Two different methods for immobilization of DNA probes on glass surfaces have been comparatively investigated. Glass slides were derivatized with 3-aminopropyltriethoxysilane. Then 5'-amino-modified- 21 mer oligodeoxynucleotide probes were covalently immobilized onto the surface via 1,4-phenylene diisothiocyanate and glutaraldehyde, respectively. The surface molecular hybridization of the immobilized probes with FITC- labeled complementary target sequences was monitored with an inverted fluorescence microscope (Model IX70) coupled with a cooled-CCD. There was hardly and difference in the fluorescence intensity between the spots on the chips, obtained at high solution concentrations of DNA probes by the two immobilization methods respectively, indicating that these methods were satisfactory under the conditions used. However, the fluorescent signal for the chips obtained at low concentrations of DNA probes by the 1,4- phenylene diisothiocyanate method was stronger than that by the glutaraldehyde method, indicating that the former method was better than the latter at low solution concentrations of DNA probes. The detection limit for the target sequence was 1.5×10^-9 mol/L. Some experimental conditions, including the time for DNA probe immobilization, hybridization time and temperature, were examined. The results obtained would be useful for the development of genochips.

Key words: GLASS, NUCLEIC ACIDS, ISOCYANATE P, PROBES, FIXATION, HYBRIDIZATION