Quantitative analysis and characterization of biofunctionalized fluorescent silica particles

A strategy for the production and subsequent characterization of biofunctionalized silica particles is presented. The particles were engineered to produce a bifunctional material capable of both (a) the attachment of fluorescent dyes for particle encoding and (b) the sequential modification of the surface of the particles to couple oligonucleotide probes. A combination of microscopic and analytical methods is implemented to demonstrate that modification of the particles with 3-aminopropyl trimethoxysilane results in an even distribution of amine groups across the particle surface. Evidence is provided to indicate that there are negligible interactions between the bound fluorescent dyes and the attached biomolecules. A unique approach was adopted to provide direct quantification of the oligonucleotide probe loading on the particle surface through X-ray photoelectron spectroscopy, a technique which may have a major impact for current researchers and users of bead-based technologies. A simple hybridization assay showing high sequence specificity is included to demonstrate the applicability of these particles to DNA screening.     

Surface acidity and basicity of functionalized silica particles

Tetraethoxysilane has been co-hydrolyzed with functionalized organosilanes in a modified Stöber process to produce silica particles with amino, carboxylate or dihydroimidazole groups on the surface. The effects of reaction conditions and the loading of the functionalized organosilane on particle size was examined by TEM. Fluorescence spectroscopy of the surface amino groups covalently modified with fluorescamine, and the surface carboxylate groups with 4-bromomethyl-6,7-dimethoxycoumarin, demonstrated that these functional groups were accessible for further reaction. Changes in surface acidity and basicity caused by the presence of functional groups (amine, dihydroimidazole, carboxylate) on the particle surface were determined using an indicator titration technique. Particles with surface imidazole and amine groups and particles with surface carboxylate groups have enhanced basicity and acidity, respectively. Dihydroimidazole-modified silica had greater surface basicity than the amine-modified silica. The effect on basicity and acidity increases as the amount of added functionalized silane increases. However, this increase is nonlinear with respect to the increase in added functionalized silane. Particles with both surface dihydroimidazole and carboxylate groups demonstrated reduced surface basicity and acidity.     

Synthesis and characterization of amino-functionalized silica nanoparticles

Monodispersed raw silica nanoparticles (RSNPs) with the particle size of 40 nm were successfully fabricated by condensation reaction of tetraethylorthosilicate in methanol with high concentration ammonia (1.2 M). The RSNPs were treated with the coupling agent 3-aminopropyltrimethoxysilane (APTMS) for grafting amine groups on the surface to obtain the amino-functionalized silica nanoparticles (ASNPs). The chemical structure and surface morphology of RSNPs and ASNPs were characterized by Fourier-transform infrared spectra, solid-state NMR spectra and scanning electron microscopy. In addition, a method to quantify the grafted amine groups on the surface of ASNPs was developed by using the ninhydrin assay. The ninhydrin analysis showed that 60 mol % of the APTMS molecules were immobilized on the surface, that is, 4.4 amine groups per nm2 of surface area were bonded on nonporous ASNPs. The weight loss of particles obtained from thermogravimetry analysis indicated the amount of grafted amine groups and was used as a reference to compare with the value determined from ninhydrin method.     

Nitric oxide-releasing fumed silica particles: synthesis,characterization, and biomedical application

The preparation, characterization, and preliminary biomedical application of various nitric oxide (NO)-releasing fumed silica particles (0.2-0.3 microm) are reported. The tiny NO-releasing particles are synthesized by first tethering alkylamines onto the surface of the silica using amine-containing silylation reagents. These amine groups are then converted to corresponding N-diazeniumdiolate groups via reaction with NO(g) at high pressure in the presence of methoxide bases (e.g., NaOMe). N-Diazeniumdiolate groups were found to form more readily with secondary amino nitrogens than primary amino nitrogens tethered to the silica. Different alkali metal cations of the methoxide bases, however, have little effect on the degree of N-diazeniumdiolate formation. The N-diazeniumdiolate moieties attached on the silica surface undergo a primarily proton-driven dissociation to NO under physiological conditions, with an "apparent" reaction order somewhat greater than 1 owing to local increases in pH at the surface of the particles as free amine groups are generated. The rates of N-diazeniumdiolate dissociation are further related to the parent amine structures and the pH of the soaking buffer. The N-diazeniumdiolate groups also undergo slow thermal dissociation to NO, with zero-order dissociation observed at both -15 and 23 degrees C. It is further shown that the resulting NO-releasing fumed silica particles can be embedded into polymer films to create coatings that are thromboresistant, via the release of NO at fluxes that mimic healthy endothelial cells (EC). For example a polyurethane coating containing 20 wt % of NO-releasing particles prepared with pendant hexane diamine structure (i.e., Sil-2N[6]-N(2)O(2)Na) is shown to exhibit improved surface thromboresistivity (compared to controls) when used to coat the inner walls of extracorporeal circuits (ECC) employed in a rabbit model for extracorporeal blood circulation.     

Controlled synthesis of stable poly(vinyl formamide-co-vinyl amine)/silica hybrid particles by interfacial post-cross-linking reactions

 The chemical synthesis and the physicochemical properties of stable poly(vinyl formamide-co-vinyl amine)/silica hybrid particles are presented. Copolymers of poly(vinyl formamide) (PVFA) and poly(vinyl amine) (PVAm) and their protonated forms were adsorbed onto silica from aqueous solutions. The influences of the pH strength and the ion concentration of the aqueous solution as well as the copolymer composition (degree of hydrolyzation of PVFA), and the molecular mass on the adsorption process were investigated by electrokinetic measurements, potentiometric titration, and quantitative elemental analyses. Silica surface-charge neutralization is achieved at a pH strength above 10 for highly hydrolyzed (95%) PVFA polymers. Decreasing the amino content in the PVAm chain shifts successively both the point of zero charge and the isoelectric point to lower pH values. PVFA-co-PVAm layers onto silica are adsorbed weakly. To fix these layers irreversibly, cross-linking reactions with (4,4′-diisocyanate)diphenyl methane were carried out on the surface of solid PVFA-co-PVAm/silica hybrid particles suspended in acetone. The cross-linking reaction, which is connected with the conversion of amino groups, is also a tool to control the surface charge of the PVFA-co-PVAm/silica hybrids. X-ray photoelectron spectroscopy and solid-state ¹³C cross-polarization magic-angle spinning NMR spectroscopy were used to obtain information on the number of and the structure of the functionalized polyelectrolyte layers on silica. The success of cross-linking was also shown by the results of these spectroscopic methods. Received: 28 June 1999 /Accepted: 27 August 1999     

Surface modification of silica nanoparticles to reduce aggregation and nonspecific binding

In this article, a systematic study of the design and development of surface-modification schemes for silica nanoparticles is presented. The nanoparticle surface design involves an optimum balance of the use of inert and active surface functional groups to achieve minimal nanoparticle aggregation and reduce nanoparticle nonspecific binding. Silica nanoparticles were prepared in a water-in-oil microemulsion and subsequently surface modified via cohydrolysis with tetraethyl orthosilicate (TEOS) and various organosilane reagents. Nanoparticles with different functional groups, including carboxylate, amine, amine/phosphonate, poly(ethylene glycol), octadecyl, and carboxylate/octadecyl groups, were produced. Aggregation studies using SEM, dynamic light scattering, and zeta potential analysis indicate that severe aggregation among amine-modified silica nanoparticles can be reduced by adding inert functional groups, such as methyl phosphonate, to the surface. To determine the effect of various surface-modification schemes on nanoparticle nonspecific binding, the interaction between functionalized silica nanoparticles and a DNA chip was also studied using confocal imaging/fluorescence microscopy. Dye-doped silica nanoparticles functionalized with octadecyl and carboxylate groups showed minimal nonspecific binding. Using these surface-modification schemes, fluorescent dye-doped silica nanoparticles can be more readily conjugated with biomolecules and used as highly fluorescent, sensitive, and reproducible labels in bioanalytical applications.     

Self-organized fluorescent nanosensors for ratiometric Pb2+ detection

Silica nanoparticles (60 nm diameter) doped with fluorescent dyes and functionalized on the surface with thiol groups have been proved to be efficient fluorescent chemosensors for Pb2+ ions. The particles can detect a 1 microM metal ion concentration with a good selectivity, suffering only interference from Cu2+ ions. Analyte binding sites are provided by the simple grafting of the thiol groups on the nanoparticles. Once bound to the particles surface, the Pb2+ ions quench the emission of the reporting dyes embedded. Sensor performances can be improved by taking advantage of the ease of production of multishell silica particles. On one hand, signaling units can be concentrated in the external shells, allowing a closer interaction with the surface-bound analyte. On the other, a second dye can be buried in the particle core, far enough from the surface to be unaffected by the Pb2+ ions, thus producing a reference signal. In this way, a ratiometric system is easily prepared by simple self-organization of the particle components.     

Directed self-assembly of functionalized silica nanoparticles on molecular printboards through multivalent supramolecular interactions

Silica nanoparticles functionalized with beta-cyclodextrin (CD) host molecules (5) have been prepared by reacting carboxylic active ester-terminated silica nanoparticles (4) with CD heptamine. Silica nanoparticles functionalized with glucosamine (6), having similar surface properties as 5 but lacking the host-guest recognition motif, were used to perform blank experiments. The CD-functionalized silica nanoparticles 5 were determined by TEM to be 55 +/- 6 nm in size. They exhibited pH-dependent aggregation, which is explained by the presence of free amino and carboxylic acid groups on the particle surface, which was corroborated by zeta potential measurements. The functionalization with CD was further confirmed by host-guest studies in solution and at CD-functionalized silicon substrates. The addition of an adamantyl-terminated dendrimer, capable of multivalent host-guest binding with CD, led to strong aggregation of the CD particles 5, but not of the glucosamine-functionalized 6. Furthermore, 5 gave strong adsorption to CD monolayers on silicon onto which adamantyl-terminated dendrimers were adsorbed, whereas 6 did not. The good discrimination between dendrimer-covered and uncovered areas of the CD monolayer substrates allowed the directed self-assembly of the silica particles 5 onto dendrimer-patterned areas created by microcontact printing.     

Surface properties of submicrometer silica spheres modified with aminopropyltriethoxysilane and phenyltriethoxysilane

The surface of submicrometer silica spheres are modified with aminopropyl and phenyl groups through a one-step process. Various experimental techniques, i.e., scanning electron microscopy (SEM), quasi-elastic light scattering (QELS), differential scanning calorimetry (DSC), thermogravimetry (TG), zeta potential measurement, nitrogen sorption, and water vapor and organic dye adsorption are used to comprehensively characterize the pure (TEOS particles) and modified silica particles. The SEM micrographs of the particles demonstrate that the modified particles are spherical with uniform size and shape. The particles modified with aminopropyl groups (APTES particles) show the highest isoelectric point (IEP) and the highest weight loss at 780 degrees C because of the basic nature of aminopropyl groups and the higher reactivity of aminopropyltriethoxysilane. The particles modified with the phenyl groups (PhTES particles) show the lowest water vapor adsorption because their surface is more hydrophobic than that of TEOS and APTES particles. The organic dye (brilliant blue FCF or BBF) adsorption experiments demonstrate that the adsorption capacity of the particles increases greatly after acidification. This is caused by the protonation of silanol groups and amine groups on the particle surface, which presents an enhanced electrostatic attraction with BBF anions. The APTES particles exhibit the highest dye adsorption due to the hydrophobic attractions and the enhanced electrostatic attractions from aminopropyl groups.     

Conjugated polyelectrolyte-grafted silica microspheres

A direct method for preparation of conjugated polymer-grafted silica particles is reported. Silica particles (0.3 and 5 mum diameter) are treated with a 3-(trimethoxysilyl)propylamine derivative that is functionalized with an aryl iodide unit. A solution step-growth polymerization reaction is performed in solution that contains a dispersion of the aryl iodide-functionalized particles. The reaction is a Pd(0)-catalyzed (Sonogashira) A-B-type polymerization of an oligo(ethylene glycol)-fuctionalized diiodobenzene and a bis(propyloxy)sulfonate-substituted diethynylbenzene. The overall process affords silica particles that feature a surface graft layer of an anionic poly(phenylene ethynylene)-type conjugated polyelectrolyte. The particle surface modification process was monitored by infrared (FTIR) spectroscopy, and the polymer-grafted silica particles were characterized by thermogravimetric analysis, scanning and transmission electron microscopy, confocal fluorescence microscopy, and absorption and fluorescence spectroscopy. The conjugated polyelectrolyte-grafted silica particles are highly fluorescent, and a Stern-Volmer quenching study of the particles' fluorescence with electron-transfer- and energy-transfer-type quenchers shows that the quenching response depends on the type of quenching mechanism.     

ATRP技术用于热敏性高聚物在硅胶表面的接枝

在超细硅胶表面引入原子转移自由基聚合(ATRP)的引发基团,通过ATRP技术使N-异丙基丙烯酰胺(NIPAM)在硅胶表面接枝聚合,合成得到了具有温敏性的核-壳复合微粒.通过FTIR,TG,EA,SEM,DSC等分析方法对接枝前后的复合粒子进行了分析与表征,结果证明聚N-异丙基丙烯酰胺(PNIPAM)接在了硅胶表面.TG分析得出PNIPAM在硅胶表面的接枝率达到25.2%;DSC分析表明复合硅胶具有温度敏感性,在34.1℃时发生相转变行为;GPC分析得出从复合硅胶表面"劈下"的聚合物PNIPAM的数均分子量约为8000,分子量分布为1.06.复合微粒表面均匀平坦,显示出活性聚合的优越性.     

Novel fluorescent silica nanoparticle probe for ultrasensitive immunoassays

The preparation and utilization of a novel particulate label based on fluorescent hybrid silica (FHS) nanoparticles are reported in this article. These nanoparticles have shown several unique advantages over existing dye molecules, quantum dots, and latex-based fluorescent particles in easy preparation, good photostability and high sensitivity. A high molar ratio of the fluorescent molecules present in the core to biomolecules on the particle surface was achieved by using the well-developed silica surface immobilization chemistry for biomolecular linking. A fluoroimmunoassay method for detecting trace level Hepatitis B Surface Antigen (HBsAg) was developed. The calibration graph for HBsAg was linear over the range 0.5-220 ng/ml with a detection limit of 0.1 ng/ml. The sensitivity is greatly increased when compared with the corresponding immunoassay performed with direct fluorophore labeling. The present work shows that these FHS nanoparticles are high-quality markers for biochemical assays.     

Light‐Triggered,Spatially Localized Chemistry by Photoinduced Electron Transfer

It is of immense interest to exert spatial and temporal control of chemical reactions. It is now demonstrated that irradiation can trigger reactions specifically at the surface of a simple colloidal construct, obtained by adsorbing polyethyleneimine on fluorescent colloidal particles. Exciting the fluorescent dye in the colloid affords photoinduced electron transfer to spatially proximal amine groups on the adsorbed polymer to form free radical ions. It is demonstrated that these can be harnessed to polymerize acrylic acid monomer at the particle surface, or to break up colloidal assemblies by cleaving a cross‐linked polymer mesh. Formation of free radical ions is not a function of the size of the colloid, neither is it restricted to a specific fluorophore. Fluorophores with redox potentials that allow photoinduced electron transfer with amine groups show formation of free radical ions.     

Light‐Triggered,Spatially Localized Chemistry by Photoinduced Electron Transfer

It is of immense interest to exert spatial and temporal control of chemical reactions. It is now demonstrated that irradiation can trigger reactions specifically at the surface of a simple colloidal construct, obtained by adsorbing polyethyleneimine on fluorescent colloidal particles. Exciting the fluorescent dye in the colloid affords photoinduced electron transfer to spatially proximal amine groups on the adsorbed polymer to form free radical ions. It is demonstrated that these can be harnessed to polymerize acrylic acid monomer at the particle surface, or to break up colloidal assemblies by cleaving a cross‐linked polymer mesh. Formation of free radical ions is not a function of the size of the colloid, neither is it restricted to a specific fluorophore. Fluorophores with redox potentials that allow photoinduced electron transfer with amine groups show formation of free radical ions.     

Adsorption of poly(vinyl formamide-co-vinyl amine) onto silica particle surfaces and stability of the formed hybrid materials

In order to produce silica/polyelectrolyte hybrid materials the adsorption of the polyelectrolyte poly(vinyl formamide-co-vinyl amine), P(VFA-co-VAm) was investigated. The adsorption of the P(VFA-co-VAm) from an aqueous solution onto silica surface is strongly influenced by the pH value and ionic strength of the aqueous solution, as well as the concentration of polyelectrolyte. The adsorption of the positively charged P(VFA-co-VAm) molecules on the negatively charged silica particles offers a way to control the surface charge properties of the formed hybrid material. Changes in surface charges during the polyelectrolyte adsorption were studied by potentiometric titration and electrokinetic measurements. X-ray photoelectron spectroscopy (XPS) was employed to obtain information about the amount of the adsorbed polyelectrolyte and its chemical structure. The stability of the adsorbed P(VFA-co-VAm) was investigated by extraction experiments and streaming potential measurements. It was shown, that polyelectrolyte layer is instable in an acidic environment. At a low pH value a high number of amino groups are protonated that increases the solubility of the polyelectrolyte chains. The solvatation process is able to overcompensate the attractive electrostatic forces fixing the polyelectrolyte molecules on the substrate material surface. Hence, the polyelectrolyte layer partially undergoes dissolving process.     

Synthesis of monodisperse fluorescent core-shell silica particles using a modified Stober method for imaging individual particles in dense colloidal suspensions

Core-shell silica particles, with a diameter of 1.5 mum, containing a dye fluorescein isothiocyanate (FITC), are synthesized by the hydrolysis and condensation of tetraethylorthosilicate (TEOS). Sodium dodecyl sulfate (SDS) is added to synthesize fluorescent core particles with the diameter of approximately 1 mum. In the addition of SDS, the surface charge reduced by counterions (Na+) of the surfactant leads to a higher degree of aggregation of the primary particles and the formation of larger secondary particles. The particle growth kinetics confirms the aggregation growth model for the synthesis of monodisperse silica particles, and also shows the dependence of final particle size on colloidal stability resulting from the addition of SDS. Light and X-ray scattering data reveal that the final particles have compactly packed structures with smooth surfaces. The seeded growth technique is then used to form a silica shell layer on the fluorescent core. The added amount of water and NH4OH has significant effects on shell formation. Finally, the final core-shell silica particles are modified by chemisorption of octadecanol at the surface to be dispersed in organic solvents. Octadecyl-coated silica particles are sterically stabilized in silica index-matching solvents such as chloroform and hexadecane to directly image separate particles using confocal microscopy. In chloroform, the organophilic silica particles disperse well, whereas in hexadecane they form a volume-filling gel structure at room temperature.     

Synthesis and Characterization of Silica Gel Particles Functionalized with Bioactive Materials

Bioactive materials (having an amino acid, Ac-Tryptophan, A or a peptide, Ac-Trp-Ala-Ala, B) were anchored onto silica particles. A photoactive linker (N,N′-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (DPN)) was initially attached to the particles and next the amino acids were bound by using both Zr/P chemistry and 1,3-diisopropylcarbodiimide/1-hydroxybenzotriazole coupling. In A derivative extensive complexation of Trp with DPN was observed. Photolysis studies showed the presence of excited state reactions on the silica particles, moreover the radical species (DPN^.−, TrpH^.+, Trp^.) remained alive ∼100 times longer on the particle surface than in solution. These studies show that the formation rate of these radicals is a function of the supramolecular structures of the peptide and of the amino acid derivative on the silica particle. This paper was presented in the 5th Brazilian Meeting on Adsorption, held at Natal, Brazil, 18–21 July, 2004.     

Synthesis of poly(ethylene glycol) (PEG)-grafted colloidal silica particles with improved stability in aqueous solvents

The known grafting procedures of colloidal silica particles with poly(ethylene glycol) (PEG) lead to grafting layers that detach from the silica surface and dissolve in water within a few days. We present a new grafting procedure of PEG onto silica with a significant improvement of the stability of the grafting layers in aqueous solvents. Moreover, the procedure avoids any dry states or other circumstances leading to strong aggregation of the particles. To achieve the improved water stability, St?ber silica particles are first pre-coated with a silane coupling agent (3-aminopropyl)triethoxysilane (APS) to incorporate active amine groups. The water solubility of the pre-coating layer was minimized using a combination of APS with bis-(trimethoxysilylpropyl)amine (BTMOSPA) or bis-(triethoxysilyl)ethane (BTEOSE). These pre-coated particles were then reacted with N-succinimidyl ester of mono-methoxy poly(ethylene glycol) carboxylic acid to form PEG-grafted silica particles. The particles form stable dispersions in aqueous solutions as well as several organic solvents.     

Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption

Control over selective recognition of biomolecules on inorganic nanoparticles is a major challenge for the synthesis of new catalysts, functional carriers for therapeutics, and assembly of renewable biobased materials. We found low sequence similarity among sequences of peptides strongly attracted to amorphous silica nanoparticles of various size (15-450 nm) using combinatorial phage display methods. Characterization of the surface by acid base titrations and zeta potential measurements revealed that the acidity of the silica particles increased with larger particle size, corresponding to between 5% and 20% ionization of silanol groups at pH 7. The wide range of surface ionization results in the attraction of increasingly basic peptides to increasingly acidic nanoparticles, along with major changes in the aqueous interfacial layer as seen in molecular dynamics simulation. We identified the mechanism of peptide adsorption using binding assays, zeta potential measurements, IR spectra, and molecular simulations of the purified peptides (without phage) in contact with uniformly sized silica particles. Positively charged peptides are strongly attracted to anionic silica surfaces by ion pairing of protonated N-termini, Lys side chains, and Arg side chains with negatively charged siloxide groups. Further, attraction of the peptides to the surface involves hydrogen bonds between polar groups in the peptide with silanol and siloxide groups on the silica surface, as well as ion-dipole, dipole-dipole, and van-der-Waals interactions. Electrostatic attraction between peptides and particle surfaces is supported by neutralization of zeta potentials, an inverse correlation between the required peptide concentration for measurable adsorption and the peptide pI, and proximity of cationic groups to the surface in the computation. The importance of hydrogen bonds and polar interactions is supported by adsorption of noncationic peptides containing Ser, His, and Asp residues, including the formation of multilayers. We also demonstrate tuning of interfacial interactions using mutant peptides with an excellent correlation between adsorption measurements, zeta potentials, computed adsorption energies, and the proposed binding mechanism. Follow-on questions about the relation between peptide adsorption on silica nanoparticles and mineralization of silica from peptide-stabilized precursors are raised.     

Asymmetrically modified silica particles: a simple particulate surfactant for stabilization of oil droplets in water

Spherical silica particles that are able to assemble at a phase boundary of a dual-phase mixture of water and an immiscible organic solvent were prepared by a partial modification of their surface hydroxyl groups with an alkylsilylation agent. Scanning electron microscopic observation of these particles in which their remaining surface hydroxyl groups had been selectively modified with colloidal gold particles revealed that each particle has an asymmetric surface structure: one side of the surface is hydrophilic and the other is hydrophobic. We found that these particles could form a micellar structure in water in the presence of an organic solution of a toluene/polystyrene mixture. The micellar structure was evidenced by formation of golf-ball-like polystyrene particles with dimples imprinting morphologies of the hydrophobic part of modified silica particles.