On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/µL, 1 GU/µL, and 5 × 10(3) GU/µL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

Development and characterization of a mobile photoacoustic sensor for on-line soot emission monitoring in diesel exhaust gas.

Upcoming regulations for vehicle exhaust emission demand substantial reduction of particle emission in diesel exhaust. To achieve these emission levels, the car manufacturing industry is developing new combustion concepts and exhaust after-treatment techniques such as the use of catalysts and particle filters. Many of the state-of-the-art analytical instruments do not meet the required detection limits, in combination with a high temporal resolution necessary for engine optimization. This paper reports a new detection system and the first results of its application to on-line diesel exhaust soot measurements on a engine test bench (MAN diesel engine facility Nürnberg, Germany). The instrument is based on differential photoacoustic (PA) spectroscopy of black carbon aerosol. It contains two identical PA cells, one for the measurement of the aerosol particles and one which analyses the particle-free gas. Thus, a potential cross-sensitivity to gaseous absorbers in the exhaust gas can be excluded. The PA cells were characterized in a laboratory set-up, with water vapor as reference gas and artificial soot generated by a spark discharge generator. The detection limit was found to be 2 microg m(-3) BC (for diesel soot) with a sampling rate of 3 Hz. The temporal response of the system was found to be in the order of 1 s. After full characterization of the cells, the system was transferred into a mobile 19"-rack. Characterization of the mobile sensor system under real-world conditions was performed during several measurement campaigns at an engine test bench for heavy-duty diesel engines. Results for the limit of detection, the time resolution, accuracy, repeatability, and robustness of the sensor system are very promising with regards to a routine application of the system in engine development.

Monitoring polycyclic aromatic hydrocarbon metabolites in human urine: extraction and purification with a sol-gel glass immunosorbent.

A new, rapid method for selective extraction of hydroxylated polycyclic aromatic hydrocarbons metabolites (OH-PAHs) in human urine was developed using an immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix. Resulting chromatograms after immunoextraction of urine samples and HPLC analysis of the extracts were free from matrix interferences. The LODs for the determination of OH-PAHs in these difficult samples were in the low-ppt range (1-16 ng/L). In addition to its high selectivity, the immunosorbent proved to be robust and reusable. Obtained recoveries in spiked urine samples ranged from 83 to 107% for the hydroxyphenanthrene and hydroxypyrene compounds under investigation, while recovery for 3-hydroxybenzo[a]pyrene was only 45-62%. In a biomonitoring study, the SGG immunosorbent was successfully used for trace-level analysis of OH-PAHs in 20 human urine samples. Results were compared to data obtained by an independent reference analysis method and revealed good correlation between both methods.

On-line and in-situ detection of polycyclic aromatic hydrocarbons (PAH) on aerosols via thermodesorption and laser-induced fluorescence spectroscopy.

A fiber optical sensor system for the determination of polycyclic aromatic hydrocarbons (PAH) on aerosols by laser-induced, time-resolved fluorescence is combined with a thermodesorption device. The sensor system is based on an aerosol flow cell, which is fibre-optically coupled to a pulsed nitrogen laser for excitation and the detection system. Time-resolved fluorescence emission spectra are detected by a monochromator equipped with a photomultiplier and a fast digital storage oscilloscope. The analytical figures of merit of the thermodenuder are reported for benzo[a]pyrene, benzo[b]fluoranthene, and benzo[ghi]-perylene on ultrafine soot and NaCl aerosols. By thermodesorption of the PAH, problems due to quenching of the PAH fluorescence by the bulk aerosol material or excimer formation on the aerosol surface were avoided. For the PAH under study, the sensitivity was improved considerably and detection limits between 110 and 850 ng m(-3) were attained, while a response time of 2-3 min was achieved with the thermodenuder. A calibration for PAH on ultrafine soot and NaCl aerosols was established independent of the aerosol substrate.

Analysis of atrazine, terbutylazine and their N-dealkylated chloro and hydroxy metabolites by solid-phase extraction and gas chromatography-mass spectrometry and capillary electrophoresis-ultraviolet detection.

Solid-phase extraction (SPE) with the styrene-divinylbenzene adsorbent LiChrolut EN was investigated for the extraction of the s-triazine herbicides atrazine and terbutylazine, their polar N-dealkylated degradation products deethylatrazine (DEA), deisopropylatrazine (DIA) and deethylterbutylazine (DET) and for the hydrophilic hydroxytriazine degradation products (HTDPs) hydroxyatrazine (HA), hydroxyterbutylazine (HT), deethylhydroxyatrazine (DEHA), deisopropylhydroxyatrazine (DIHA) and deethyldeisopropylhydroxyatrazine (ameline). The optimum pH value for the extraction of the HTDPs from fortified tap water at 2 micrograms/l is 3.0. Recovery values with 200 mg LiChrolut EN are > 80% for HA, HT, DEHA and 30% for DIHA from 200 ml spiked tap and river water. Atrazine, terbutylazine, DEA, DIA and DET are quantitatively extracted by LiChrolut EN. The chlorotriazines are analyzed by GC-MS and the HTDPs by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) with an acetate buffer at pH 4.6 or a sodium borate-sodium dodecyl sulfate buffer at pH 9.3. The combined method of SPE enrichment and CE analysis allows the determination of HTDPs in the low microgram/l range.

Immunological determination of triazine pesticides bound to soil humic acids (bound residues).

An immunological method for the determination of triazine herbicides covalently bound to soil humic acids has been developed. A sandwich-immunoassay has been performed, based on both polyclonal humic acid-antibodies and monoclonal triazineantibodies. A peroxidase-labelled third antibody has been used for the photometric detection. A triazine-humic acid conjugate served as calibration standard. The coupling density for this conjugate has been determined by measuring the difference of free amino groups both with ninhydrin and with the trinitrobenzene sulfonic acid method. In addition, the coupling density has been confirmed by scintillation counting using a (14)C-atrazine derivative. Due to nonspecific interactions between antibody proteins and humic acids, different blocking steps had to be performed. Finally, the assay has been applied to a triazine contaminated soil sample. Humic acids (including bound residues) have been extracted by diluted sodium carbonate solution. Concentrations of bound atrazine residues have been found in the range of 2 mg/kg soil on fields where triazine herbicides has been applied over a period of 21 years. These results are comparable to both the applied amount and the nonextractable fraction.