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.
Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution format. We demonstrated a simple, high-throughput, and non-PCR based DNA assay for quantifying antibiotic resistance gene tetQ. Fe3O4/Eu:Gd2O3 nanoparticles (NPs) synthesized by spray pyrolysis were biofunctionalized by passive adsorption of NeutrAvidin. Following immobilization of biotinylated probe DNA on the particles' surfaces, target dsDNA and signaling probe DNA labeled with Cy3 were hybridized with NPs-probe DNA. Hybridized DNA complexes were separated from solution by a magnet, while non-hybridized DNA remained in solution. A linear quantification (R2 = 0.99) of a target tetQ gene was achieved based on the normalized fluorescence (Cy3/NPs) of DNANP hybrids. A real-time qPCR assay was used for evaluation of the NPs assay sensitivity and range of quantification. The quantity of antibiotic resistance tetQ genes in activated sludge microcosms, with and without addition of tetracycline or triclosan has been determined, indicating the potential of the optimized assay for monitoring the level of antibiotic resistance in environmental samples. In addition, the tetQ gene copy numbers in microcosms determined by NPhybridization were well correlated with the numbers measured by real-time qPCR assay (R2 = 0.92).
Fluorescence techniques rely on measurement of relative fluorescence units and require calibration to obtain reliable
and comparable quantitative data. Fluorescent immunoassays are a very sensitive and convenient method of choice
for rapid detection of biotoxins, such as ricin. Here we present the application of magnetic luminescent nanoparticles
(MLNPs) with a magnetic core of Fe3O4 and a fluorescent shell of Eu:Gd2O3 as carriers for a nanobead-immunoassay for the detection of ricin with internal calibration. A sandwich immunoassay for ricin was performed
on the surface of the MLNPs. The particles were functionalized with capture polyclonal antibodies. Anti-ricin
antibodies labeled with Alexa Fluor dye were used as the detecting antibodies. After magnetic extraction, the
amount of ricin bound to the particle surface was quantified and related to the fluorescence signal of the
nanoparticles. In this new platform, the MLNPs have three main functions: (1) a probe for the specific extraction of
the target analyte from the sample; (2) a carrier in the quantitative immunoassay with magnetic separation; and (3)
an internal standard in the fluorescence measurement of the dye reporter. The MLNPs serve as an internal control
for the total analysis including extraction and assay performance. This approach eliminates the experimental error
inherent in particle extraction and measurement of absolute organic dye fluorescence intensities. All fluorescent
measurements were performed in a microplate reader. The standard curve for ricin had a dynamic range from 20
ng/ml to 100 μg/ml with a detection limit of 5 ng/ml. The configuration that has been developed can be easily
adapted to a high throughput miniaturized system.
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 quantum dots (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.
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.
Fluorescent properties and low production cost makes lanthanide oxide nanoparticles attractive labels in biochemistry. Nanoparticles with different fluorescent spectra were produced by doping of oxides such as Y2O3 and Gd2O3 with different lanthanide ions (Eu, Tb, Sm) giving the possibility for multicolor labeling. Protein microarrays have the potential to play a fundamental role in the miniaturization of biosensors, clinical immunological assays, and protein-protein interaction studies. Here we present the application of fluorescent lanthanide oxide nanoparticles as labels in microarray-based immunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to the highly potent insecticides pyrethroids. A novel polymer-based protocol was developed for biochemical functionalization of the nanoparticles. Microarrays of antibodies were fabricated by microcontact printing in line patterns onto glass substrates and immunoassays were successfully performed using the corresponding functionalized nanoparticles. The applicability of the fluorophore nanoparticles as reporters for detection of antibody-antigen interactions has been demonstrated for phenoxybenzoic acid (PBA)/anti-PBA IgG. The sensitivity of the competitive fluorescent immunoassay for PBA was similar to that of the corresponding ELISA.
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.
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.
Nanoparticles made of lanthanide oxides are promising fluorophores as a new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long lifetime and lack of photobleaching. We demonstrate for first time the application of these nanoparticles to the visualization of protein micropatterns. Europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles were synthesized by spray pyrolysis and were characterized by means of laser-induced fluorescent spectroscopy and TEM. Their main emission peak is at 612 nm. And their size distribution is from 5 nm to 500 nm. The nanoparticles were coated with avidin through physical adsorption. Biotinylated Bovine Serum Albumin (BSA-b) was patterned on a silicon wafer using a micro-contact printing technique. The BSA-b - patterned wafer was incubated in a solution containing the avidin-coated nanoparticles. The specific interaction between biotin and avidin was studied by means of fluorescent microscopy and atomic-force microscopy (AFM). The fluorescent microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process - non-specific binding of the avidin-coated nanoparticles to bare substrate was negligible. The fluorescent pattern did not suffer any photobleaching during the observation process which demonstrates the suitability of Eu:Gd2O3 nanoparticles as fluorescent labels with extended excitation periods - organic dyes, including chelates, suffer bleaching over the same period. More detailed studies were preformed using AFM at a single nanoparticle level. The specific and the non-specific binding densities of the particles were qualitatively evaluated.
The use of polystyrene nanoparticles with europium chelate has been demonstrated as fluorescent reporters in an immunoassay for atrazine. The limit of detection with the nanoparticles was similar to that achieved with a conventional ELISA. It was shown that as the particle size decreased the time required for binding decreased and the sensitivity of the assay increased. This suggests that the use of smaller particles would greatly speed up the reaction and simultaneously increase sensitivity. However, the detection system used sets limits to the particle size as well. There is clearly a point where our detection system would not be sensitive enough to detect the emission from small particles. Therefore, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size.
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.
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