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In this study, a new chemiluminescent method based on the dependence of luminol light emission induced by free radicals in airborne particulate matter (PM) is proposed as a screening assay for the rapid characterization of samples from different sources based on their redox properties. This parameter is considered critical for assessing particulate matter toxicity and its impacts on human health. We propose a cell-free, luminescent assay to evaluate the redox potential of particulate matter directly on the filters employed to collect it. A joint chemometric approach based on Principal Component Analysis and Hotelling Analysis was applied to quickly sort out ambient particulate samples with a significantly different light emission profile caused by Luminol reaction. Based on Spearman correlation analysis, the association of the samples light emission intensity with their chemical composition and emission sources was attempted. The overall methodology was tested with certified reference materials and applied to two series of particulate matter samples previously subjected to thorough chemical speciation and subsequent source apportionment. The results show the effectiveness of the luminescent method, allowing the quick assessment of particulate matter oxidative potential, but providing further evidence on the complexity of the oxidative potential determination in this kind of samples. The chemometric processing of the whole dataset clearly highlights the distinct behavior among the two series of samples, the certificate standard reference materials, and the blank controls, supporting the suitability of the approach.

A molecular diagnostic technique (polymerase chain reaction enzyme-linked immunosorbent assay [PCR-ELISA]) for detection of Erwinia amylovora was developed. The protocol is based on the immunoenzymatic determination of PCR products. For in vitro amplification, we used previously published primers able to detect the cryptic plasmid pEA29, which is ubiquitous in E. amylovora. Amplicons were labeled with 11-digoxigenin (DIG)-dUTP during the amplification reaction, captured by hybridization to a biotinylated oligonucleotide in streptavidin-coated ELISA microplates, and then detected with anti-DIG-Fab′-peroxidase conjugated antibodies. The specificity of the assay was verified using E. amylovora strains from different host plants and geographical origins in addition to other plant-associated bacteria (either phytopathogenic or saprophytic) belonging to the genera Erwinia, Pseudomonas, and Agrobacterium. In detection threshold experiments with pure cultures, as few as 30 and 3 CFU/reaction tube were detected when the ABTS (colorimetric) and ECL (chemiluminescent) detection assays, respectively, were used. PCR-ELISA coupled with chemiluminescent detection was able to detect as few as 4 × 102 CFU/g of artificially infested pear twigs. The assay was further shown to be suitable for detection of E. amylovora in naturally infected plant organs, and the results were compared to those obtained using standard PCR assays with electrophoretic separation of amplicons.

Tap water filtering devices are widely employed to improve odor and taste of tap water, or to obtain refrigerated or sparkling drinking water. The presence of disinfectants-resistant bacteria in tap water is responsible of the biofilm formation inside tubes and tanks. The consequent contamination of dispensed water is a well-known hygiene problem because of the quite constant presence of potentially pathogenic bacteria likes P. aeruginosa. In this study, we tested the technical feasibility and effectiveness of the addition to different commercial devices of a packaged polysulphone fibers filter. We aimed to find a simple solution to implement the quality of the delivered water. Water contamination levels were determined in a wide selection of microfiltered water dispensers and we selected among them a representative group of 10 devices, new or in use. The packaged ultrafilter was introduced in about half of them, to monitor, when possible, in parallel the contamination levels and flow rate of a couple of identical units, with and without the filter. The placement of the dialysis filters resulted feasible at different positions along the water circuits of the variously designed filtration units. Delivered water resulted completely free from bacteria when the filter was placed exactly at, or very close to, the outlet in spite of the inner surfaces contamination. This performance was not obtained in presence of a more or less long tract of water circuits downstream the ultrafilter: a significant but not complete reduction of the plate count numbers was observed. The filters worked in continue over the whole study period, ten months, showing exactly the same efficiency. Moreover, the flow rate in presence of the filter was quite unaffected. The addition of this kind of filter to already in use water dispensers was technically easy, and its use can be recommended in all cases a simple but reliable water sanitization is requested.

A quantitative chemiluminescent method, enabling indirect identification of the peroxide-based explosives TATP (triacetone triperoxide) and HMTD (hexamethylene triperoxide diamine) has been developed. Treatment of these compounds with acidic solutions produced peroxides, which were transformed into radical derivatives by horseradish peroxidase (HRP) and then quantified by measuring the light emitted during their oxidation of luminol. The method was first developed in the microplate format and later optimized for a portable luminometer, to enable rapid application of the assay directly on site. When the portable luminometer was used each analysis took only 5-10 min. The method had good selectivity, sensitivity, and reproducibility; in the microplate format the limits of detection (LOD) and quantification (LOQ) were 40 and 50 ng mL(-1), respectively, for both TATP and HMTD. When the portable luminometer was used the LOD and LOQ were 50 and 100 ng mL(-1), respectively, for both compounds. Introduction of light emission-enhancing compounds did not improve the analytical performance of the assay. Imprecision (CV values) was always below 10%. Recovery varied rapidly with time, with an average value of 78% after 5 min. No false-positive result was detected on measurement of a variety of samples; this is an important feature for analysis on site. The method was applied both to contaminated materials and to fortified soil samples, simulating operational conditions.

To identify the explosive used in a terrorist attack, or to get an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particular true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates as well as the influence of various non specific parameters such as: the tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The CL-ELISA showed a good sensitivity (LOD and IC50 values in the ng mL-1 range) and a good reproducibility (CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an “Improvised Explosive Device”. Three extraction procedures have been tested on these samples, all employing methanol as the solvent. The Lateral Flow Immuno Assay (LFIA), developed by using the same immunoreagents, reached a Detection Limit of 1 µg mL-1 when tested on the same samples analysed by CL-ELISA.

The suitability of competitive enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection-based immobilized antigen (indirect assay) for rapid and accurate determination of chlorpyrifos in various food matrices was tested. The limit of detection (LOD) values were 1–1.75 ng mL−1, the standard curve midpoint (IC50) was 3.5 ng mL−1, and the assay duration was 1.5 h. Assay application to the analysis of honeybee extract resulted in chlorpyrifos recoveries varying between 62 and 83% in 5–15 ng mL−1 herbicide concentration range.

It has been recently demonstrated that honeybees can be used to monitor the presence of Erwinia amylovora in the environment. The monitoring protocol involves setting up stations (consisting of three hives each) in infected areas, at the edges of infected areas, or in apparently non-infected areas. From 2000 to 2002, over 500 samples of pollen and honeybees were analysed for the fire blight pathogen by a new chemiluminescent PCR-ELISA rapid method. This monitoring programme demonstrated that honeybees can be an efficient and economic tool to detect the bacterium, even in the absence of symptoms. In fact, in several cases, E. amylovora was detected in samples from areas where no symptoms of fire blight had been observed. On the basis of results presented at the 9th International Workshop on Fire Blight, a study was carried out in 2002 to evaluate, under greenhouse conditions, the role of honeybees in the dissemination of E. amylovora from experimentally inoculated pear flowers and contaminated hives to healthy pear flowers and honeybee matrixes. Our results show that, after contamination, honeybees can act as carriers of live bacterial cells of E. amylovora for only 48 hours. After this period, no viable E. amylovora cells were found in non-inoculated pear flowers visited by insects, on honeybee bodies or intestines, or in any of the analysed beehive products (honey, pollen and wax). These and previous results provide useful data to establish prophylactic operating procedures to allow beehive movement in disease-free areas. The quarantine procedures mandate closing the hives and keeping them in a temperature controlled chamber (between 15 and 20°C) for 48 hours, or for 24 hours only if they have been treated with oxalic acid, which inhibits E. amylovora.

In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. Both were conjugate-coated formats based on identical monoclonal antibodies and homologous protein conjugates. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of horseradish peroxidase allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing extracts of apple-strawberry baby food and simply diluted fruit juices, both spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more. Results obtained by ELISAs correlated well, both in terms of accuracy and precision, with those obtained by a liquid chromatography–electrospray mass spectrometry (LC/ESI/MS/MS) analysis, used as reference method to validate the immunoassays results. The limits of detection reached by using the chemiluminescent assay were 0.03, 0.007 and 0.004 ng ml −1 for carbofuran, carbaryl and methiocarb, respectively.

The analytical performances of a manual and a partially automated chemiluminescent (CL) assay, of total antioxidant capacity (TAC) were assessed. In both cases the light emitting reaction involved luminol, horseradish peroxidase and hydrogen peroxyde, but the emission kinetics and the parameters taken into account to calculate TAC values were completely different. The major characteristics expressing the quality of the two analytical methods, i.e. inaccuracy, repeteability and reproducibility, sensitivity, time required for the analysis and detection limit, were estimated by using standard solutions of Trolox. The reliability of the automated method, in comparison with the more validated manual one, was demonstrated testing food samples such as honey, wine and dietary supplements and performing a statistical analysis of the results. The comparison of the two series of data by t-test resulted in p values in the range 0.1-0.01. The time required for the analysis of each sample was reduced to one third using the automated method.

All aerobic organisms have developed different mechanisms for neutralising the free radicals, mostly produced by the monoelectronic reduction of O(2), and preventing the severe damages these can provoke. The efficiency of these mechanisms can be assessed, in different matrices, by a simple and direct chemiluminescent assay (CL) based on luminol oxidation catalysed by horseradish peroxidase. Light emission is mediated by the production of free radicals and it is inhibited after a sample addition in a way that is directly proportional to the sample total content of molecules displaying antioxidant activity. The performances of this chemiluminescent assay were compared with those of two spectrophotometric methods already applied in clinical practice. First spectrophotometric method measures, like CL assay, the total antioxidant capacity, whereas the second one determines free thiol groups content. The chemiluminescent assay has a linearity interval between 0.60 and 9.46 mumol l(-1) of Trolox (y=34.91x+3.10; r=0.999; n=5) with an imprecision, expressed as CV, of 3.8% and an inaccuracy, expressed as percentage recovery, of 109%. The first spectrophotometric method, based on the same reference standard, the Trolox molecule, has a linearity interval between 0.2 and 2.5 mmol l(-1) of Trolox (y=-0.01x+4.54; r=0.95; n=5); the thiol groups assay has a linearity interval between 0.1 and 1 mmol l(-1) of l-cysteine (y=1.68x-47.09; r=0.998; n=5). Different clinical samples of plasma from healthy individuals, obese subjects and patients with liver diseases were tested. Interesting correlations were obtained among the three methods, but no significant correlations emerged between antioxidant capacity and clinical parameters. Significant differences were there only between men and women among obese subjects and between drinkers and non-drinkers among liver disease patients.

A heterogeneous chemiluminescent (CL) flow immunoassay for DDT was optimized comparing different types of immunoaffinity supports: beads, nylon coils and membranes (membranes HyBondN+). In order to characterize solid immunoaffinity supports two basic immunoassay formats were performed, using (1) enzyme-labeled secondary and (2) enzyme-labeled specific monoclonal antibodies (MAbs). In both formats, hapten DDT5 conjugated to ovalbumin immobilized on solid supports according to the appropriate immobilization procedure, enzyme label (horseradish peroxidase, HRP) and luminescent detection (luminol/H 2 O 2 / p -iodophenol) were used. The lowest limit of detection (LOD), 1 nM p , p -DDT, was obtained with a membrane-based flow immunoassay with HRP-labeled specific antibody. Beads and packed tubing were discarded as appropriate supports because of the difficulties encountered for reproducible packing and the occurrence of light scatterring (beads), which seriously compromised the performance and reproducibility of the flow immunoassay.

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4¿-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL¿1, respectively. The LOD of LFIA was 0.1 ¿g mL¿1. A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL¿1, with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL¿1 and 17.4 ng mL¿1, respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 ¿g mL¿1) and by the Directive 2004/19/EC (0.6 ¿g mL¿1), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays, This work was supported by a grant from the University of Bologna (RFO-Focused Fundamental Research Projects 2000-2011) grants.

The analysis of several organophosphorus and carbamate pesticide residues in the bodies of honeybees using gas chromatography (GC) and gel permeation chromatography (GPC) clean-up is described. Freeze-dried or lyophilized insect samples were blended with diatomaceous earth (Extrelut) then underwent elution with methylene chloride. This extraction method has shown good recovery on various spike standard levels. Samples are cleaned up by GPC with a Bio Beads SX 3 column and a cyclohexane-ethylacetate (1:1) eluant. Organophosphorus and carbamate compounds are quantified using capillary gas chromatography. Good linearity ranges were observed for all compounds. The extraction process was rapid and results were good, despite the complexity of the matrix on which it was applied. It allowed a reduction both in cost and the consumption of solvents, thereby safeguarding the health of the analyst and the environment. Environmental monitoring using bees was confirmed to be a valid procedure.

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from the catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L−1 and from 0.7 to 250 U L−1 for CLMrho and CLMb respectively, at 25°C. 5 and 0.7 U L−1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous dete...

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o-phenylendiamine as substrates. Recovery of the assay was 97-112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.

[EN] Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen-antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2¿gmL -1 TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5¿gmL -1 TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion). © 2012 Elsevier B.V., This work was supported by grants from the Italian Ministry of Instruction, University and Research (PRIN 2007AWK85F "Development of integrated analytical instruments based on luminescence principles for multiplexed analysis in POCT (Point-Of-Care Testing) applications" and PRIN 2006033429 "New Analytical Tools for Security and Criminal Investigations: Trace Detection and Identification of Explosives and Related Compounds") and from the University of Bologna ("Fundamental Oriented Research").

A chemiluminescence-based flow method for the determination of some organophorus and carbamate pesticides based on inhibition of acetylcholinesterase was developed. Acetylcholinesterase in solution or immobilized on methacrylate beads (Eupergit C) was coupled to choline oxidase and peroxidase immobilized on Eupergit C. In this system choline formed by acetylcholinesterase was oxidized by choline oxidase and the H2O2 produced was determined via the luminol/peroxidase luminescent reaction. The detection limits (3 σ) for Paraoxon and Aldicarb were 0.75 μg 1−1 and 4 μg 1−1, respectively, when soluble acetylcholinesterase was used under the following optimized experimental conditions: 56 μM luminol in working solution, sample volume 60 μl, flow-rate 0.3 ml min−1 and 60 min incubation time. The flow sensor device using all the three enzymes in the immobilized form had a higher detection limit of 125 μg 1−1 for Paraoxon. The mid-range relative standard deviation (n=10) using 1 mM standard substrate solution was 3.7%. The recovery from contaminated samples (soil, vegetables) varied from 81 to 108%. The results obtained by the developed methods were in good agreement with those obtained by a commonly used colorimetric test.

The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 μm. Other applications, such as the alkaline phosphatase localization in 8 μm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 μm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.

An indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the determination of benzene, toluene, ethylbenzene, and ortho-, meta-, para-xylenes (BTEX) in soil and water was developed. The assay was optimized concerning the coating conjugate concentration, anti-BTEX antiserum dilution, incubation time effect on primary and secondary antibody incubation, and temperature effect on the competitive step and tolerance to different organic solvents. The IC(50) and lower limit of the detection (LDD(90)) values were 4.6 and 0.5 microg mL(-1), respectively. While water samples could be analyzed directly, soil has to be extracted and diluted prior to immunochemical measurements. BTEX could be recovered from spiked soil with a yield higher than 60% using 5-min ultrasonication with methanol. Finally, the assay was applied to soil and water samples collected at a contaminated site in Italy, and was compared to GC-MS.

A Chemiluminescente Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide imidacloprid in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-imidacloprid antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 14.8 ng mL-1 and 0.11 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 0.1 – 2600 ng mL-1. Cross reactivity of some related compounds such as three imidacloprid metabolites, 6-chloro nicotinic acid, 5-hydroxy-imidacloprid and imidacloprid olefin and one other chloronicotinoid insecticide, acetamiprid were tested. The assay was then applied to honeybee extracts obtained by using the liquid-liquid extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 23.7 ng mL-1 and a LOD 1.6 ng mL-1. The average recovery value from honeybee extracts spiked with 100 and 1000 ng mL-1 of imidacloprid were 73% and 76% respectively. Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy. Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and was founded that only five of the 27 samples were positives, with low concentrations of imidacloprid ranging between 1.2 and 15.4 ng g-1.

Involvement of coenzyme Q (CoQ) in anti-oxidant activities, in addition to its major redox role, has frequently been suggested in recent years. In order to elucidate if CoQ could really be engaged in scavenging free radicals produced endogenously in a biological system, an experimental system was developed in which beef heart mitochondria in the presence of a saturating NADH concentration and of rotenone produce free radicals. The presence of oxygen-reactive forms was easily detected by a luminol-dependent chemiluminescence process. The chemiluminescence assay showed that short-chain CoQ homologues can act as pro-oxidants, enhancing free radical effects, while exogenous coenzyme Q 10 could scavenge free radicals, especially at very low concentration. In this system, exogenous CoQ 10 was more effective than α-tocopherol at the same concentration in scavenging free radicals. The molecular mechanism that leads to this ability is still nuclear, but these results are of biochemical and biomedical importance because they indicate that CoQ may act as an anti-oxidant in situations mimicking physiopathological conditions. This direct chemiluminescent method is promising for studies of biochemical processes which involve active oxygen species.

Previously, a bioluminescent flow sensor was developed for the determination of the content of l -lactate in biological fluids (serum, plasma, ventricular fluid) by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. Based on a similar scheme, a sensor has now been developed for d -(−)-lactate. The co-immobilization of alanine aminotransferase (ALT) with d -LDH improved the lactate transformation to 40–60%. The response was linear from 1 to 100 μmol 1 −1 at 25 °C for the LDH-ALT reactor. The intra- and inter-assay relative standard deviations were less than 75% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and applications to d -(−)-lactate determination in serum, milk and microorganism extracts are reported together with those for l -(+)-lactate.

International audience; A Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.

Cling films are prepared by adding (5–30% w/w) adipates, citrates, and phthalates. The adverse effects on human health of the plasticizers have been demonstrated, and then their content in food contact plastics and possible migration to foodstuffs must be carefully checked. To offer an easier tool to perform these controls, a Soxhlet extraction by ethyl acetate and a multi residue chromatography analysis, with Flame Ionization Detector, have been optimised for the application in evaluating both the real content of plasticizers in films and freezing bags, and their migrated amounts in simulants. The average extraction yield was 95 ± 10%. The chromatographic analysis was able to determine 16 different compounds, clearly separated, taking only 25 min to complete the assay. The detection limits ranged from 0.07 to 0.7% (w/w). The total amount of plasticizers in the packaging products was comprised between 3 and 10% (w/w). Extracted and migrated amounts were compared, and they resulted in good agreement. No migration of Phthalates, whose presence has been demonstrated in some samples, has been observed. It has been also demonstrated that a prolonged contact (till 60 days) did not increase the amount of migrated plasticizers, and then the risks for health.

The total antioxidant capacity (TAC) of five different wines (four red and one white) was determined in five different steps of winemaking carried out in a commercial wine cellar by a chemiluminescence (CL) assay. The CL method is suitable to determine the antioxidant capacity of beverages, and preliminary trials showed that the TAC immediately after the bottle was opened was greater than the day after (about 25% decrease). Immediate analysis or a correct sample storage is therefore necessary. The wines were characterized by different levels of total phenolics and TAC: these differences were related to grape composition and winemaking technologies. The TAC values were the highest immediately after devatting. The TAC suffered the highest decrease (30–50%) after the clarification procedure, which may be due to the fining agents used and to oxygen contact, then remained more or less constant in the subsequent steps. Copyright © 2006 John Wiley & Sons, Ltd.

In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. The sensitivity of immunochemical methods was expressed as detection limit, linear working range, and I50 value. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of HRP allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing simply diluted fruit juices, spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more.

Total antioxidant capacity was measured in red wine samples by two analytical methods: chemiluminescent (CL) assay based on the luminol/peroxidase system and a spectrophotometric (SP) method based on the use of crocin. The imprecision, expressed as coefficient of variation (CV) for each method, was similar (1.2%), whereas the detection limit (LOD) was 35 and 0.3 µmol of Trolox for SP and CL methods, respectively. In addition, the chemiluminescent method has the advantage of being simple, rapid, sensitive and inexpensive. Different samples of red wine were analysed and both methods showed that the antioxidant capacity is related to the total polyphenol content and varies over time depending on storage conditions.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40°C followed by a 1[ratio ]10 dilution with UHT (‘XOD-free’) milk. The assay was carried out at 25°C. The response obtained from XOD standard solutions in milk was linear from 0·1 to 500 enzyme units (U) l−1, but for the actual milk samples values ranged only from 1 to 135 U l−1. The detection limit at 2 SD was 0·1 U l−1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6–12%.

A highly rapid chemiluminescent assay for the determination of superoxide dismutase (SOD) activity in erythrocytes was developed. The inhibition of the luminescent emission caused by the decrease of generated superoxide anions was measured. The aim of this work was to verify the application of a non amplified luminol SOD luminescent assay (CLM) in erythrocytes starting from an amplified method already used for the determination of XOD activity in milk (CLME). Both the assays had a detection limit of 3 10 2 9 7 10 3 U:ml of SOD at 2s level, and a linear range of activity from 5.2 to 0.03 U:ml of SOD. The imprecision of assays (repeatability) presented coefficients of variations ranging from 3.1 to 7.9% for the CLME method and from 0.6 to 17.7% for CLM method. Both luminescent techniques were compared using a spectrophotometric kit, that had a detection limit of 0.3 U:ml, and showed good agreement. © 2000 Elsevier Science B.V. All rights reserved.

A new synthetized stable trifluoro-substituted acridan (2′, 3′, 6′-trifluorophenyl 10-methylacridan-9-carboxylate known as Lumigen PS-3) has been applied as chemiluminescent substrate of the horseradish peroxidase (HRP) enzyme (neutral isoenzyme C) in a dot blot hybridization assay for the detection of B19 Parvovirus DNA. The dot blot hybridization assay uses digoxigenin-labeled DNA probes which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with HRP. The results obtained using PS-3 reagent or the luminol-based enhanced chemiluminescence detection system (ECL Amersham and Renaissance DuPont NEN kits) were compared. A high-performance, low-intensity-light imaging luminograph apparatus to collect light emission was used. The detection systems using the different chemiluminescent substrates gave sensitive and reproducible results for calibration graphs, with high precision (relative standard deviation 5–18%). With the chemiluminescent assay it was possible to detect 0.5, 1, or 2 pg of target homologous DNA, using PS-3, ECL, or Renaissance (RE) reagents, respectively, while colorimetry had a detection limit of 5 pg. When clinical samples were analyzed the positive reference sera and the PCR-positive products gave light emissions with values higher than background at 2 σ level, while the negative samples gave a signal comparable to the background noise for all chemiluminescent reagents. The PS-3 reagent detected one more dilution (1/256) than ECL and RE (1/128) of positive reference sera.

In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.

A direct chemiluminescent dot blot hybridization assay for the detection of B19 Parvovirus DNA is described. The hybridization test uses digoxigenin-labelled probes which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (AP) or with horseradish peroxidase (HRP). The chemiluminescent signal, obtained from an enzyme-triggerable dioxetane for Ap or from the luminol-amplified reaction for HRP, is directly measured by placing the spot cut from the nylon solid support in a cuvette and inserting it into a luminometer. Both the enzymatic systems using this direct chemiluminescent detection gave reproducible results for calibration graphs and positive clinical samples, with higher reproducibility and long lifetime emission (15 days) for the AP-dioxetane system. This direct method allowed up to 0.2 pg of homologous target DNA to be revealed. The results obtained with the quantitative chemiluminescent assay described were compared with those obtained in a hybridization assay with colorimetric detection or photographic chemiluminescent detection and good agreement among the tests was found.

Immobilized enzymes are widely used in the clinical laboratory to assay several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable, disposable and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow sensors based on the use of nylon tube coil or epoxy methacrylate column as solid support. For in-vivo determination a suitable microdialysis probe inserted directly into brain or blood allows continuous measurement of extracellular lactate levels by means of a bioluminescent flow detector system. This procedure performs more measurements in the same time interval than other systems (HPLC), e.g. to give a detailed description of the effects of ischemia, or other pathological events, on lactate concentration in the brain.

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.

A continuous-flow bioluminescence method for determinations of branched-chain l -amino acids in serum and urine is described. Serum can be analyzed directly after simple filtration. Response is linear for 20-2000 pmol in the biological matrix. Leucine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bacterial bioluminescence enzymes. The stability of the immobilized is high (lifetime > two months) and more than 900 samples can be analyzed with the use of a few mg of enzymes. The results obtained agree well with those obtained by ion-exchange chromatography (amino acid analyzer).

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.

Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.