FRET-based homogeneous immunoassay on a nanoparticle-based photonic crystal.

The potential of fluorescence resonance energy transfer (FRET) in a photonic crystal (PC) nanostructured array to enhance the speed and sensitivity of a protein-based immunoassay was tested. Forty-nanometer carboxylated particles conjugated with donor-labeled capture antibodies were trapped by electrophoresis and used as a FRET energy donor. The PC array was able to enhance fluorescent excitation and emission by phase matching. To provide a proof of concept for this FRET-based homogeneous assay on a PC chip, an immunoassay was tested with a simple immunoglobulin G (IgG)-based reaction. A standard curve was generated by testing two different antibody reaction times: 20 min. and 1 min. The results were compared directly to those obtained from a FRET assay that used a modern, high-sensitivity plate reader with a 96-well plate and a reaction time of 1 h. The rabbit-IgG detection limits of the FRET-based homogeneous assay on the PC were 0.001 and 0.1 µg/mL for incubation times of 20 and 1 min, respectively; the sensitivities were 10(3) and 10 times better than the 96-well plate reader, respectively. Thus, FRET on a PC immunoplatform was shown to be a facile, effective, rapid, and sensitive detection technology.

Potential toxicity of up-converting nanoparticles encapsulated with a bilayer formed by ligand attraction.

The cellular toxicity of nanoparticles that were capped with a bilayered ligand was studied using an up-converting (UC) phosphor material as a representative nanoparticle (NP). The results indicate that although UC NPs are known to be nontoxic, the toxicity of the NPs depends strongly on ligand coordination conditions, in addition to the other commonly known parameters such as size, structure, surface charge etc. Oleate-capped hydrophobic NaYF4:Yb,Er NPs were surface modified to yield three extreme conditions: bare particles that were stripped of the oleate ligands; particles with covalently bound poly(ethylene glycol) (PEG) ligands; and particles with an bilayer of PEG-oleate ligands using the oleate surface group that was remained after synthesis. It was found that the bare particles and the covalent PEG NPs induced little toxicity. However, particles that were rendered biocompatible by forming a bilayer with an amphiphilic ligand (i.e., PEG-oleate) resulted in significant cell toxicity. These findings strongly suggest that the PEG-oleate group dissociated from the bilayered oleate-capped NPs, resulting in significant toxicity by exposing the hydrophobic oleate-capped NPs to the cell. Based on results with bare particles, the NaLnF4:Yb,Er (Ln = Y, Gd) up-converting phosphors are essentially less-toxic. Capping and functionalizing these particles with ligand intercalation may, however, not be a suitable method for rendering the NPs suitable for bioapplication as the ligand can potentially dissociate upon cellular interaction, leading to significant toxicity.

Nanotechnology and toxicology.

The Pacific Basin Consortium session on Nanotechnology and toxicology brought together experts from biology and the physical sciences and engineering to discuss the environmental and health impacts of nanotechnology and nanomaterials in particular. The discussion included new findings in the area of inhalation toxicology as well as aquatic toxicology. Opportunities for engineering new forms of particles for toxicology studies were also presented.

Photonic crystal lab-on-a-chip for detecting staphylococcal enterotoxin B at low attomolar concentration.

Nanoscale wells have been fabricated in a chip to construct a photonic crystal that is used for enhanced immunoassays of a common food-borne toxin, Staphylococcal enterotoxin B (SEB). The nanostructure of the photonic crystal (PC) in the array enhanced the fluorescent signal due to a guided mode resonance. Nanoparticles were used as the solid substrate for attachment of capture antibodies; the particles were then isolated in individual wells of the chip by using an electrophoretic particle entrapment system (EPES). The standard curve generated from the chip consisted of two log-linear regions: the first region with a greater sensitivity, limited by the Kd of the antibody, resembling the 96-well plate ELISA and the other that shows greater than six orders of linearity extending to attomolar concentrations, which is unique to the device we have developed. SEB dissolved in phosphate buffered saline was resolved to levels as low as 35 aM with 10(6)-fold better limit of detection than a conventional 96-well-ELISA. Different concentrations of SEB spiked into milk were tested to assess the reliability of the device and the efficacy of the extended log-linear regime in a "real" food matrix. The presence of the milk did not significantly alter the limit of detection. With very low amounts of sample (less than 10 µL) and fast read-out time, the PC-based system shows great promise for the detection of a wide range of target molecules with close to a single molecule level of sensitivity.

Novel lanthanide-labeled metal oxide nanoparticles improve the measurement of in vivo clearance and translocation.

The deposition, clearance and translocation of europium-doped gadolinium oxide nanoparticles in a mouse lung were investigated experimentally. Nanoparticles were synthesized by spray flame pyrolysis. The particle size, crystallinity and surface properties were characterized. Following instillation, the concentrations of particles in organs were determined with inductively coupled plasma mass spectrometry. The protein corona coating the nanoparticles was found to be similar to the coating on more environmentally relevant nanoparticles such as iron oxide. Measurements of the solubility of the nanoparticles in surrogates of biological fluids indicated very little propensity for dissolution, and the elemental ratio of particle constituents did not change, adding further support to the contention that intact nanoparticles were measured. The particles were intratracheally instilled into the mouse lung. After 24 hours, the target organs were harvested, acid digested and the nanoparticle mass in each organ was measured by inductively coupled plasma mass spectrometry (ICP-MS). The nanoparticles were detected in all the studied organs at low ppb levels; 59% of the particles remained in the lung. A significant amount of particles was also detected in the feces, suggesting fast clearance mechanisms. The nanoparticle system used in this work is highly suitable for quantitatively determining deposition, transport and clearance of nanoparticles from the lung, providing a quantified measure of delivered dose.

Accelerated immunoassays based on magnetic particle dynamics in a rotating capillary tube with stationary magnetic field.

A rapid and simple magnetic particle-based immunoassay has been demonstrated in a capillary mixing system. Antibody-coated micrometer size superparamagnetic polystyrene (SPP) particles were used in an assay for rabbit IgG in a sandwich (noncompetitive) format. The kinetics of the assay was compared between a plate-based system and a single capillary tube. The interaction between the antigen (R-IgG) and the antibody (anti-R-IgG) that was carried by the SPP particles in a rotating capillary was tested under a stationary magnetic field. Competing magnetic and viscous drag forces helped to enhance the interaction between the analyte and the capture antibodies on the particles. The dimensionless Mason number (Mn) was employed to characterize the magnetic particle dynamics; a previously determined critical Mason number (Mn(c)) was employed as a guide to the appropriate experimental conditions of magnetic field strength and rotational speed of the capillary. The advantage of the rotating capillary system included a short assay time and a reduced reactive volume (20 µL). The results show that the immunoassay kinetics were improved by the formation of chains of the SPP particles for the conditions that corresponded to the critical Mason number.

Upconversion in NaYF(4):Yb, Er nanoparticles amplified by metal nanostructures.

Upconversion (UC) fluorescence in NaYF(4):Yb, Er nanoparticles amplified by metal nanostructures was compared in two nanostructure geometries: gold nanoshells surrounding nanoparticles and silver nanostructures adjacent to the nanoparticles, both placed on a dielectric silica surface. Enhanced UC luminescence signals and modified lifetimes induced by these two metals were observed in our study. The UC luminescence intensities of green and red emissions were enhanced by Ag nanostructures by a factor of approximately 4.4 and 3.5, respectively. The corresponding UC lifetimes were reduced ∼ 1.7-fold and ∼ 2.4-fold. In NaYF(4):Yb, Er nanoparticles encapsulated in gold nanoshells, higher luminescence enhancement factors were obtained (∼9.1-fold for the green emission and ∼ 6.7-fold for the red emission). However, the Au shell coating extended the red emission by a factor of 1.5 and did not obviously change the lifetime of green emission. The responsible mechanisms such as plasmonic enhancement and surface effects are discussed.

Alveolar epithelial cell injury due to zinc oxide nanoparticle exposure.

RATIONALE: Although inhalation of zinc oxide (ZnO) nanoparticles (NPs) is known to cause systemic disease (i.e., metal fume fever), little is known about mechanisms underlying injury to alveolar epithelium. OBJECTIVES: Investigate ZnO NP-induced injury to alveolar epithelium by exposing primary cultured rat alveolar epithelial cell monolayers (RAECMs) to ZnO NPs. METHODS: RAECMs were exposed apically to ZnO NPs or, in some experiments, to culture fluid containing ZnCl2 or free Zn released from ZnO NPs. Transepithelial electrical resistance (R(T)) and equivalent short-circuit current (I(EQ)) were assessed as functions of concentration and time. Morphologic changes, lactate dehydrogenase release, cell membrane integrity, intracellular reactive oxygen species (ROS), and mitochondrial activity were measured. MEASUREMENTS AND MAIN RESULTS: Apical exposure to 176 µg/ml ZnO NPs decreased R(T) and I(EQ) of RAECMs by 100% over 24 hours, whereas exposure to 11 µg/ml ZnO NPs had little effect. Changes in R(T) and I(EQ) caused by 176 µg/ml ZnO NPs were irreversible. ZnO NP effects on R(T) yielded half-maximal concentrations of approximately 20 µg/ml. Apical exposure for 24 hours to 176 µg/ml ZnO NPs induced decreases in mitochondrial activity and increases in lactate dehydrogenase release, permeability to fluorescein sulfonic acid, increased intracellular ROS, and translocation of ZnO NPs from apical to basolateral fluid (most likely across injured cells and/or damaged paracellular pathways). CONCLUSIONS: ZnO NPs cause severe injury to RAECMs in a dose- and time-dependent manner, mediated, at least in part, by free Zn released from ZnO NPs, mitochondrial dysfunction, and increased intracellular ROS.

Oxidative injury in the lungs of neonatal rats following short-term exposure to ultrafine iron and soot particles.

Greater risk of adverse effects from particulate matter (PM) has been noted in susceptible subpopulations, such as children. However, the physicochemical components responsible for these biological effects are not understood. As critical constituents of PM, transition metals were postulated to be involved in a number of pathological processes of the respiratory system through free radical-medicated damage. The purpose of this study was to examine whether oxidative injury in the lungs of neonatal rats could be induced by repeated short-term exposure to iron (Fe) and soot particles. Sprague Dawley rats 10 d of age were exposed by inhalation to two different concentrations of ultrafine iron particles (30 or 100 microg/m(3)) in combination with soot particles adjusted to maintain a total particle concentration of 250 microg/m(3). Exposure at 10 d and again at 23 d of age was for 6 h/d for 3 d. Oxidative stress was observed at both Fe concentrations in the form of significant elevations in glutathione disulfide (GSSG) and GSSG/glutathione (GSH) ratio and a reduction in ferric/reducing antioxidant power in bronchoalveolar lavage. A significant decrease in cell viability associated with significant increases in lactate dehydrogenase (LDH) activity, interleukin-1-beta (IL-1beta), and ferritin expression was noted following exposure to particles containing the highest Fe concentration. Iron from these particles was shown to be bioavailable in an in vitro assay using the physiologically relevant chelator, citrate. Data indicate that combined Fe and soot particle exposure induces oxidative injury, cytotoxicity and pro-inflammatory responses in the lungs of neonatal rats.

GAS-PHASE FLAME SYNTHESIS AND PROPERTIES OF MAGNETIC IRON OXIDE NANOPARTICLES WITH REDUCED OXIDATION STATE.

Iron oxide nanoparticles of reduced oxidation state, mainly in the form of magnetite, have been synthesized utilizing a new continuous, gas-phase, nonpremixed flame method using hydrocarbon fuels. This method takes advantage of the characteristics of the inverse flame, which is produced by injection of oxidizer into a surrounding flow of fuel. Unlike traditional flame methods, this configuration allows for the iron particle formation to be maintained in a more reducing environment. The effects of flame temperature, oxygen-enrichment and fuel dilution (i.e. the stoichiometric mixture fraction), and fuel composition on particle size, Fe oxidation state, and magnetic properties are evaluated and discussed. The crystallite size, Fe(II) fraction, and saturation magnetization were all found to increase with flame temperature. Flames of methane and ethylene were used, and the use of ethylene resulted in particles containing metallic Fe(0), in addition to magnetite, while no Fe(0) was present in samples synthesized using methane.

Age specific responses to acute inhalation of diffusion flame soot particles: cellular injury and the airway antioxidant response.

Current studies of particulate matter (PM) are confounded by the fact that PM is a complex mixture of primary (crustal material, soot, metals) and secondary (nitrates, sulfates, and organics formed in the atmosphere) compounds with considerable variance in composition by sources and location. We have developed a laboratory-based PM that is replicable, does not contain dust or metals and that can be used to study specific health effects of PM composition in animal models. We exposed both neonatal (7 days of age) and adult rats to a single 6-h exposure of laboratory generated fine diffusion flame particles (DFP; 170 µg/m(3)), or filtered air. Pulmonary gene and protein expression as well as indicators of cytotoxicity were evaluated 24 h after exposure. Although DFP exposure did not alter airway epithelial cell composition in either neonates or adults, increased lactate dehydrogenase activity was found in the bronchoalveolar lavage fluid of neonates indicating an age-specific increase in susceptibility. In adults, 16 genes were differentially expressed as a result of DFP exposure whereas only 6 genes were altered in the airways of neonates. Glutamate cysteine ligase protein was increased in abundance in both DFP exposed neonates and adults indicating an initiation of antioxidant responses involving the synthesis of glutathione. DFP significantly decreased catalase gene expression in adult airways, although catalase protein expression was increased by DFP in both neonates and adults. We conclude that key airway antioxidant enzymes undergo changes in expression in response to a moderate PM exposure that does not cause frank epithelial injury and that neonates have a different response pattern than adults.

Quantitative gene monitoring of microbial tetracycline resistance using magnetic luminescent nanoparticles.

A magnetic/luminescent nanoparticles (MLNPs) based DNA hybridization method was developed for quantitative monitoring of antibiotic resistance genes and gene-expression in environmental samples. Manipulation of magnetic field enabled the separation of the MLNPs-DNA hybrids from the solution and the fluorescence of MLNPs normalized the quantity of target DNA. In our newly developed MLNPs-DNA assay, linear standard curves (R(2) = 0.99) of target gene was determined with the detection limit of 620 gene copies. The potential risk of increased bacterial antibiotic resistance was assessed by quantitative monitoring of tetracycline resistance (i.e., tetQ gene) in wastewater microcosms. The gene abundance and its expression showed a significant increase of tetQ gene copies with the addition of tetracycline, triclosan (TCS), or triclocarban (TCC). A real-time PCR assay was employed to verify the quantification capability of the MLNPs-DNA assay and accordingly both assays have shown strong correlation (R(2) = 0.93). This non-PCR based MLNPs-DNA assay has demonstrated its potential for gene quantification via a rapid, simple, and high throughput platform and its novel use of internal calibration standards.

Observed velocity fluctuations in monodisperse droplet generators.

On-chip monodisperse droplet generation and analysis is ideal for low concentration and single molecule applications because it enables reagents to be partitioned into identical nanoliter or smaller reactor volumes while allowing real-time optical interrogation. Typically, these systems operate in a continuous droplet generation mode to maximize sample throughput. We have observed that on-chip droplet production for water-in-oil emulsions causes downstream droplet velocity fluctuations similar to those reported for gas-in-liquid emulsion systems with a periodicity equivalent to the droplet generation rate. This phenomenon can affect the accuracy of data collection due to variation in photon integration times as the moving droplets are observed. Here we perform high speed imaging and frequency domain analysis to describe the periodic velocity fluctuations due to monodisperse droplet generation.

Monodisperse droplet generation and rapid trapping for single molecule detection and reaction kinetics measurement.

On-chip monodisperse droplet analysis systems are ideal for low concentration and single molecule applications because they partition reagents into identical picoliter or smaller reactor volumes that can be observed in real-time. We present a novel trapping method with droplet stopping times of approximately 38 ms that is applicable to most on-chip droplet generators. The technique greatly extends optical interrogation times without droplet motion or coalescence; and will allow on-chip single molecule detection of nanoparticle emitters with simple optics. The method maintains droplet monodispersity without chemistry-altering surfactants, and has been shown to preserve a stationary droplet stream through repetitive high-temperature thermal cycling with no additional energy input required to maintain droplet position.

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.

Effect of cerium oxide nanoparticles on inflammation in vascular endothelial cells.

Because vascular endothelial cell inflammation is critical in the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs) to ultrafine particles induces an inflammatory response. To test the hypothesis, we incubated HAECs for 4 h with different concentrations (0.001-50 microg/ml) of CeO(2) nanoparticles and subsequently measured mRNA levels of the three inflammatory markers intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-8, and monocyte chemotactic protein (MCP-1) using real-time polymerase chain reaction (PCR). Ceria nanoparticles caused very little inflammatory response in HAECs, even at the highest dose. This material is apparently rather benign in comparison with Y(2)O(3) and ZnO nanoparticles that we have studied previously. These results suggest that inflammation in HAECs following acute exposure to metal oxide nanoparticles depends strongly on particle composition.

Uptake and inflammatory effects of nanoparticles in a human vascular endothelial cell line.

The mechanisms governing the correlation between exposure to nanoparticles and the increased incidence of cardiovascular disease remain unknown. Nanoparticles appear to cross the pulmonary epithelial barrier into the bloodstream, raising the possibility of direct contact with the vascular endothelium. Because endothelial inflammation is critical for the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs*) to nanoparticles induces an inflammatory response and that this response depends on the composition of the particles. To test this hypothesis, we incubated HAECs for 1 to 8 hours with different concentrations (0.001-50 microg/mL) of iron oxide (Fe2O3), yttrium oxide (Y2O3), cerium oxide (CeO2), and zinc oxide (ZnO) nanoparticles. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we subsequently measured messenger RNA (mRNA) levels of three markers of inflammation: intercellular cell adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1). The particles were well characterized in terms of size, surface area, composition, and crystal structure. To determine the interactions of nanoparticles with HAECs, we used inductively coupled plasma-mass spectrometry (ICP-MS) to measure the concentration of internalized particles. Our data indicate that the delivery of nanoparticles to the HAEC surface and their uptake within the cells correlate directly with the concentration of particles in the cell culture medium. Transmission electron microscopy (TEM) revealed that the Fe2O3, Y2O3, and ZnO nanoparticles are internalized by HAECs and are often found within intracellular vesicles (the CeO2 particles were not imaged). Fe2O3 nanoparticles did not provoke an inflammatory response in HAECs at any of the concentrations tested, CeO2 particles elicited no response at low concentrations and a weak response above 10 microg/mL, and Y2O3 and ZnO nanoparticles elicited a pronounced inflammatory response above a threshold concentration of 10 microg/mL. We used fluorescent markers to identify the production of reactive oxygen species (ROS) in cells; the results showed that Y2O3 and ZnO particles at the highest concentrations may lead to the production of ROS. At the highest concentration, ZnO nanoparticles caused significant loss of cell adherence. These results demonstrate that inflammation in HAECs after acute exposure to metal oxide nanoparticles depends on the concentration and composition of the particles.

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.

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.

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.