A fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle as a label for the ultrasensitive detection of cancer cells

A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine 6G or tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained 90 nm silica particles was about 61%. Compared with the fluorescent core–shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO₄ oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.     

Solvation dynamics of coumarin 153 in alcohols confined in silica nanochannels

Solvation dynamics in alcohols confined in silica nanochannels was examined by time-resolved fluorescence spectroscopy using coumarin 153 (C153) as a fluorescent probe. Surfactant-templated mesoporous silica was fabricated inside the pores of an anodic alumina membrane. The surfactant was removed by calcination to give mesoporous silica (Cal-NAM) containing one-dimensional (1D) silica nanochannels (diameter, 3.1 nm) whose inner surface was covered with silanol groups. By treating Cal-NAM with trimethylchlorosilane, trimethylsilyl (TMS) groups were formed on the inner surface of the silica nanochannels (TMS-NAM). Fluorescence dynamic Stokes shifts of C153 were measured in alcohols (ethanol, butanol, hexanol, and decanol) confined in the silica nanochannels of Cal- and TMS-NAMs, and the time-dependent fluorescence decay profiles could be best fitted by a biexponential function. The estimated solvent relaxation times were much larger than those observed in bulk alcohols for both Cal- and TMS-NAMs when ethanol or butanol was used as a solvent, indicating that the mobility of these alcohol molecules was restricted within the silica nanochannels. However, hexanol or decanol in Cal- and TMS-NAMs did not cause a remarkable increase in the solvent relaxation time in contrast to ethanol or butanol. Therefore, it was concluded that a relatively rigid assembly of alcohols (an alcohol chain) was formed within the silica nanochannels by hydrogen bonding interaction and van der Waals force between the surface functional groups of the silica nanochannels and alcohol molecules and by the successive interaction between alcohol molecules when alcohol with a short alkyl chain (ethanol or butanol) was used as a solvent.     

Synthesis of monodisperse, rodlike silica colloids with tunable aspect ratio

Although the experimental study of spherical colloids has been extensive, similar studies on rodlike particles are rare because suitable model systems are scarcely available. To fulfill this need, we present the synthesis of monodisperse rodlike silica colloids with tunable dimensions. Rods were produced with diameters of 200 nm and greater and lengths up to 10 μm, resulting in aspect ratios from 1 to ～25. The growth mechanism of these rods involves emulsion droplets inside which silica condensation takes place. Due to an anisotropic supply of reactants, the nucleus grows to one side only, resulting in rod formation. In concentrated dispersions, these rods self-assemble in liquid crystal phases, which can be studied quantitatively on the single particle level in three-dimensional real-space using confocal microscopy. Isotropic, paranematic, and smectic phases were observed for this system.     

Encapsulation of Inorganic Particles by Dispersion Polymerization in Polar Media: 2. Effect of Silica Size and Concentration on the Morphology of Silica–Polystyrene Composite Particles

Following a previous work (Bourgeat-Lami, E., and Lang, J.,J. Colloid Interface Sci.197, 293 (1998)), encapsulation of silica beads has been achieved by dispersion polymerization of styrene in an aqueous ethanol medium using poly(N-vinyl pyrrolidone) as stabilizer. Silica beads, prepared according to the Stöber method, were coated prior to polymerization by grafting 3-(trimethoxysilyl)propyl methacrylate onto the surface. A great number of silica beads per composite particle were previously found using beads that had diameters between 49 and 120 nm. In the present work, larger silica beads with diameters between 191 and 629 nm are investigated. We demonstrate by transmission electron microscopy that, consequently, only a small number of silica beads are contained in the composite particles. By counting the composite particles containing precisely zero, one, two, three, four, and more than four silica beads, it clearly appears that the encapsulation of only one silica bead can be obtained simply by increasing the size of the beads. Under our experimental conditions, the optimal bead diameter for achieving composite particles containing only one silica bead turns out to be around 450 nm. We show that increasing the silica bead size above this value results in an increased number of composite particles without silica beads. In contrast, the number of composite particles with two, three, four, or more than four silica beads increases with decreasing silica bead size. In addition to the above variations in composition of the composite particles, changes in particle shapes were also observed as a function of the size of the silica beads and the styrene concentration in the polymerization medium. Hypotheses concerning these variations are presented.     

Confinement Effect of Organic Nanotubes Toward Green Fluorescent Protein (GFP) Depending on the Inner Diameter Size

Transportation, release behavior, and stability of a green fluorescent protein (GFP, 3×4 nm) in self‐assembled organic nanotubes with three different inner diameters (10, 20, and 80 nm) have been studied in terms of novel nanocontainers. Selective immobilization of a fluorescent acceptor dye on the inner surface enabled us to not only visualize the transportation of GFP in the nanochannels but to also detect release of the encapsulated GFP to the bulk solution in real time, based on fluorescence resonance energy transfer (FRET). Obtained diffusion constants and release rates of GFP markedly decreased as the inner diameter of the nanotubes was decreased. An endo‐sensing procedure also clarified the dependence of the thermal and chemical stabilities of the GFP on the inner diameters. The GFP encapsulated in the 10 nm nanochannel showed strong resistance to heat and to a denaturant. On the other hand, the 20 nm nanochannel accelerated the denaturation of the encapsulated GFP compared with the rate of denaturation of the free GFP in bulk and the encapsulated GFP in the 80 nm nanochannels. The confinement effect based on rational fitting of the inner diameter to the size of GFP allowed us to store it stably and without denaturation under high temperatures and high denaturant concentrations.     

Responsive colloidal systems: reversible aggregation and fabrication of superhydrophobic surfaces

We report on a method of fabricating stimuli-responsive core-shell nanoparticles using block copolymers covalently bound to a silica nanoparticle surface. We used the "grafting to" approach to graft amphiphilic block copolymer brushes of poly(styrene-b-2-vinylpyridine-b-ethylene oxide) and poly(styrene-b-4-vinylpyridine) onto silica nanoparticles with two different diameters: colloidal silica 200 nm in diameter and fumed silica 15 nm in diameter. We used the pH-responsive properties of the grafted brush to regulate the interactions between the particles, and between the particles and their environment. We show that this behavior can be applied for a reversible formation of particle aggregates, and can be used to tune and stabilize the secondary aggregates of particles of the appropriate size and morphology in an aqueous environment. The suspensions of the particles form a textured hydrophilic coating on various substrates upon casting and the evaporation of water. Heating above the polymer's glass transition temperature or treatment in acidic water result in back and forth switching between superhydrophobic and hydrophilic surfaces, respectively.     

Size-controlled, one-pot synthesis, characterization, and biological applications of epoxy-organosilica particles possessing positive zeta potential

Epoxy-organosilica particles made from 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (EpoMS) as a single silica source were synthesized by means of a one-pot method. We evaluated three sets of synthesis conditions, including traditional Stober conditions and two variations. Although the traditional conditions did not afford EpoMS particles, the variations did. The size distributions of the particles were evaluated by means of transmission electron microscopy. The mean diameters and size distributions of the particles depended on the EpoMS concentration, and the best coefficient of variation for the size distribution was 5.9%. The surface of the particles had unique properties, such as a positive zeta potential. The particles bound strongly to proteins as well as to DNA. The particles made from EpoMS, allowing particles internally functionalized with fluorescent dye to be prepared by means of a one-pot synthesis. EpoMS particles doped and tuned with fluorescent dye showed strong fluorescence signals and distinct peaks on flow cytometry, and the fluorescent particles could be used to label cells. The labeled cells showed clear fluorescence under a fluorescence microscope, and electron microscopy showed many particles in the cytoplasm. This is the first report describing the synthesis of epoxy-organosilica particles with a positive zeta potential and describing differences in the characteristics of particle formations due to changes in synthesis conditions. We also discuss the advantages of EpoMS particles, as well as the potential biological applications of these particles.     

Aggregation of silica nanoparticles directed by adsorption of lysozyme

The interaction of the globular protein lysozyme with silica nanoparticles of diameter 20 nm was studied in a pH range between the isoelectric points (IEPs) of silica and the protein (pH 3-11). The adsorption affinity and capacity of lysozyme on the silica particles is increasing progressively with pH, and the adsorbed protein induces bridging aggregation of the silica particles. Structural properties of the aggregates were studied as a function of pH at a fixed protein-to-silica concentration ratio which corresponds to a surface concentration of protein well below a complete monolayer in the complete-binding regime at pH > 6. Sedimentation studies indicate the presence of compact aggregates at pH 4-6 and a loose flocculated network at pH 7-9, followed by a sharp decrease of aggregate size near the IEP of lysozyme. The structure of the bridged silica aggregates was studied by cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The structure factor S(q) derived from the scattering profiles displays characteristic features of particles interacting by a short-range attractive potential and can be represented by the square-well Percus-Yevick potential model, with a potential depth not exceeding 3k(B)T.     

Label‐Free Analysis of Single Viruses with a Resolution Comparable to That of Electron Microscopy and the Throughput of Flow Cytometry

Viruses are by far the most abundant biological entities on our planet, yet existing characterization methods are limited by either their speed or lack of resolution. By applying a laboratory‐built high‐sensitivity flow cytometer (HSFCM) to precisely quantify the extremely weak elastically scattered light from single viral particles, we herein report the label‐free analysis of viruses with a resolution comparable to that of electron microscopy and the throughput of flow cytometry. The detection of single viruses with diameters down to 27 nm is described. T7 and lambda bacteriophages, which differ in size by as little as 4 nm, could be baseline‐resolved. Moreover, subtle structural differences of the same viral particles can be discriminated. Using monodisperse silica nanoparticles as the size reference standards, the virus sizes measured by the HSFCM are in agreement with the equivalent particle diameters derived from their structural dimensions. The HSFCM opens a new avenue for virus characterization.     

Biphasic synthesis of Au@SiO2 core-shell particles with stepwise ligand exchange

We report the synthesis of well-dispersed core-shell Au@SiO(2) nanoparticles with minimal extraneous silica particle growth. Agglomeration was suppressed through consecutive exchange of the stabilizing ligands on the gold cores from citrate to L-arginine and finally (3-mercaptopropyl)triethoxysilane. The result was a vitreophilic, stable gold suspension that could be coated with silica in a biphasic mixture through controlled hydrolysis of tetraethoxysilane under L-arginine catalysis. Unwanted condensation of silica particles without gold cores was limited by slowing the transfer across the liquid-liquid interface and reducing the concentration of the L-arginine catalyst. In-situ dynamic light scattering and optical transmission spectroscopy revealed the growth and dispersion states during synthesis. The resulting core-shell particles were characterized via dynamic light scattering, optical spectroscopy, and electron microscopy. Their cores were typically 19 nm in diameter, with a narrow size distribution, and could be coated with a silica shell in multiple steps to yield core-shell particles with diameters up to 40 nm. The approach was sufficiently controllable to allow us to target a shell thickness by choosing appropriate precursor concentrations.     

Heteroaggregation between Al2O3 submicrometer particles and SiO2 nanoparticles: experiment and simulation

The aggregation process of a two-component dilute system (3 vol %), made of alumina submicrometer particles and silica nanoparticles, is studied by Brownian dynamics simulations. Alumina and silica particles have very different sizes (diameters of 400 and 25 nm, respectively). The particle-particle interaction potential is of the DLVO form. The parameters of the potential are extracted from the experiments. The simulations show that the experimentally observed aggregation phenomena between alumina particles are due to the silica-alumina attraction that induces an effective driving force for alumina-alumina aggregation. The experimental data for silica adsorption on alumina are very well reproduced.     

石英纳米通道单磷脂囊泡的电阻-脉冲分析方法

本文报告了用于检测单囊泡及其粒径分析的石英纳米通道电阻-脉冲分析方法. 采用圆柱形石英纳米通道可检测粒径为100~300 nm的单磷脂囊泡和直径为170~400 nm的聚苯乙烯纳米颗粒. 单囊泡和纳米颗粒的迁移可通过检测各自产生的方波电流脉冲信号, 并由此确定颗粒尺寸. 结果表明,采用石英纳米通道电阻-脉冲分析方法得到的颗粒/囊泡粒径与采用动态光散射法和扫描电子显微法得到的结果完全一致. 这种基于电子的分析方法具有快速简单的特点,所用的自制微传感器廉价耐用. 石英通道的应用还可与其它分析方法如电流分析法和荧光显微法联用,以获得生物囊泡及人工囊泡更完全的信息.     

Mesoporous silica spheres from colloids

A novel method has been developed to synthesize mesoporous silica spheres using commercial silica colloids (SNOWTEX) as precursors and electrolytes (ammonium nitrate and sodium chloride) as destabilizers. Crosslinked polyacrylamide hydrogel was used as a temporary barrier to obtain dispersible spherical mesoporous silica particles. The influences of synthesis conditions including solution composition and calcination temperature on the formation of the mesoporous silica particles were systematically investigated. The structure and morphology of the mesoporous silica particles were characterized via scanning electron microscopy (SEM) and N2 sorption technique. Mesoporous silica particles with particle diameters ranging from 0.5 to 1.6 microm were produced whilst the BET surface area was in the range of 31-123 m2 g-1. Their pore size could be adjusted from 14.1 to 28.8 nm by increasing the starting particle diameter from 20-30 nm up to 70-100 nm. A simple and cost effective method is reported that should open up new opportunities for the synthesis of scalable host materials with controllable structures.     

Efficient synthesis of poly(2-vinylpyridine)-silica colloidal nanocomposite particles using a cationic azo initiator

Colloidal poly(2-vinylpyridine)-silica nanocomposite particles can be efficiently prepared by emulsion polymerization at 60 degrees C using a commercial 20 nm aqueous silica sol as the sole stabilizing agent. Unlike previously reported colloidal nanocomposite syntheses, transmission electron microscopy studies indicate very high silica aggregation efficiencies (88-99%). The key to success is simply the selection of a suitable cationic azo initiator. In contrast, the use of an anionic persulfate initiator leads to substantial contamination of the nanocomposite particles with excess silica sol. The cationic azo initiator is electrostatically adsorbed onto the anionic silica sol at submonolayer coverage, which suggests that surface polymerization may be important for successful nanocomposite formation. Moreover, the 2-vinylpyridine can be partially replaced with either styrene or methacrylic comonomers to produce a range of copolymer-silica nanocomposite particles. The poly(2-vinylpyridine)-silica nanocomposite particles have a well-defined core-shell morphology, with poly(2-vinylpyridine) cores and silica shells; mean diameters typically vary from 180 to 220 nm, and mean silica contents range from 27 to 35% by mass.     

圆二色性测定蛋白质在超细粒子上吸附的构象变化

研究了蛋白质在超细粒子固体表面吸附过程中构象的变化;选择的吸附剂粒径很小,可以直接应用圆二色性光谱测定蛋白质吸附层的二级结构变化;研究了pH、固体吸附表面在蛋白质吸附过程中对蛋白质构象的影响,得到了较好的实验数据,为进一步进行蛋白质吸附机理的理论研究提供了基础。     

Synthesis and characterization of dually labeled Pickering-type stabilized polymer nanoparticles in a downscaled miniemulsion system

Dual fluorescently labeled polymer particles were prepared in a downscaled Pickering-type miniemulsion system. Stable dispersions were obtained and the size of the hybrid particles could be varied between ca. 180 and 430 nm. Silica nanoparticles were employed as sole emulsifier, which were labeled by a fluorescein dye (FITC) or (encapsulated) quantum dots, and the polymer core was labeled by a perylene derivative. Downscaling of the Pickering-type miniemulsion system is intriguing by itself as it allows the use of precious nanoparticles as emulsifiers. Here, silica particles with a fluorescent core and an overall diameter between 20 and 40 nm were prepared and employed as stabilizer. The dual excitation and emission of both dyes was tested by fluorescence measurements and confocal laser scanning microscopy (cLSM).     

Dynamics of molecular diffusion of rhodamine 6G in silica nanochannels

We describe a method to study diffusion of rhodamine 6G dye in single silica nanochannels using arrays of silica nanochannels. Dynamics of the molecules inside single nanochannel is found from the change of the dye concentration in solution with time. A 10(8) decrease in the dye diffusion coefficient relative to water was observed. In comparison to single fluorescent molecule studies, the presented method does not require fluorescence of the diffusing molecules.     

The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: local fluorescence study at the single-molecule level

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.     

Detection of nonfluorescent molecules using differential interference contrast thermal lens microscope for extended nanochannel chromatography

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of ~100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples <1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of ~fL volume.     

Surface Assisted Nucleation and Growth of Polymer Latexes on Organically-Modified Inorganic Particles

Summary: Polymer latex particles were synthesized in the presence of inorganic particles, which had been organically-modified to promote favorable interactions with growing macromolecules. The organic modification was performed using three different routes: (1) surface covalent grafting of vinyl trialkoxysilanes, (2) surface adsorption of polyethylene glycol-based macromonomers, and (3) bulk modification through ion exchange with cationic monomers or cationic initiators. Two types of mineral particles were studied: commercial and self-prepared silica particles (with diameters from 80 nm to 1 µm), and commercial laponite clay particles with a cation exchange capacity of 0.75 meq · g⁻¹. Emulsion polymerization was performed in the presence of styrene or butyl acrylate monomers. The morphologies of the nanocomposite particles were observed by (cryogenic) transmission electron microscopy and correlated to the organic modification procedure.