The development of bioaffinity chromatography columns that are based on the entrapment of biomolecules within the pores of sol-gel-derived monolithic silica is reported. Monolithic nanoflow columns are formed by mixing the protein-compatible silica precursor diglycerylsilane with a buffered aqueous solution containing poly(ethylene oxide) (PEO, MW 10,000) and the protein of interest and then loading this mixture into a fused-silica capillary (150-250-microm i.d.). Spinodal decomposition of the PEO-doped sol into two distinct phases prior to the gelation of the silica results in a bimodal pore distribution that produces large macropores (>0.1 microm), to allow good flow of eluent with minimal back pressure, and mesopores (approximately 3-5-nm diameter) that retain a significant fraction of the entrapped protein. Addition of low levels of (3-aminopropyl)triethoxysilane is shown to minimize nonselective interactions of analytes with the column material, resulting in a column that is able to retain small molecules by virtue of their interaction with the entrapped biomolecules. Such columns are shown to be suitable for pressure-driven liquid chromatography and can be operated at relatively high flow rates (up to 500 microL x min(-1)) or with low back pressures (<100 psi) when used at flow rates of 5-10 microL x min(-1). The clinically relevant enzyme dihydrofolate reductase was entrapped within the bioaffinity columns and was used to screen mixtures of small molecules using frontal affinity chromatography with mass spectrometric detection. Inhibitors present in compound mixtures were retained via bioaffinity interactions, with the retention time being dependent on both the ligand concentration and the affinity of the ligand for the protein. The results suggest that such columns may find use in high-throughput screening of compound mixtures.

At present, there is no direct experimental evidence that primary silica particles, which exist only transiently for a few seconds during the Sto ¨ber silica synthesis, can be stable in aqueous solutions. In the present work, we show that primary silica particles are formed spontaneously after the dissolution of diglycerylsilane (DGS) in aqueous solutions and remain stable for prolonged periods of time. By using time-resolved fluorescence anisotropy (TRFA), we demonstrate that this unique property of DGS is ascribed to the slow kinetics of silica particle growth in diluted sols at pH ∼ 9.0. The anisotropy decay of the cationic dye rhodamine 6G (R6G), which strongly adsorbs to silica oligomers and nanoparticles in DGS sols, could be fit to three components: a fast (picosecond) scale component associated with free R6G, a slower (nanosecond) rotational component associated with R6G bound to primary silica particles, and a residual (nondecaying) anisotropy component associated with R6G that was bound to secondary or larger particles that were unable to rotate on the time scale of the R6G emission lifetime (4 ns). The data show that, under conditions where fast hydrolysis is obtained, the initial size of the nuclei depends on the silica concentration, with larger nuclei being present in more concentrated sols, while the rate of growth of primary particles depends on both silica concentration and solution pH. At low silica concentrations and high pHs, it was possible to observe the growth of stable, nonaggregating primary silica particles by a mechanism involving rapid nucleation followed by monomer addition. The presence of stable primary particles was confirmed by atomic force microscopy (AFM) imaging. At higher silica concentrations and lower pHs, there was an increase in the initial size of the nuclei formed, which subsequently grew to a larger radius (>4.5 nm) or aggregated with time, and in such cases, nucleation and aggregation occurred simultaneously in the early stage of silica formation. The data clearly show the power of time-resolved fluorescence anisotropy decay measurements for probing the growth of silica colloids and show that this method is useful for elucidating the mechanism of particle formation and growth in situ.

Recently, Johansson and co-workers provided the first direct evidence for the existence of nonfluorescent bodipy (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) H dimers in double-labeled proteins and fluorescent J dimers in labeled lipid vesicles et al. J. Am. Chem. Soc. 2002, 124, 196), allowing for the calculation of many of the properties of the dimers. Herein, we report on the use of molecular confinement within a sodium silicate derived glass to provide a highly reproducible system wherein nonfluorescent bodipy H dimers can be formed from the free probe essentially quantitatively without any interference from higher-order aggregates or fluorescent J dimers. The formation of the H dimer followed an unexpected first order kinetic process. On the basis of analysis of the fluorescence anisotropy of the entrapped monomer, it was concluded that the H-dimer formation was promoted by adsorption of monomers onto the silica surface (rate limiting step), followed by rapid dimerization. Using exciton coupling theory, it was determined that the H dimer consisted of two strongly coupled monomers that were stacked in a parallel orientation with a distance of 7.6 Å between the monomer units. The transition dipole moment of the monomer was determined to be 26.6 × 10 -30 C m (8.1 D), the emission quantum yield of the H dimer was found to be close to zero, and the Fo ¨rster distance for energy transfer between the monomer and H dimer was calculated to be 56 ( 2 Å. All of these values are in excellent agreement with those determined by Johansson et al.