Specificity and sensitivity of fluorescence labeling of surface species.

FLOSS (fluorescence labeling of surface species) enables one to identify and quantify very low concentrations of surface functional groups. Unlike most surface analytical techniques, FLOSS can provide absolute, as well as relative, surface coverage determination. However, as with any other surface derivatization technique, FLOSS provides a lower limit to surface coverage. The specificity of FLOSS for a particular functional group is the key to this application. In one FLOSS protocol, amine-modified dyes are used to label surface aldehyde groups. However, amine-modified dyes, in principle, can bind to both aldehyde and carboxyl groups, limiting specificity. In this paper, we report that the FLOSS protocol devised results in less than 0.5 % of the carboxyl-modified dyes binding to the surface amine groups. Therefore, the presence of carboxyl groups on the surface should have a limited effect on the detection of aldehyde groups by amine-modified dye. Quenching of fluorescence can potentially affect quantitative measurements. To address this issue, the densities of surface functional groups of CHO-, NH2-, and epoxy-coated glass surfaces were quantified using FLOSS and compared to surface densities estimated by other methods. The FLOSS technique was extended to glass surfaces by using visible absorbing and emitting dyes. The lower detection limit is on the order of 10(9) groups/cm2.