TY - JOUR
T1 - Immobilisation of oligo-peptidic probes for microarray implementation
T2 - Characterisation by FTIR, Atomic Force Microscopy and 2D fluorescence
AU - Soultani-Vigneron, S.
AU - Dugas, V.
AU - Rouillat, M. H.
AU - Fédollière, J.
AU - Duclos, M. C.
AU - Vnuk, E.
AU - Phaner-Goutorbe, M.
AU - Bulone, V.
AU - Martin, J. R.
AU - Wallach, J.
AU - Cloarec, J. P.
PY - 2005/8/5
Y1 - 2005/8/5
N2 - Proteomic microarrays show a wide range of applications for the investigation of DNA-protein, enzyme-substrate as well as protein-protein interactions. Among many challenges to build a viable "protein microarray", the surface chemistry that will allow to immobilised various proteins to retain their biological activity is of paramount importance. Here we report a chemical functionalisation method allowing immobilisation of oligo-peptides onto silica surface (porous silica, glass, thermal silicon dioxide). Substrates were first derivatised with a monofunctional silane allowing the elaboration of dense and uniform monolayers in highly reproducible way. Prior to the oligo-peptides grafting, this organic layer was functionalised with an amino-polyethyleneglycol. The coupling step of oligo-peptides onto functionalised supports is achieved through activation of the C-terminal function of the oligo-peptides. Chemical surface modifications were followed by FTIR spectroscopy, AFM measurements and fluorescence scanning microscopy. A systematic study of the oligo-peptide grafting conditions (time, concentration, solvent) was carried out to optimise this step. The oligo-peptides grafting strategy implemented in this work ensure a covalent and oriented grafting of the oligo-peptides. This orientation is ensured through the use of fully protected peptide except the terminal primary amine. The immobilized peptides will be then deprotected before biological recognition. This strategy is crucial to retain the biological activity of thousands of oligo-probes assessed on a microarray.
AB - Proteomic microarrays show a wide range of applications for the investigation of DNA-protein, enzyme-substrate as well as protein-protein interactions. Among many challenges to build a viable "protein microarray", the surface chemistry that will allow to immobilised various proteins to retain their biological activity is of paramount importance. Here we report a chemical functionalisation method allowing immobilisation of oligo-peptides onto silica surface (porous silica, glass, thermal silicon dioxide). Substrates were first derivatised with a monofunctional silane allowing the elaboration of dense and uniform monolayers in highly reproducible way. Prior to the oligo-peptides grafting, this organic layer was functionalised with an amino-polyethyleneglycol. The coupling step of oligo-peptides onto functionalised supports is achieved through activation of the C-terminal function of the oligo-peptides. Chemical surface modifications were followed by FTIR spectroscopy, AFM measurements and fluorescence scanning microscopy. A systematic study of the oligo-peptide grafting conditions (time, concentration, solvent) was carried out to optimise this step. The oligo-peptides grafting strategy implemented in this work ensure a covalent and oriented grafting of the oligo-peptides. This orientation is ensured through the use of fully protected peptide except the terminal primary amine. The immobilized peptides will be then deprotected before biological recognition. This strategy is crucial to retain the biological activity of thousands of oligo-probes assessed on a microarray.
KW - AFM
KW - Biotin
KW - Fluorescence
KW - FTIR
KW - Microarray
KW - Oligo-peptide immobilization
KW - Streptavidin
UR - http://www.scopus.com/inward/record.url?scp=22244456710&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2005.04.019
DO - 10.1016/j.jchromb.2005.04.019
M3 - Article
C2 - 15908289
AN - SCOPUS:22244456710
VL - 822
SP - 304
EP - 310
JO - Journal of Chromatography B-Analytical Technologies in The Biomedical and Life Sciences
JF - Journal of Chromatography B-Analytical Technologies in The Biomedical and Life Sciences
SN - 1570-0232
IS - 1-2
ER -