Immobilisation of oligo-peptidic probes for microarray implementation: characterisation by FTIR, atomic force microscopy and 2D fluorescence.
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.
Soultani-Vigneron
S
S
LEOM UMR CNRS 5512, Ecole Centrale de Lyon, 69134 Ecully, France.
Dugas
V
V
Rouillat
M H
MH
Fédollière
J
J
Duclos
M C
MC
Vnuk
E
E
Phaner-Goutorbe
M
M
Bulone
V
V
Martin
J R
JR
Wallach
J
J
Cloarec
J P
JP
D019921:Biotinylation; D005453:Fluorescence; D005898:Glass / Q000737:chemistry; D018625:Microscopy, Atomic Force; D009842:Oligopeptides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties
D009842:Oligopeptides; D012997:Solvents; D012822:Silicon Dioxide