Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5′-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42°C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.

Aminosilane-treated molecular layers on glass surfaces are frequently used as functional platforms for biosensor preparation. All the amino groups present on the surface are not available in reactive forms, because surface amino groups interact with remaining unreacted surface silanol groups. Such nonspecific interactions might reduce the efficiency of chemical immobilization of biomolecules such as DNA, enzymes, antibodies, etc., in biosensor fabrication. To improve immobilization efficiency we have used additional surface silanization with alkylsilane (capping) to convert the remaining silanol groups into Si-O-Si linkages, thereby liberating the amino groups from nonspecific interaction with the silanol groups. We prepared different types of capped amine surface and evaluated the effect of capping on immobilization efficiency by investigating the fluorescence intensity of Cy3-NHS (N-hydroxysuccinimide) dye that reacted with amino groups. The results indicate that most of the capped amine surfaces resulted in enhanced efficiency of immobilization of Cy3-NHS compared with the untreated control amine surface. We found a trend that trialkoxysilanes had greater capping effects on immobilization efficiency than monoalkoxysilanes. It was also found that the aliphatic chain of alkylsilane, which does not participate in the capping of the silanol, had an important function in enhancing immobilization efficiency. These results would be useful for preparation of an amine-modified surface platform, with enhanced immobilization efficiency, which is essential for developing many kinds of biosensors on a silica matrix.

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.