NIR-to-visible up-conversion nanomaterials have been investigated in many promising applications including nextgeneration displays, solar cells, and biological labels. When doped with different trivalent lanthanide ions, NaYF4 nanoparticles can produce up-converted emission from visible to infra-red wavelengths. However, the quantum yield of this class of materials is low. Noble metals in the vicinity of the phosphor can increase the phosphorescence by local field enhancement due to plasmonic resonances, and by modification of the radiative rate of the phosphor. Most previous studies have investigated the phenomenon by placing nanophosphors onto a metal substrate, or by fabrication of nano structures with spacers such as polymers, dielectric materials (silica). By contrast, we have studied the interaction between the luminescence and the surface plasmon using a core-shell type nanostructure where a uniform shell of silver is shown to grown on doped-NaYF4 nanophosphors by Ostwald ripening. We further demonstrate the proximity effect of metal-enhanced luminescence by exciting an undoped NaYF4 shell. The result shows a significant synergistic enhancement of up-conversion luminescence due to the active shell as spacer layer. In addition, we have shown this novel nanostructure may be useful in surface-enhanced Raman spectroscopy (SERS).
Some lanthanide-doped nanoparticles can absorb X-ray radiation and emit in the visible to near infrared spectrum via a down-conversion mechanism, making them a potentially valuable agent for in vivo imaging studies. We have studied a series of Gd3+ and Eu3+compositions in lanthanide fluorides to optimize the emission from Eu3+ upon X-ray excitation. The optimum concentration of Eu3+ that produced the most intense emission in NaGdF4 was found to be 15% molar concentration. The impact of the crystallographic phases (i.e. cubic or hexagonal) on the optical emission was investigated. Furthermore, an attempt to include a sensitizer (i.e Ce3+) in NaGdF4:Eu resulted in a reduction in the emission following X-ray excitation. A surface coating of NaGdF4:Eu nanoparticles with a gold shell showed a similar decrease in luminescence intensity by a factor of two although the gold shell offers other advantages in biomedical applications.
Up-converting nanophosphors consisting of Er activator with Yb sensitizer in a NaYF4 matrix have been studied for
heating and temperature sensing. We show the response of the nanothermometer to a pump laser operating at 1064 nm
with a pulse width of 5-7 nS with a 20 Hz repetition rate. The heating pulse (pump) is probed by the change in the ratio
of the two characteristic green emissions centered around 525 and 545 nm. A quasi continuous probe laser operating at
80 MHz and 980 nm is employed to study the effect of the pump laser on the phosphor. The emission is characterized by
a spectrophotometer attached to a gated intensifier and a charge coupled device. The time gated measurement shows that
the heating produced by 1064 nm pulse is easily resolved with time gating, and the read-out of temperature is deciphered
based on the temperature calibration performed with the emission lines. It was found that an increasing the energy of the
heating pulse caused a drop in the total green intensity, which had a direct correlation to the increase in the temperature.
The signal transduction of the thermal characteristics of the phosphor was delayed in time from the arrival of the heating
pulse by about two-three decades in the unit of microseconds. The gold coated phosphor also showed a response to the
heating pulse, but the enhancement of both the 525 and 545 nm emissions from the gold shell, to varying extents made
the deconvolution of temperature quite involved.
A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high
sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into
nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is
superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing
kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The
nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass
slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene
(PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was
connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a
ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit-
IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could
quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to
a single nanoparticle in a nanowell.
Core-shell nanoparticles with an upconverting phosphorescent, lanthanide core and plasmonic gold shell are employed
for Förster Resonance Energy Transfer (FRET). FRET is demonstrated from a highly efficient upconverting fluoride
nanoparticle doped with Ytterbium (Yb) and Erbium (Er) ions to Streptavidin conjugated with Tetramethyl rhodamine
fluorophore.
A nanoarray, integrated with an electrophoretic system, was developed to trap nanoparticles into their corresponding
nanowells. This nanoarray overcomes the complications of losing the function and activity of the protein binding to the
surface in conventional microarrays by using minimum amounts of sample. The nanoarray is also superior to other
biosensors that use immunoassays in terms of lowering the limit of detection to the femto- or atto-molar level. In
addition, our electrophoretic particle entrapment system (EPES) is able to effectively trap the nanoparticles using a low
trapping force for a short duration. Therefore, good conditions for biological samples conjugated with particles can be
maintained. The channels were patterned onto a bi-layer consisting of a PMMA and LOL coating on conductive indium
tin oxide (ITO)-coated glass slide by using e-beam lithography. The suspensions of 170 nm-nanoparticles then were
added to the chip that was connected to a positive voltage. On top of the droplet, another ITO-coated-glass slide was
covered and connected to a ground terminal. Negatively charged fluorescent nanoparticles (blue emission) were
selectively trapped onto the ITO surface at the bottom of the wells by following electric field lines. Numerical modeling
was performed by using commercially available software, COMSOL Multiphysics to provide better understanding about
the phenomenon of electrophoresis in a nanoarray. Simulation results are also useful for optimally designing a nanoarray
for practical applications.
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.
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.
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).
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.
Lasing from spherical microdroplets ejected into a liquid medium with lower refractive index is observed in a
microchannel. A microfabricated device that combines the droplet production and excitation/detection has been
utilized. Droplets of 50 µm-diameter containing a fluorescent dye were first detected and then excited through
multimode fibers after their production at a T-junction. Images show intense lasing emission around the droplet rim.
Spectra from the droplets exhibit morphology dependent resonances (MDRs) which are red shifted relative to the
bulk fluorescence emission from the dyes. The dependence of resonant peak intensities on the pump beam power is
nonlinear.
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.
A microdroplet or a latex microsphere often acts as an optical cavity that supports Morphology Dependent Resonances (MDRs) at wavelengths where the droplet circumference is an integer multiple of the emission wavelength. 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 fluorescent beads (donor) and streptavidin-Alexa Fluor 555 (acceptor) conjugate was used to observe the energy transfer between the dye pairs. Strong coupling of acceptor emission into optical resonances shows that the energy transfer is efficiently mediated through these resonances.
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.
The detection of single bacterial cells and novel absorbing labels has been demonstrated through optical resonances in microdroplets. The setup enables high throughput detection of single Escherichia coli (E. Coli) cells without any direct labeling although Rhodamine 6G (R6G) was used as the signal transduction mechanism. A micro droplet acts as an optical cavity that supports Morphology Dependent Resonances (MDRs) at wavelengths where the droplet circumference is an integer multiple of the emission wavelength. The cells inside the droplet have a direct effect on the fluorescence lasing spectrum of R6G fluorescence by means of scattering and local refractive index change. The change in the lasing spectrum can be observed at the concentrations where each droplet has as little as one cell. C60 fluorescence quenching has also been demonstrated in microdroplets. R6G in ethanol (10μM) was used for the fluorescence spectrum measurements. Quenching of the optical resonances was observed when C60 dissolved in ethanol was mixed with the R6G-Ethanol solution. Quenching can be observed at C60 concentrations of 1μM in the final solution. The background signal was also checked by repeating the experiment with only R6G and only C60 in the solvent, assuring that the signal reduction was due to the addition of C60 in to the solution. This quenching mechanism may have many applications in multiplexing in bioassays.
Carbon 60 has been used in a functionalized form in a bioassay for a common herbicide, atrazine. It was found that the C60 is a very effective quencher of fluorescence from a number of common dyes. C60 was conjugated to atrazine for use in an immunoassay in which fluorescence from rhodamine was measured. Quenching of the rhodamine emission provided a detection scheme in this assay that yielded a very good limit of detection. The C60 quenching scheme can be used with a wide variety of fluorescent dyes, permitting the potential use of a range of small, cheap excitation sources.
New fluorophores that can be excited using visible or near-infrared radiation are of considerable interest for application in environmental and complex bioassays, where background fluorescence is exacerbated by ultra-violet or blue excitation. Useful labels for biomolecules include infrared emitting semiconductor nanoparticles that can be blue-shifted into the near-infrared and visible through quantum confinement effects, oxides of iron, and rare earth oxides. In this work, the synthesis of 6 nm average diameter lead selenide nanocrystals (well below the Bohr exciton diameter of 92 nm) through a reverse micelle technique; and the synthesis of iron and europium oxides with particles less than 5 nm in diameter by pulsed laser ablation is reported. The europium oxide nanoparticles' emission showed a large Stokes shift (144 nm or 216 nm, depending on excitation wavelength); a narrow, symmetric emission line at 610 nm (FWHM of 8 nm); and long lifetime (300 μs). The Eu2O3 nanoparticles, which were coated with silica for functionalization, displayed a greatly enhanced sensitivity over a conventional ELISA (0.025 ng ml-1 vs. 0.1 ng ml-1) when run in an atrazine immunoassay.
The use of micro droplets as a medium for sensitive detection in fluorescence-based immunoassays has been explored in two contexts. The competitive immuno-reaction of a pesticide hapten, esfenvalerate, with its antibody was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-micrometers diameter orifice. Fluorescence from Rhodamine 6G was excited by the second harmonic of a Nd:YAG laser and detected by a 1/8 m imaging spectrograph with a 512 X 512 thermoelectrically cooled, charged-coupled device (CCD) camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased due to quenching. When a sample of esfenvalerate was added to the droplets, esfenvalerate and esfenvalerate- rhodamine conjugate competed for binding with the anti- esfenvalerate antibody. The release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. The assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. Micro droplets also exhibit strong cavity-dependent optical behavior that gives rise to lasing action. Lasing from Rhodamine fluorescence was quenched by the addition of a second dye, oxonol, that absorbs in the spectral region where the Rh 6G fluorescence peaked. A very strong impact on the droplet resonances was observed, leading to the possible use of quenching as an assay for oxonol-labeled haptens.
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