RESUMEN
A novel diagnostic tool has been developed for the characterization of intracellular pH (pHi) in the model organism Caenorhabditis elegans. This tool exploits the chemical stability of colloidal silica and the pH sensitivity of certain fluorescent dyes. Once ingested, the fluorescent colloidal dispersion yields a reliable visual indication of pH without the use of chemical fixatives or damaging the nematode. The pH-sensitive silica nanoparticles were visualized by confocal microscopy, and the fluorescence spectra from the internally referenced colloidal particulates were measured. By comparing the fluorescence profile of colloids in wild-type (N2) and mutant (eat-3) C. elegans against a calibration series, the intestinal pHi could be established in each population. The rapid characterization of pHi using this inexpensive nonintrusive technique has significant implications for disease research where C. elegans is used as a model organism.
Asunto(s)
Caenorhabditis elegans/citología , Espacio Intracelular/química , Imagen Molecular/métodos , Nanopartículas , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Colorantes Fluorescentes/química , GTP Fosfohidrolasas/genética , Concentración de Iones de Hidrógeno , Microscopía Confocal , Mutación , Dióxido de Silicio/química , Espectrometría de FluorescenciaRESUMEN
A multiplexed screening methodology for the rapid development of antifouling polymer surfaces is presented. An array of protein resistant polymer layers with high grafting (>100 mg m(-2)) were polymerized on optically encoded particles. Multiplexed analysis showed a 97% reduction in nonspecific protein adsorption for all polymer layers created.
Asunto(s)
Incrustaciones Biológicas/prevención & control , Polímeros/química , Polímeros/farmacología , Adsorción , Animales , Bovinos , Compuestos de Organosilicio/química , Polimerizacion , Proteínas/química , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Simulación de Dinámica Molecular , Oligopéptidos/química , Polietilenglicoles/química , Secuencia de Aminoácidos , Conformación Proteica , Soluciones , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Inmunoensayo/métodos , Microesferas , Biosíntesis de Péptidos , Dióxido de Silicio/química , Amoníaco/químicaRESUMEN
The concept of optically encoding particles for solid phase organic synthesis has existed in the literature for several years. However, there remains a significant challenge to producing particles that are capable of withstanding harsh solvents and reagents whilst maintaining the integrity and range of the optical encoding. In this study, a new generation of fluorescently encoded support particles was used for both solid phase peptide synthesis and on-particle analysis of proteolysis in a multiplexed, flow cytometric assay. The success of the assay was demonstrated through the use of a model protease, trypsin. Our results show that the use of solid supports with high peptide yield, high swellability in water and high penetration of the enzyme into the interior of the particle is not absolutely necessary for proteolysis assays.
Asunto(s)
Citometría de Flujo/métodos , Biblioteca de Péptidos , Péptidos/química , Colorantes Fluorescentes/química , Hidrólisis , Microesferas , Compuestos de Organosilicio/química , Péptido Hidrolasas/metabolismo , Péptidos/síntesis químicaRESUMEN
This paper reports on the synthesis of uniformly dye-doped organosilica particles with narrow size distribution. The particle size can be controlled from tenths of nanometers up to several micrometers, whilst still maintaining monodispersity. Microparticles were observed to swell in various solvents up to approximately 2.5 times their original volume, suggesting the presence of a gel-like internal structure. As shown by confocal microscopy, this morphological control of particle swelling has important implications for the encoding of the nano/micro particles with organic dyes, such as rhodamine B isothiocyanate. Swelling allows the dye to penetrate the organosilica matrix and produce uniformly dye-doped nano- and microparticles. Finally, we suggest a coagulation model for the particle formation which significantly differs from conventional Stöber synthesis.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Microesferas , Nanopartículas , Compuestos de Organosilicio/química , Tamaño de la Partícula , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Rodaminas/químicaRESUMEN
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.
Asunto(s)
Emulsiones , Compuestos Orgánicos/química , Dióxido de Silicio , Colorantes/química , Concentración de Iones de Hidrógeno , Hidrólisis , Espectroscopía de Resonancia Magnética/métodos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microesferas , Potenciometría , SolucionesRESUMEN
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.