Thiol-functionalized organosilica microspheres were synthesized via a two-step process: (1) acid-catalyzed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPTMS), followed by (2) base-catalyzed condensation, which led to the rapid formation of emulsion droplets with a narrow size distribution. These droplets continued to condense to form solid microspheres. Solution (29)Si NMR and optical microscopy were applied to study the mechanism of this novel synthetic route. Solid-state (29)Si NMR, SEM, zeta potential titration, and Coulter counter measurements were used to study the bulk and surface properties and to determine the particle size distributions of the final microspheres. Compared to conventional Stöber silica particles, these microspheres were shown to have a lower degree of cross-linking (average degree of condensation, r = 1.25), a larger average size (up to 6 microm), and a higher isoelectric point (pH = 4.4). Confocal microscopy of dye-labeled microspheres showed an even distribution of dye molecules throughout the interior, characteristic of a readily accessible and permeable organosilica network. These findings have implications for the production of functionalized solid supports for use in catalysis and biological applications, such as optically encoded carriers for combinatorial synthesis.
Organosilica microspheres synthesised via a novel surfactant-free emulsion-based method show applicability towards optical encoding, solid-phase synthesis and high-throughput screening of bound oligonucleotide and peptide sequences.
Poly(ethylene glycol) (PEG) is used as an inert spacer in a wide range of biotechnological applications such as to display peptides and proteins on surfaces for diagnostic purposes. In such applications it is critical that the peptide is accessible to solvent and that the PEG does not affect the conformational properties of the peptide to which it is attached. Using molecular dynamics (MD) simulation techniques, we have investigated the influence of a commonly used PEG spacer on the conformation properties of a series of five peptides with differing physical-chemical properties (YGSLPQ, VFVVFV, GSGGSG, EEGEEG, and KKGKKG). The conformational properties of the peptides were compared (a) free in solution, (b) attached to a PEG-11 spacer in solution, and (c) constrained to a two-dimensional lattice via a (PEG-11)(3) spacer, mimicking a peptide displayed on a surface as used in microarray techniques. The simulations suggest that the PEG spacer has little effect on the conformational properties of small neutral peptides but has a significant effect on the conformational properties of small highly charged peptides. When constrained to a two-dimensional surface at peptide densities similar to those used experimentally, it was found that the peptides, in particular the polar and nonpolar peptides, aggregated strongly. The peptides also partitioned into the PEG layer. Potentially, this means that at high packing densities only a small fraction of the peptide attached to the surface would in fact be accessible to a potential interaction partner.
The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment, multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (MES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.
A new generation of optically encoded organosilica microspheres, suitable for both solid phase synthesis and multiplexed microsphere-based assays, has recently been described. One of the challenges of producing this type of dual-purpose solid support is that the particles must maintain their morphology as well as their encoding during exposure to the solvents used for solid phase synthesis. In this article, organosilica microspheres are subjected to ammonia treatment methods for enhancing the condensation of the silica matrix and their subsequent resilience toward organic solvents and peptide synthesis reagents is described. The instability of the untreated supports toward organic synthesis reagents was found to be associated with the swelling and permeability of these microspheres in organic solvents. Post-synthesis ammonia treatment resulted in reduced permeability, as demonstrated by dye uptake studies. The treated microspheres exhibited enhanced stability against organic synthesis conditions and were characterized via a variety of techniques including electron microscopy, (29)Si-nuclear magnetic resonance (NMR) and optical microscopy. The ammonia-treated supports were subjected to an Fmoc peptide synthesis procedure and successfully applied in a model microsphere-based flow cytometric immunoassay.
The synthesis, characterization, and use of dendron-like poly(ethylene glycol)-lysine (PEG-Lys) copolymers as an intermediate layer for biomolecular diagnostic signal enhancement is presented. Solid phase Fmoc-peptide synthesis was used to synthesize polymers with one, two, and three PEG-Lys comonomer units in both a linear and first and second-generation dendronic structure directly onto organosilica microspheres. The microsphere surface loadings (number of free amine sites) were modified and quantified through an innovative use of the protecting groups of coupled amino acids. Surfaces with 0.1-100% of the original loading corresponding to 0.3-270 nmol/m2 of free amines were achieved. The influence of polymer structure and surface loading (grafting density) on the signal-to-noise of the microsphere-based molecular diagnostic was assessed measuring the difference in the signal of a model protease digestion assay and reduction in the nonspecific adsorption of bovine serum albumin. Increasing the polymer grafting density and the addition of dendronic branching were both found to increase the assay signal and reduce the nonspecific protein adsorption.
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