Development of a multichannel flow-through chemiluminescence microarray chip for parallel calibration and detection of pathogenic bacteria

Pathogen detection is important for health and safety reasons. Several outbreaks all over the world have shown the need for rapid, qualitative, quantitative, and, particularly, multianalyte detection systems. Hence, a multichannel flow-through chemiluminescence microarray chip for parallel detection of pathogenic bacteria was developed. The disposable chip made of acrylonitrile–butadiene–styrene (ABS) copolymer was devised as a support for a multiplexed sandwich immunoassay. Calibration and measurement was possible in one experiment, because the developed chip contains six parallel flow-through microchannels. Polyclonal antibodies against the pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila were immobilized on the chip by microcontact printing in order to use them as specific receptors. Detection of the captured bacteria was carried out by use of specific detection antibodies labelled with biotin and horseradish peroxidase (HRP)–streptavidine conjugates. The enzyme HRP generates chemiluminescence after adding luminol and hydrogen peroxide. This signal was observed by use of a sensitive CCD camera. The limits of detection are 1.8 × 10⁴ cells mL⁻¹ for E. coli O157:H7, 7.9 × 10⁴ cells mL⁻¹ for L. pneumophila, and 2.0 × 10⁷ cells mL⁻¹ for S. typhimurium. The overall assay time for measurement and calibration is 18 min, enabling very fast analysis.      

Automated analytical microarrays: a critical review

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. Figure MCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays     

Microwave‐Assisted Free Radical Polymerizations and Copolymerizations of Styrene and Methyl Methacrylate

Summary: Radical homopolymerizations and copolymerizations of styrene were performed in toluene and N,N‐dimethylformamide (DMF) as solvents using different initiators with and without microwave irradiation. Only the homopolymerization of styrene under microwave irradiation in DMF with DtBP showed significantly enhanced styrene conversion whereas other initiators resulted in no or only slight increase of styrene conversion under microwave irradiation. In any case, DMF was required to gain in styrene conversion under microwave irradiation. Significantly higher monomer conversions were observed under otherwise comparable conditions in the copolymerization of styrene and methyl methacrylate (MMA) in DMF. Microwave‐induced selectivity of monomers was not observed in copolymerizations.

Yield of styrene polymerizations under varying reaction conditions initiated by DtBP.     

Simultaneous determination of four different antibiotic residues in honey by chemiluminescence multianalyte chip immunoassays

We are presenting the first method for identification and quantification of antibiotic derivatives in honey samples using regenerable antigen microarrays in combination with an automated flow injection system. The scheme is based on an indirect competitive immunoassay format using monoclonal antibodies bound to the surface of the microarray. The surface of glass slides was coated with epoxy-activated poly(ethylene glycol) and enables direct immobilization of the antibiotic derivatives. The antigen/antibody interaction on the surface of the chip can be detected by chemiluminescence (CL) read-out via CCD camera. The method allows for fast analysis of the four analytes simultaneously and without purification or extraction. An effective data evaluation method also was developed to warrant unambiguous identification of the spots and to establish grey levels of CL intensities. The software developed enables fast and automated processing of the CL images. Dose?Cresponse curves were obtained for the derivatives of enrofloxacin, sulfadiazine, sulfamethazine and streptomycin. Spiking experiments revealed adequate recoveries within the dynamic ranges of the calibration curves of enrofloxacin (92%?±?6%), sulfamethazine (130%?±?21%), sulfadiazine (89%?±?20%) and streptomycin (93%?±?4%).

Surface-enhanced Raman scattering detection of bacteria on microarrays at single cell levels using silver nanoparticles

We describe a method for the synthesis of SERS-active silver nanoparticles (AgNPs) directly on the surface of bacteria (bacteria@AgNPs), specifically of E. coli cells. This straightforward strategy allows for the sensitive determination of bacteria on a microarray platform. Antibodies were used as selective receptors on the microarray surface. The Raman signal of bacteria@AgNPs is about 10 times higher than that obtained previously with microarrays based on mixing bacteria and AgNPs (bacteria+AgNPs). The optimum SERS enhancement of bacteria@AgNPs is obtained under 633-nm laser excitation, and this most likely is due to the plasmonic interaction of aggregated AgNPs. The method allows for an identification and quantification even of single E. coli bacteria. In our perception, this straightforward approach represents a most valuable tool for the detection of E. coli and, conceivably, of other bacteria, and thus has a large potential in environmental monitoring, medical diagnosis, and in food safety and quality control.

Synthesizing AgNPs directly on the surface of bacteria is demonstrated to be a highly efficient approach for a label-free readout of bacteria microarrays by surface-enhanced Raman scattering (SERS), resulting in signals about 10 times higher than previously reported results.

     

NO2 measurement artifacts in the presence of soot

Nitrogen dioxide is a regulated pollutant, which is measured routinely. Since it can be formed during combustion processes, it is often measured in the presence of soot. This study investigates the possible artifact formation due to the interaction of soot and NO₂ in the sampling lines and instrument prefilters. The transfer of varying NO₂ concentrations through filters and tubes coated with different kinds of soot was investigated by using a dedicated photoacoustic soot and NO₂ analyzer (TwinPAS). The effects of flow rate, temperature, relative humidity, tubing respectively filter material, soot reactivity, and passivation on the NO₂ measurement artifacts have been investigated. We found significant lags (up to 2 min) of the NO₂ transfer as well as total NO₂ losses of up 10 %.     

In-source fragmentation of partially oxidized mono- and polycyclic aromatic hydrocarbons in atmospheric pressure chemical ionization mass spectrometry coupled to liquid chromatography

Partially oxidized derivatives of polycyclic aromatic hydrocarbons (PAHs) are known to be important environmental pollutants. For the identification of these substances in complex mixtures, e.g. atmospheric aerosol samples, liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC/APCI-MS) has been found to be a suitable analytical technique. In this study 31 derivatives of mono- and polycyclic aromatic hydrocarbons with up to five condensed aromatic rings carrying different functional groups (carboxyl, dicarboxylic anhydride, lactone, hydroxyl, and carbonyl) were characterized by LC/APCI-MS. Each substance was measured in positive and negative ion detection mode at four different fragmentor voltages (90 to 190 V). For the first time, the results show that characteristic and well-interpretable fragmentation patterns can be obtained for these classes of compounds by in-source collision-induced dissociation in a single quadrupole LC/APCI-MS system. For each class of compounds typical spectral features and optimum measurement conditions are reported, and fragmentation pathways are proposed. The study demonstrates the applicability of LC/APCI-MS for the determination of most of the investigated compounds at trace levels, and it provides a database for the identification of unknown partially oxidized aromatic hydrocarbons. Copyright 1999 John Wiley & Sons, Ltd.     

Characterization of a monoclonal TNT-antibody by measurement of the cross-reactivities of nitroaromatic compounds

The characterization of a commercially available monoclonal antibody directed against the explosive 2,4,6-trinitrotoluene (TNT) is reported. The cross-reactivities of various nitroaromatic compounds have been determined by competitive enzyme-linked immunosorbent assay (ELISA). Byproducts and metabolites of TNT were examined as well as the azo dye Disperse Blue 79 and its major metabolites (2-bromo-4,6-dinitroaniline and 2-chloro-4,6-dinitroaniline, respectively). By investigation of the cross-reactivities of different spacer derivatives of TNT it could be demonstrated that the bridge-recognition of the antibody is not very pronounced. N-(2,4,6-Trinitrophenyl)-methylamine shows the highest cross-reactivity (240%) of the examined compounds. Additionally, affinity constants of several nitroaromatic compounds have been determined. The affinity constant of TNT has been calculated to 1.3 × 10⁹ L/mol from the minimal midpoint (IC50 value) of the standard curve. The detection limit achieved for TNT was 0.06 μg/L; the midpoint of the optimized assay was 0.34 μg/L. Received: 17 July 1998 / Revised: 28 December 1998 / Accepted: 1 January 1999     

Comparison of liquid chromatography-mass spectrometry interfaces for the analysis of polar metabolites of benz[a]pyrene

 The performance of two liquid chromatography-mass spectrometry (LC/MS) interfacing techniques, thermospray (TSP) and atmospheric pressure chemical ionization (APCI), for the analysis of benzo[a]pyrene (BaP) metabolites (hydroxy, epoxy and quinone derivatives) was compared. Interface and detection parameters such as source temperature, eluent composition or flow rate were optimized using negative ion mode. In TSP, the main ions are mostly [M]^-, [M−H₂O]^- or [M+CH₃COO]^-, whereas APCI gives mainly the [M]^- and [M−H]^- ions. Quantification was carried out by flow injection. Calibration graphs were linear in the range of 10 ng to 1000 ng in TSP and 0.1 ng to 10 ng in APCI. Detection limits were in the range of 1 ng to 20 ng in TSP and 0.002 ng to 0.2 ng in APCI. The presence of BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione was confirmed in environmental samples of air particulate matter. Received: 6 January 1997/Accepted: 18 April 1997