tu Rs cHEBcAL ELSEVIER Sensors and Actuators B 29 (1995) 324 327 Fixation of DNA directly on optical waveguide surfaces for molecular probe biosensor development M.A. Karymov, A.A. Kruchinin, Yu.A. Tarantov, I.A. Balova, L.A. Remisova, Yu.G. Vlasov Department o[ ChemistJ3,. St. Petersburg University, Peterh(~/i St. Petersburg 198904, Russia Abstract Covalent attachment of DNA molecules to the surface of silicon nitride without an intermediate polymer layer is described. It is shown that the silicon nitride has a sorption capacity no less than 200 pg/mm2, hybridization efficiency being close to 100%. The expected value of refractive index changes in the molecular layer under hybridization is estimated as sufficient for the operation of two kinds of waveguide-containing transducers. Key'a'ords': DNA fixation; Optical waveguide sensors: Molecular probe biosensor 1. Introduction A biosensor with a DNA probe can be used for real time identification of genome DNA, for example, in clinical diagnostics. The basis of this biosensor opera- tion is the complementary coupling between the molecules of interest in the analyte and the molecules with the specific nucleotide sequence (the probe), immo- bilized onto a solid support surface. This reaction leads to an increase in the refractive index near the support surface that can be detected by a sensitive refractome- ter. It is attractive to use for this purpose an integrated optics device with evanescent field build-up in the reac- tion region. Since the expected refractivity changes have a small value it is desirable to locate the interacting molecules as close as possible to the waveguide surface. Previously we developed a method for DNA-probe immobilization on the surface of an intermediate poly- mer layer with a thickness of several micrometers [1]. In this communication a method of DNA covalent bonding directly on silicon nitride surfaces without an intermediate polymer layer is described. 2. Experimental 2.1. Probe molecules and supports Replicative and single-stranded forms of bacterio- 0925-4005/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 0925-4005(95)01701 -V phage M 13 DNA with about 6000 nucloeotides pre- pared by a routine method were used in the experi- ments. The replicative form was labeled by nick translation with [~_32p] dNTP and had a specific activ- ity of about 3 × 109cpm/gg. An LS Beckman 100C counter was used to determine the DNA sorption ca- pacity of the supports and the immobilized DNA hy- bridization efficiency. Silicone nitride layers on silicon wafers employed in integrated optics devices were chosen as the support for the probe molecules. The silicon nitride layers were produced by chemical vapor deposition (CVD) in a low pressure reaction chamber. Two types of silicon nitride were used in the experiments: de- posited from a chloride ammonia mixture (first type) and deposited from a silane-ammonia mixture (second type). The thickness of all layers was in the range 80- 120 nm. 2.2, Immobilization procedure and reagents DNA immobilization was carried out according to the procedure development in our laboratory. This procedure is intended for the treatment of different kinds of supports, having hydroxyl or amino groups on the surface. Its principal stages are a prelimin- ary support surface treatment (surface activation), its modification by a bifunctional reagent and covalent