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The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes [10.sup.6] smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet morion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of ~18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

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 is examined. 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.

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:[Gd.sub.2][O.sub.3]) nanoparticles, as labels in immunoassay microarrays. The Eu:[Gd.sub.2][O.sub.3] 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 [micro]m). 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 [micro]g [L.sup.-1] PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and highthroughput screening.

A method for simply and cheaply preparing inorganic phosphor nanoparticles of [Eu.sub.2][O.sub.3] as labels in biology has been demonstrated with a simple microwave-assisted surface chemistry. The capping process adds a silane layer to the surface of the particles and provides amine groups that can be used for biological conjugation. The surface layer also protects the particles during conjugation chemistry. The particles retain their desirable optical properties that are typical of europium, that is, a spectrally narrow, red emission and a long fluorescence lifetime. The application of the nanoparticle labels in an immunoassay yields very good sensitivity in an immunoassay for atrazine (sub-parts-per-billion detection limit) without optimization of the detection system. The microwave functionalization technique will permit a broad range of inorganic nanophase phosphors to be used in high-throughput assays for environmental monitoring.

An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 [micro]m) produced by a piezoelectric generator system with 10-[micro]m-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the I[C.sub.50] of the LIF-microdroplet QFIA is 0.45 [micro]g [L.sup.-1] reaching a LOD of 0.04 [micro]g [L.sup.-1]. The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 [micro]g [L.sup.-1] in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 [micro]g [L.sup.-1], with a dynamic range between 4 and 149.5 [micro]g [L.sup.-1] in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.

Faster mixing of chlorinated hydrocarbons is found to yield lower Reynolds numbers and greater amounts of flouranthene, a toxic combustion byproduct. Lower Reynolds number flames produced more aromatic and chlorinated aromatic species compared to higher Reynolds number flames. The most number of chlorinated species in the postflame gases were chloronaphthalene, while methyl chloride was effectively destroyed in all the Reynolds numbers used. Methyl chloride and methane were combusted in a combustion wind tunnel with a coflow of air with varied Reynolds numbers used ranging from 3,500 to 7,200.

Positive and negative ion mass spectra of arsenic trioxide ([As.sub.2][O.sub.3]) and arsenic pentaoxide ([As.sub.2][O.sub.5]) have been obtained by single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 nm was used for the simultaneous desorption and ionization of the solid sample. High-mass cluster ions that are unique to the oxidation state of each oxide sample appear in the negative ion mass spectra. The [As.sub.2][O.sub.3] produces [As.sub.3][O.sub.[5.sup.-]], while the [As.sub.2][O.sub.5] yields [As.sub.3][O.sub.[8.sup.-]]. The formation of unique negative cluster ions presents the capability for arsenic oxidation state speciation by laser desorption/ionization mass spectrometry. The ability of time-of-flight mass spectrometry to examine the relative amounts of each arsenic oxide present in a series of mixtures is discussed. Application of our speciation technique to a model incinerator sample is demonstrated.

A wind tunnel technique employing different operating configurations was developed to model controlled agricultural burning conditions to allow for the determination of emission factors and flame structure. The correlation between the operating configuration and total suspended particulate (TSP) matter emissions and flame structure was determined. Average emission factors for TSP matter varied from 0.505% to 0.727% for the four tunnel configurations. Flame structure was analyzed via local temperature measurements, gas concentrations and soot volumes.

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 Gd2O3, 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:Gd2O3 crystallographic structure and cell dimensions to be consistent with a predominantly monoclinic phase. [ABSTRACT FROM AUTHOR]

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photo-bleaching 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-encapsulatecl 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 × 10 μm). 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 μg 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. [ABSTRACT FROM AUTHOR]

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.

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

Current in vitro methods to assess nanomaterial cytotoxicity involve various assays to monitor specific cellular dysfunction, such as metabolic imbalance or inflammation. Although high throughput, fast, and animal-free, these in vitro methods suffer from unreliability and lack of relevance to in vivo situations. New approaches, especially with the potential to reliably relate to in vivo studies directly, are in critical need. This work introduces a new approach, single cell mechanics, derived from atomic force microscopy-based single cell compression. The single cell based approach is intrinsically advantageous in terms of being able to directly correlate to in vivo investigations. Its reliability and potential to measure cytotoxicity is evaluated using known systems: zinc oxide (ZnO) and silicon dioxide (SiO2) nanoparticles (NP) on human aortic endothelial cells (HAECs). This investigation clearly indicates the reliability of single cell compression. For example, ZnO NPs cause significant changes in force vs relative deformation profiles, whereas SiO2 NPs do not. New insights into NPs-cell interactions pertaining to cytotoxicity are also revealed from this single cell mechanics approach, in addition to a qualitative cytotoxicity conclusion. The advantages and disadvantages of this approach are also compared with conventional cytotoxicity assays.

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.

Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e., antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g., enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a-well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 attomolar (aM) in less than 10 μL of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early stage detection of biomarkers.

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.