An activated GOPS-poly-L-lysine-coated glass surface for the immobilization of 60mer oligonucleotides

Abstract

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly-L-lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly-L-lysine to a glycidoxy-modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 104 and 5.0 × 104 after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS-PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above-mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large-scale manufacture. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.