DNA-directed immobilisation of glycomimetics for glycoarrays application: comparison with covalent immobilisation, and development of an on-chip IC50 measurement assay.
Glycoarrays are powerful tools for the understanding of protein/carbohydrate interactions and should find applications in the diagnosis of diseases involving these interactions. Immobilisation of the carbohydrate probe is a key issue in the elaboration of high performance devices. In the present study, we have compared the fluorescent signal intensity and determined the lower detection limit of glycoconjugates immobilised at two concentrations (0.5 and 25 microM) by DNA-directed immobilisation (DDI), to glycoconjugates covalently immobilised on the solid support (borosilicate glass slide). At 0.5 microM, DDI led to a stronger fluorescence signal (by a factor of 4.5) and to a lower detection limit (20 nM) than covalent immobilisation (higher than 200 nM). We also report the development of an IC(50) measurement assay of DDI immobilised glycoconjugates. We found that the relative affinity per galactose residue of RCA 120 for glycoconjugates bearing one or three galactose residues was different by a factor of 23 when measured under IC(50) conditions or by direct fluorescence reading.
Zhang
Jing
J
Institut des Nanotechnologies de Lyon, UMR 5270 CNRS, Ecole Centrale de Lyon, INSA Lyon, Université Claude Bernard Lyon 1, Equipe Chimie et Nanobiotechnologies, 36 Avenue Guy-de-Collongue, 69134 Ecully, France.
Pourceau
Gwladys
G
Meyer
Albert
A
Vidal
Sébastien
S
Praly
Jean-Pierre
JP
Souteyrand
Eliane
E
Vasseur
Jean-Jacques
JJ
Morvan
François
F
Chevolot
Yann
Y
D015374:Biosensing Techniques / Q000295:instrumentation*; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004867:Equipment Design; D019544:Equipment Failure Analysis; D054794:Glycomics / Q000295:instrumentation* / Q000379:methods; D056656:Lab-On-A-Chip Devices; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D002241:Carbohydrates; D004247:DNA