Dosi Dosev

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.

Fluorescence upconversion in Sm-doped Gd2O3

We report the observation of efficient fluorescence upconversion in Sm-doped Gd2O3 nanopowders prepared by the spray pyrolysis method. The blue upconversion emission was observed with low-power continuous-wave excitation at 514, 561, 594, and 633nm and with a pulsed femtosecond at 710nm, in a laser scanning confocal microscope. This result indicates that Sm-doped Gd2O3 has the potential as a fluorescent label that may be excited in red, yellow, and green with blue emission.

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.

Quantum Dots as Reporters in Multiplexed Immunoassays for Biomarkers of Exposure to Agrochemicals

Abstract The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3‐phenoxybenzoic acid (PBA) and atrazine‐mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using QDs (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.

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

AbstractRapid 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. FigureSchematic diagram of PKD (Polycystic Kidney Disease) SNPs detection assay using feline genomic DNA in magnetic/luminescent nanoparticle-based DNA hybridization

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.

Time-gated detection of europium nanoparticles in a microchannel-based environmental immunoassay

Using MEMs (Micro Electro Mechanical system) fabrication techniques, it is possible make a micro-sized instrument for optical detection of trace amounts of chemical species in aqueous solutions. The red-emitting Eu2O3 nanoparticle is suitable for a biolabel for such species because of its long fluorescence lifetime and narrow emission bandwidth. The europium nanoparticles are excited by a laser pulse. Their long-lived emission allows the detected signal to be separated from the laser pulse both spectrally and temporally. The background signal can also be eliminated in this manner. The instrument we present, is assembled with silicon and glass layers with a 200μm deep channel. A Nd:YAG pumped optical parametric oscillator (OPO) is used as the excitation source. The measurement sensitivities using two detectors, a PMT (Photo Multiplier Tube) and an APD (Avalanche Photodiode), are compared. The underlying fundamental principles and the micro-fabrication steps for the instrument and detection are discussed.

Chemical and biological sensing through optical resonances in pendant droplets

A microdroplet can act as a high quality factor optical cavity that supports Morphology Dependent Resonances(MDRs). Enhanced radiative energy transfer through these optical resonances can also be utilized as a transduction mechanism for chemical and biological sensing. Enhancement in radiative energy transfer is observed when a donor/acceptor pair is present in the resonant medium of a microcavity. Here, we demonstrate avidin-biotin binding and its detection through a FRET pair as a potential application for ultra-sensitive detection for fluoroimmunoassays. The binding interaction between the biotinylated donor molecules and streptavidin-acceptor conjugate was used to observe the energy transfer between the dye pairs. The radial modes of MDRs extend to approximately 0.6 r0 inside the droplet. As a result, the fluorescent emission around the center is not coupled to the optical resonances losing sensitivity. To address this problem, we prepared water-in-oil emulsions of avidin and biotin containing solutions. The water phase contains the streptavidin-Alexa Fluor 610 and the oil phase contains biotinylated fluorescent bead. Streptavidin-biotin binding reaction occurs at the water-oil interface. The water phase accumulates at the droplet air interface due to higher specific density enhancing the resonance coupling. Water and oil phase are index-matched to avoid scattering problems. As a result, a large portion of the avidin-biotin complex was localized at the pendant droplet and air interface. Strong coupling of acceptor emission into optical resonances shows that the energy transfer is efficiently mediated through these resonances.

Fast and precise detection of ricin with microcapillary sensor system

Ricin is an easily available toxin which can be used as a bio-terror agent. Fast and inexpensive methods for its detection in different samples are needed. Recently we have developed a novel fluorescent sandwich immunoassay for ricin using magnetic-luminescent nanoparticles (MLNPs) as carriers in a microcapillary system for incubation and detection. Antiricin antibody coated MLNPs that were dispersed in buffer solution were introduced in the capillary tube and immobilized inside using an external electromagnet. Then the sample containing ricin was injected while the MLNPs were mixed by an alternating magnetic field. After the incubation, washing solution and secondary antibody conjugated with Alexa-fluorescent were injected into the capillary while the MLNPs were constantly mixed. After the final wash, the particles were immobilized for detection. The total analysis time was reduced to less than forty minutes which is about 8-10 fold improvement in comparison with the plate-based protocols. This system is promising for the development of a portable biosensor and can be used for the detection of other analytes of interest.

Spray pyrolysis synthesis of particles possessing magnetic and luminescent properties: application of magnetic/luminescent particles in immunoassays

Many types of fluorescent nanoparticles have been investigated as alternatives to conventional organic dyes in biochemistry. In addition, magnetic beads are another type of particle that 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:Gd2O3). Measurements on a Vibrating Sample Magnetometer showed an overall paramagnetic response of these composite particles. Fluorescence spectroscopy showed spectra typical of the Eu ion in a Gd2O3 host; a narrow emission peak centered near 615 nm. Our synthesis method offers low-cost, high-rate synthesis allowing a wide range of biological applications of magnetic/fluorescent core/shell particles. We demonstrate an immunoassay using the magnetic and fluorescent properties of the particles for separation and detection purposes.