclic ligands, polyazamacrocycles are recognized as excellent chelating agents for a wide variety of metal cations. Due to the presence of secondary amines, these macrocycles can be N-functionalized by different coordinating groups what allows the preparation of a wide range of ligands for the complexation of a large variety of metals. The ChASaM team has for some years the expertise in the further functionalization of the macrocycle via a carbon atom of the macrocycle backbone, what is called C-functionalization of the macrocycle. It is thus possible to introduce an additional function on the macrocycle skeleton to bind it covalently to an antibody.
The research project therefore aims the synthesis and characterization of new biomarkers based on polyazamacrocycles, suitably functionalized and capable of complexing a large number of metals, for the development of immunoassays used in mass cytometry (Scheme 1).
Scheme 1.Project principle: (a) design of new ligands for metal detection; (b) grafting of the ligands on antibodies; (c) and (d) metal and isotope detection by mass cytometry.
The development of mass cytometry to chelation of additional isotopes would therefore strengthen the supremacy of this technology and increase its use in the field of health to analyze or predict many genetic diseases or cancers. Scientific advances in the field of medicine and biology would so be remarkable and the economic interest would be undeniable through a generalization of the technology in all laboratories worldwide.