The analytical signal produced from a site selectively templated and tagged xerogel (SSTTX) based sensor is more completely elucidated. In an SSTTX, target analyte (TA) binding sites are created within a xerogel and a fluorescent reporter molecule (e.g., nitrobenzo-2-oxa-1,3-diazole, NBD) is covalently and selectively positioned within the template site. TA binding modulates the NBD cybotactic region, yielding a TA concentration-dependent change in the NBD reporter fluorescence. Our results reveal that there are two NBD sub-populations within the SSTTX. One sub-population is associated with misformed template sites that bind TA poorly and the NBD reporter molecules are heavily quenched by an adjacent amine residue that is intentionally positioned within close proximity to the NBD reporter molecule. The second NBD sub-population is associated with a properly formed template site that binds TA; the adjacent amine is still present but it is not as close to the NBD in comparison to the misformed site. When TA is present, the TA-responsive site binds TA, the TA disrupts the adjacent amine–NBD interaction (dequenching), and the NBD fluorescence increases. All NBD quenching occurs via photo-induced electron transfer (PET) from the adjacent amines to the excited NBD reporter molecules. As the mole fraction ratio of n-octyltrimethoxysilane (C8-TMOS) to tetramethoxysilane (TMOS) within an SSTTX increases, the misformed site mole fraction decreases and the dissociation constant (Kd) for TA binding to the TA-responsive site decreases by 13-fold.
Vol. 5, Iss. 11 Virtual Journal for Biomedical Optics
Ellen L. Holthoff and Frank V. Bright, "Photophysics Associated with Site Selectively Templated and Tagged Xerogel Sensor Platforms," Appl. Spectrosc. 64, 714-719 (2010)