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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.

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.

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.

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]