A microemulsion preparation of nanoparticles of europium in silica with luminescence enhancement using silver.

A facile one-pot microemulsion method has been developed for the synthesis of spherical silver core-silica shell (Ag@SiO2) nanoparticles with europium chelates doped in the shell through a silane agent. The method is significantly more straightforward than other extant methods. Measurements of the luminescent emissions from the Ag@SiO2 nanoparticles, in comparison with control silica nanoparticles without silver cores, showed that the presence of the silver cores can increase the fluorescence intensity approximately 24-fold and decrease the luminescence lifetime. This enhancement offers a potential increase in overall particle detectability with increased fluorophore photostability.

Synthesis and characterization of multifunctional silica core-shell nanocomposites with magnetic and fluorescent functionalities.

Multifunctional core-shell nanocomposites with a magnetic core and a silica shell doped with lanthanide chelate have been prepared by a simple method. First, citric acid-modified magnetite nanoparticles were synthesized by a chemical coprecipitation method. Then the magnetite nanoparticles were coated with silica shells doped with terbium (Tb(3+)) complex by a modified Stöber method based on hydrolyzing and condensation of tetraethyl orthosilicate (TEOS) and a silane precursor. These multifunctional nanocomposites are potentially useful in a variety of biological areas such as bio-imaging, bio-labeling and bioassays because they can be simultaneously manipulated with an external magnetic field and exhibit unique phosphorescence properties.

Synthesis and bio-functionalization of multifunctional magnetic Fe(3)O(4)@Y(2)O(3):Eu nanocomposites.

A facile homogenous precipitation method has been developed for the synthesis of multifunctional, magnetic, luminescent nanocomposites with Fe(3)O(4) nanoparticles as the core and europium-doped yttrium oxide (Y(2)O(3):Eu) as the shell. The nanocomposites showed both super-paramagnetic behavior and unique europium fluorescence properties with high emission intensity. Their surface has been modified with a bifunctional ligand, p-aminobenzoic acid (PABA), and further biofunctionalized with biotin; the nanocomposites showed specific targeting for avidin-coupled polystyrene beads.

Luminescent lanthanide nanoparticles as labels in DNA microarrays for quantification of methyl tertiary butyl ether degrading bacteria.

We report application of lanthanide nanoparticles for DNA quantification in a microarray platform as a substitute for conventional organic fluorophores. A non-PCR based DNA microarray assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether (MTBE) was demonstrated. Probe DNA was immobilized on a glass surface, hybridized with biotinylated target DNA and subsequently incubated with Neutravidin-biofunctionalized nanoparticles. The fluorescence spot intensities, measured by a commercial laser scanner, show a linear relationship (R2 = 0.98) with bacterial 16S rDNA over a range of target DNA concentrations, while the background fluorescence remained low. In addition, nanoparticles fluorescence shows a stronger intensity than Quasar570 (Cy3). Present sensitivity of the assay is 10 pM of target DNA. The selectivity of the DNA-nanoparticle-probes to discriminate a non-target DNA with two base pairs mismatch in the 16S rDNA gene sequence was shown. The use of Eu:Gd2O3 nanoparticles as biolabels provides a relatively non-toxic, inexpensive, rapid and sensitive alternative to the materials currently used in DNA microarrays.

Inorganic lanthanide nanophosphors in biotechnology.

Recently different types of fluorescent nanoparticles and other nanostructures have been promoted as alternatives for the fluorescent organic dyes that are traditionally used in biotechnology. Quantum dots, dye-doped polymer and silica particles have found many applications in biochemical protocols and are extensively discussed in the literature. Nanostructures based on inorganic phosphors (nanophosphors) are a new emerging class of materials with unique properties that make them very attractive for bio-application. Some results for the successful application of nanophosphors in biochemical applications have been reported. In this review we summarize the types of materials, their properties that are relevant to bio-applications, and the current status of their implementation in biotechnology.

Rapid and quantitative DNA analysis of genetic mutations for polycystic kidney disease (PKD) using magnetic/luminescent nanoparticles.

Rapid and accurate detection of genetic mutations based on nanotechnology would provide substantial advances in detection of polycystic kidney disease (PKD), a disease whose current methods of detection are cumbersome due to the large size and duplication of the mutated gene. In this study, a nanotechnology-based DNA assay was developed for detection of SNPs (single nucleotide polymorphisms) in a feline autosomal dominant PKD (ADPKD) model which can readily be adapted to diagnosis of human ADPKD type 1. Europium and terbium phosphors were doped into gadolinium crystal hosts with a magnetic core, providing stable luminescence and the possibility of magnetic manipulations in a solution-based assay. A hybridization-in-solution DNA assay was optimized for feline PKD gene SNP detection using genomic DNA extracted from feline kidney tissue and blood. This assay showed a substantial differentiation between PKD and control specimens. The nanotechnology-based DNA assay is attractive from the viewpoint of rapid availability, simple methodology, and cost reduction for clinical use to detect mutations involved in human ADPKD and other genetic diseases.

Quantitative DNA hybridization in solution using magnetic/luminescent core-shell nanoparticles.

Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution approach. We demonstrated a rapid, simple, and non-polymerase chain reaction-based DNA hybridization-in-solution assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether. Fe3O4/Eu:Gd2O3 core-shell nanoparticles synthesized by spray pyrolysis were biofunctionalized with NeutrAvidin. Following immobilization of a biotinylated probe DNA on the particles' surfaces via passive adsorption, target DNA labeled with fluorescein isothiocyanate was hybridized with probe DNA. The hybridized DNA complex was separated from solution with a magnet, while nonhybridized DNA remained in solution. The normalized fluorescence (fluorescein isothiocyanate/nanoparticles) measured with a spectrofluorometer indicated a linear quantification (R(2)=0.98) of the target bacterial 16 S rDNA. The rate of hybridization increased concurrently with the target DNA concentration. In addition, this approach differentiated between the signal outputs from perfectly complementary target and two-base mismatched target DNA in a range of concentrations, showing the specificity of the assay and the possibility for environmental applications.

Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration.

Suspension arrays present a promising tool for multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic Co:Nd:Fe(2)O(3)/luminescent Eu:Gd(2)O(3) core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay. Their optical properties enable the internal calibration of the detection system. The multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) are used as reporters. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. The concept is demonstrated for a simultaneous immunoassay for three model proteins (human, rabbit, and mouse IgGs). The method uses a standard bench plate reader. It can be applied to disease diagnostics and to the detection of biological threats.

Zeta-potential Analyses using Micro Electrical Field Flow Fractionation with Fluorescent Nanoparticles.

Increasingly growing application of nanoparticles in biotechnology requires fast and accessible tools for their manipulation and for characterization of their colloidal properties. In this work we determine the zeta-potentials for polystyrene nanoparticles using micro electrical field flow fractionation (mu-EFFF) which is an efficient method for sorting of particles by size. The data obtained by mu-EFFF were compared to zeta potentials determined by standard capillary electrophoresis. For proof of concept, we used polystyrene nanoparticles of two different sizes, impregnated with two different fluorescent dyes. Fluorescent emission spectra were used to evaluate the particle separation in both systems. Using the theory of electrophoresis, we estimated the zeta-potentials as a function of size, dielectric permittivity, viscosity and electrophoretic mobility. The results obtained by the mu-EFFF technique were confirmed by the conventional capillary electrophoresis measurements. These results demonstrate the applicability of the mu-EFFF method not only for particle size separation but also as a simple and inexpensive tool for measurements of nanoparticles zeta potentials.

Magnetic/luminescent core/shell particles synthesized by spray pyrolysis and their application in immunoassays with internal standard.

Many types of fluorescent nanoparticles have been investigated as alternatives to conventional organic dyes in biochemistry; magnetic beads also have a long history of biological applications. In this work we apply flame spray pyrolysis in order to engineer a novel type of nanoparticle that has both luminescent and magnetic properties. The particles have magnetic cores of iron oxide doped with cobalt and neodymium and luminescent shells of europium-doped gadolinium oxide (Eu:Gd(2)O(3)). Measurements by vibrating sample magnetometry showed an overall paramagnetic response of these composite particles. Luminescence spectroscopy showed spectra typical of the Eu ion in a Gd(2)O(3) host-a narrow emission peak centred near 615 nm. Our synthesis method offers a low-cost, high-rate synthesis route that enables a wide range of biological applications of magnetic/luminescent core/shell particles. Using these particles we demonstrate a novel immunoassay format with internal luminescent calibration for more precise measurements.

Optical characterization of eu-doped and undoped gd(2)o(3) nanoparticles synthesized by the hydrogen flame pyrolysis method.

Rare-earth-doped nanoparticles are promising materials for fluorescent labeling, as they are characterized by a high Stokes shift, narrow emission spectra, long lifetimes, minimized photobleaching, and low toxicity. We examined the structural and optical properties of europium-doped gadolinium oxide nanoparticles synthesized by the flame pyrolysis method, with specific emphasis on full spectral characterization and fluorescence kinetics. The emission-excitation characterization revealed the presence of predominantly monoclinic but also highly luminescent cubic phases with a prominent oxygen-to-europium charge-transfer band in the 230-260 nm range. A broad emission band in the visible region, corresponding to a similar band in undoped Gd(2)O(3), related to the matrix surface defects, was observed in time-gated spectroscopy of doped nanopowders. All of the examined nanopowders showed very short decay components, on the order of 2 ns, and much longer millisecond decay times characteristic of lanthanide ions. At intermediate times, on the order of 20-100 ns, a complex behavior of the decay was observed, indicative of progressive energy transfer to the lanthanide ion, which varied with different intrashell transitions. Structural characterization data by means of XRD measurements allowed for unambiguous determination of the Eu:Gd(2)O(3) crystallographic structure and cell dimensions to be consistent with a predominantly monoclinic phase.

Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing.

Lanthanide oxide nanoparticles are promising luminescent probes in bioanalysis, because of their unique spectral properties, photostability, and low-cost synthesis. We report for the first time the application of europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles to the optical imaging of antibody micropatterns. The nanoparticles were synthesized by spray pyrolysis and coated with antibody (IgG) molecules by physical adsorption. Our experiments showed that the Eu:Gd2O3 is a good biocompatible solid support for antibody immobilization. The antibodies (anti-rabbit IgG) immobilized on the nanoparticles had excellent biological activity in the specific recognition reaction with rabbit IgG patterned in line strips (10 micromx10 microm) on a glass substrate by use of a micro-contact printing technique. The specific immunoreaction was confirmed by two independent microscopic techniques-fluorescence and scanning electron microscopy (SEM). Both microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process with negligible non-specific binding. The nanoparticles can be used as fluorescent markers in a variety of immunosensing applications in a microscale format.

Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated Eu:Gd2O3 nanoparticles as fluorescent labels.

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photobleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulated particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 x 10 microm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 microg L(-1) PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening.

Application of luminescent Eu:Gd2O3 nanoparticles to the visualization of protein micropatterns.

Nanoparticle phosphors made of lanthanide oxides are a promising new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long luminescence lifetime, and good photostability. We demonstrate the application of these nanoparticles to the visualization of protein micropatterns. Luminescent europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles are synthesized by spray pyrolysis. The size distribution is from 5 to 200 nm. The particles are characterized by means of laser-induced fluorescent spectroscopy and transmission electron microscopy (TEM). The main emission peak is at 612 nm. The nanoparticles are coated with avidin through physical adsorption. biotinylated bovine serum albumin (BSA-b) is patterned on a silicon wafer using a microcontact printing technique. The wafer is then incubated in a solution of avidin-coated nanoparticles. Fluorescent microscopic images reveal that the nanoparticles are organized onto designated area, as defined by the microcontact printing process. The luminescent nanoparticles do not suffer photobleaching during the observation, which demonstrates their suitability as luminescent labels for fluorescence microscopy studies. More detailed studies are preformed using atomic-force microscopy (AFM) at a single nanoparticle level. The specific and the nonspecific binding densities of the particles are qualitatively evaluated.