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Accomplishing the Italian law to verify honey quality is onerous, because it requires measuring many chemical and physical parameters. On the contrary, bioluminescence-based analytical methods allow for rapid and inexpensive analysis. Bioluminescence has never been applied before to verify honey adulteration. The application of chemometrics to analytical methods based on bioluminescence has been here explored for this scope. Several honey samples were prepared, in which sugar syrup was added without exceeding legal limits: in this case, univariate analysis prescribed by the law cannot reveal the fraud. All samples were subjected to measurements of parameters prescribed by the law and also to bioluminescence analysis, executed using the Vibrio fischeri bacterium, one of the most common bioluminescent bacteria. Principal components analysis, linear discriminant analysis, and partial least square regression were applied to discriminate sugar-added honeys with respect to natural honeys, both by regulated physicochemical parameters and by bioluminescence ones. The feasibility of combining bioluminescence and multivariate analysis for a rapid screening of honey authenticity was demonstrated.

The development of methodologies for the characterization of silver nanoparticles (AgNPs) synthesized using natural products has received increasing attention, especially to monitoring its stability and size for further application. In this paper, a capillary electrophoretic (CE) method is presented for characterization of AgNPs synthesized using honey or glucose as reducing agents. A simple electrolyte solution composed of 20 mM sodium borate and 20 mM sodium dodecylsulfate (SDS) at pH 8.5 was used for separation of AgNPs within a short analysis time (

Specific and sensitive analyses tailored to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and finally foods are strongly required following the increasing attention to the environment and health protection. Our research group has been involved in monitoring activities on agrochemical presence and distribution based on beehive monitoring stations distributed throughout the area. Honeybees were employed both as biosensors concerning the pesticides affecting their viability and as “contaminant collectors” concerning all environmental pollutants. We focused our research on the development of analytical procedures able to reveal and quantify pesticides in different samples but with a special attention to the complex honeybee matrix. Specific extraction and purification procedures have been developed and some are still under optimization. The analytes of interest were determined by gas or liquid chromatographic methods and by compound-specific or group-specific immunoassays in the ELISA format, the analytical performance of which was improved by introducing a luminescent end-point detection. The range of developed chemiluminescent immunoassays was extended to include the determination of completely different pollutants such as explosives, volatile organic compounds including benzene, toluene, ethylbenzene, xylenes, and plastic components like Bisphenol A. An easier and portable format, a Lateral Flow Immuno Assay (LFIA) was added to the ELISA format to increase the application flexibility of these assays. The aspects of novelty, the specific characteristics, the analytical performance and possible future development of the different chromatographic and immunological methods are described and discussed.

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.

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos‐methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti‐mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos‐methyl assay, estimated as the IC50 value, was found to be 1.2 nmol L−1 (60 min incubation), with a linear range of 0.6–500 nmol L−1 in optimal conditions. The matrix effect on the detection of azinphos‐methyl in honeybee extract was found negligible, with the recovery values in the range 92–105%.

With the aim to evaluate the reliability of enzyme immunoassays in the determination of Aflatoxin M1 content in raw and pasteurised milk samples, in comparison with the HPLC reference method, the performances of ELISA kits were tested by analysing a large number of both artificially and naturally contaminated samples. The efficiency of the aflatoxins extraction before HPLC analysis was carefully checked in Afaltoxin M1 spiked samples and by using Aflatoxin B2 added as internal standard. Recovery (mean values: 100.1% and 97.8% for aflatoxin M1 and B2, respectively), was lowered only in freezed samples. No effect was detected in case of column overloading, presence of food preservatives or acid pH values. The ELISAs gave the same values of HPLC for the CV (coefficient of variation), recovery, and regression coefficient R2 (0.9-8% and 96.8-108%, and 0.993 respectively) analysing samples containing till 70 ng L-1 of Aflatoxin M1. When the concentration rised till 100ng L-1 a slight overestimation was observed, with CV 7-21%, recovery 96.2-115%, and R2 = 0.876. Analysing a set of 600 real samples, both raw and pasteurised milk, the overestimation by ELISA was confirmed, with a R2=0.788. The evidences in this study confirmed that ELISA is a reliable alternative assay to HPLC in the routine quality control of raw or packaged milk. Regulatory rules require to confirm positive samples by HPLC, but using ELISA no one case of false-negative determination can occur.

The capability to collect timely information about the substances employed on-site at a crime scene is of fundamental importance during scientific investigations in crimes involving the use of explosives. TNT (2,4,6-trinitrotoluene) is one of the most employed explosives in the 20th century. Despite the growing use of improvised explosives, criminal use and access to TNT is not expected to decrease. Immunoassays are simple and selective analytical tests able to detect molecules and their immunoreactions can occur in portable formats for use on-site. This work demonstrates the application of three immunochemical assays capable of detecting TNT to typical forensic samples from experimental tests: an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label, and a chemiluminescent-LFIA (CL-LFIA). Under optimised working conditions, the LOD of the colorimetric LFIA and CL-LFIA were 1 μg mL−1 and 0.05 μg mL−1, respectively. The total analysis time for LFIAs was 15 min. ELISA proved to be a very effective laboratory approach, showing very good sensitivity (LOD of 0.4 ng mL−1) and good reproducibility (CV value about 7%). Samples tested included various materials involved in controlled explosions of improvised explosive devices (IEDs), as well as hand swabs collected after TNT handling tests. In the first group of tests, targets covered with six different materials (metal, plastic, cardboard, carpet fabric, wood and adhesive tape) were fixed on top of wooden poles (180 cm high). Samples of soil from the explosion area and different materials covering the targets were collected after each explosion and analysed. In the second group of tests, hand swabs were collected with and without hand washing after volunteers simulated the manipulation of small charges of TNT. The small amount of solution required for each assay allows for several analyses. Results of immunoassays confirmed that they were suitable to detect post-blast residues in soil and target materials and post transfer residues on hands, allowing further confirmation by more selective techniques. ELISA and LFIAs results obtained from the same solution were consistently in good agreement, and were confirmed by gas chromatography coupled to mass spectrometry (GC–MS). The reported immunoassays data demonstrates the suitability of LFIAs as on-site rapid and effective assays to detect TNT traces. The CL-ELISA proved useful in obtaining very sensitive detection in forensic investigations and testing, while CL-LFIA had performances in between LFIA and CL-ELISA.

A method based on matrix solid phase dispersion (MSPD) using C18 as dispersant and dichloromethane–methanol as eluent and liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) has been developed for the simultaneous determination of imidacloprid, 6-chloronicotinic acid, carbaryl, aldicarb, aldicarb sulfoxide, and aldicarb sulfone in honeybees. The proposed method was compared with liquid–liquid extraction (LLE) combined with LC–APCI–MS analysis. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. Recovery studies were performed at different fortification levels. Average recoveries by MSPD varied from 61% of 6-chloronicotinic acid to 99% of aldicarb sulfoxide and relative standard deviations were equal or lower than 14%. Limit of detections ranged from 0.004 mg kg−1 for imidacloprid to 0.09 mg kg−1 for 6-chloronicotinic acid. Results obtained by both methods were compared, MSPD showed higher recoveries and sensitivity than LLE for most pesticides, except for carbaryl. As MSPD is easier to perform, faster, consumes less sample and organic solvents than LLE, its application for pesticide analysis in honeybees is suggested.

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.

Imidacloprid is a new insecticide with a wide range of action. Because honeybees are very sensitive to this substance, two techniques (HPLC–UV and GC–MS) which enable its detection in several matrices of both animal and vegetable origin were used to monitor its possible presence in cultivated land. In the first method quantification of imidacloprid in honeybees was achieved by use of the external standard method; the detection limit was 50 mg kg−1, the linear range 0.05–1 mg mL−1, recovery 60–83%, and the imprecision (coefficient of variation) 8.6% for repeatability and 11.8% for reproducibility. Recovery from pollen was 71–98% in the range 0.05–0.5 mg kg−1. The repeatability was 9.2–13.9%. Imidacloprid can often be found in the environment, not as a simple molecule but as a group of degradation products. The GC–MS method could be used to quantify all these species as oxidation products and to determine the initial quantity of imidacloprid by use of a conversion factor. The liquid chromatographic analysis could be used to detect, in a standard solution, 10 ng mL−1 derivatized 6-chloronicotinic acid. The linearity was good (R=0.999) over a wide concentration range (10 μg mL−1–10 ng mL−1). Several samples with different matrices (filter paper placed on an pneumatic corn seed drill, grass, flowers, honeybees, etc.) obtained during the sowing period for imidacloprid-treated corn were analyzed. The quantification limit (LOQ) was 0.005 mg kg−1 for grass and flowers, 0.002 mg kg−1 for honeybees, and 0.024 mg kg−1 for paper filters.

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 bioluminescent flow sensor, previously developed for the determination of both d- and l-lactate in clinical samples, was utilized to carry out the same assay in beer. The sensor monitored the reduced form of nicotinamide adenine dinucleotide, produced by nylon-immobilized d- and l-lactate dehydrogenase, by means of bacterial bioluminescent enzymes immobilized on a separate nylon coil. The preparation of beer samples was very simple as only a modification of pH and a dilution were necessary. The recoveries ranged from 91% to 104%, and the relative standard deviations at the 1 mmol 1–1 level were 4.6% and 6.7% for l- and d-lactate respectively. The response was linear in the range 0.1–10 mmol 1–1 for both d- and l-lactate. The total amount of lactate determined by bioluminescent biosensor (x) and by HPLC (y) showed a very good correlation (y=0.654 x+88.1, n=29, r=0.918). The flow injection system developed allowed the determination of not only the total but also the individual contents of d- and l-lactate in beer, and the timely discovery of the unwanted presence of lactic acid bacteria.

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.

A new amperometric immunosensor for 2,4,6- trinitrotoluene based on the working principle of competitive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled monolayers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our approach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determination of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidencing a good recovery rate (113 %).

[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.

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-phenylalanine (Phe) in serum by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized phenylalanine dehydrogenase (PheDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The L-PheDHs extracted from Bacillus badius, Bacillus sphaericus and Rhodococcus sp. M 4 were investigated and the performances of the three immobilized L-PheDH's were analysed. The B. badius reactor was found to give higher transformation rate and better sensitivity; the response was linear from 1 to 100 microM at 25 degrees , with a detection limit of 10 pmoles (0.5 microM). The intra- and inter-assay coefficients of variation were less than 5% and recoveries ranged from 90 to 101%. The results agreed well with those obtained with a chromatographic method for the Phe determination in serum and with the normal reference values.

A continuous-flow system was developed for the assay of magnesium(II) in serum, drugs and beverages, making use of firefly luciferase (LUC)(EC 1.13.12.7) or recombinant luciferase (r-LUC) from Escherichia coli immobilized on a nylon coil or a column of epoxy methacrylate (Eupergit) placed inside a luminometer. The flow was air segmented and 10 µl samples were analysed. The sample assay rate was 20 h–1 with no carryover. The optimum operational conditions were found to be 0.05 mmol l–1 luciferin and 0.3 mmol l–1 ATP (adenosine triphosphate) at a flow rate of 0.4 ml min–1. The detection limit of the assay was 0.05 mmol l–1 for an LUC—nylon coil and 0.01 mmol l–1 for an LUC-Eupergit column or an r-LUC—nylon coil and the response was linear from 0.05 to 6.7 and from 0.01 to 6.7 mmol l–1, respectively. Both the intra- and inter-assay relative standard deviations were lower than 7%. No interferences from ions present in the samples (Ca2+, Mn2+, Fe2+, Cu2+, Zn2+, Co2+) were found. Results obtained for serum by the present method were in good agreement with those given by spectrophotometry with Xylidyl Blue II and by atomic absorption spectrometry.

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.

The quantitation of the natural cytotoxic and anti-inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP-HPLC method with fluorimetric detection and a C(18) stationary phase was applied. Different ACN/water mobile phases were assayed, including 0-4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert-butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP-HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0 x 10(-10) to 2.0 x 10(-10) M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-Glutamate (Glu) in serum and stock cubes by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized glutamate dehydrogenase (GluDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The response was linear from 50 to 1000 μmol l−1 at 25 °C for the GluDH reactor. The intra- and inter-assay relative standard deviations were less than 7 % and recoveries ranged from 88 to 105 %. The results agreed well with those obtained with a spectrophotometry method for stock cubes and with a chromatographic method for the determination in serum.

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.

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.

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 determination of imidacloprid and its main metabolite (6-chloronicotinic acid) in honeybees was performed by liquid chromatography with post-column photochemical derivatisation in alkaline medium and fluorescence detection. The compounds were extracted from honeybees with acetone under ultrasound conditions prior to liquid-liquid partition with dichloromethane. The separation of extract components was performed using a 50 mm x 4.6 mm i.d. short column packed with 5 microm Aquasil C(18) using an acetonitrile:water gradient program as mobile phase. Injection of samples in 0.1 mol L(-1) H(3)PO(4)/KH(2)PO(4) buffer solution (pH 3) improved the chromatographic separation between the most polar components of matrix and the 6-chloronicotinic acid. Matrix components were removed to waste using an on-line clean-up method previously to post-column reaction. Limits of quantification were 0.3 and 5.0 microg L(-1) (corresponding to 1.2 and 20.0 microg kg(-1) in the honeybee sample) for imidacloprid and 6-chloronicotinic acid, respectively. The recovery was ranged from 80.2 to 91.7% with a relative standard deviation lower than 9.0%.

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.

Pesticides applied in extended agricultural fields may be controlled by means of bioindicators, such as honeybees, in which are the pesticides bioaccumulate. Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) experiments with positive (PI) and negative (NI) ion modes were optimized for the analysis of 22 organophosphorus pesticides in honeybee samples. The extraction required 3 g of sample, which was extracted with acetone. The extract was purified with coagulating solution and reextracted with Cl(2)CH(2). Pesticides studied could be detected by both ionization modes except for parathion, parathion-methyl, and bromophos, which did not give signals in PI mode, and triazophos, which was not detected in NI mode. Fragmentation voltage and vaporizer temperature were optimized to achieve the highest sensitivity. The spectra profile of each pesticide in PI mode showed the [M + H](+) ion as the main signal, whereas in NI mode only fragment ions were shown. The detection limit obtained in selected ion monitoring mode ranged from 1 to 15 microg kg(-1). The average recoveries from spiked honeybees at various concentration levels (0.5-5 mg kg(-1)) exceeded 65% with relative standard deviations of 4-15%. The method was applied to real samples, in which residues of coumaphos and dimethoate were detected.

A method based on solid-phase microextraction (SPME) followed by gas chromatography with nitrogen-phosphorus detection was developed for the purpose of determining 18 organophosphorus pesticide residues in honeybee samples (Apis mellifera). The extraction capacities of polyacrylate and poly(dimethylsiloxane) fibers were compared. The main factors affecting the SPME process, such as the absorption time profile, salt, and temperature, were optimized. The method involved honeybee sample homogenization, elution with an acetone:water solution (1:1) and dilution in water prior to fiber extraction. Moreover, the matrix effect on the extraction was evaluated. In samples spiked at the 0.2 mg kg(-1) level, the coefficient variation was between 1 and 13% and the detection limits were below 10 microg kg(-1). The SPME procedure was found to be quicker and more cost-effective than the solvent extraction method commonly used. The method was applied successfully to environmental screening. Parathion methyl was detected and confirmed in the real samples analyzed.

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.

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.

The amount of L-lactate in biological fluids (serum, plasma and cerebrospinal fluid) was determined by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced by immobilised lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilised on a separate nylon coil. The LDH catalysed the reaction of L-lactate with NAD; this reaction took place in a nylon coil that preceded the coil for the bioluminescent detection. The co-immobilisation of alanine aminotransferase (ALT) with LDH improved the lactate transformation by 117-183%. The response was linear from 0.1 to 50 micron mol l(-1) at 25 degrees C for the LDH - ALT reactor. The intra- and inter-assay coefficients of variation were less than 5% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.

A bioluminescent flow sensor was developed for the assay of creatine kinase (CK) using firefly luciferase immobilized on a nylon coil. The CK-catalysed reaction of creatine phosphate with ADP took place in a cuvette before the injection into the bioluminescent detector coil. The response was linear from 0.1 to 100 U l − at 25°C. An advantage of the flow sensor is a detection limit of less than 0.1 U l −1 , which, together with a high precision, allows determination of the CK activity in blood sera in about 5 min. The intra- and inter-assay reproducibilities (RSD) were less than 10% and the recovery range was 86–110%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.

A continuous-flow system, based on NAD(P)H:FMN oxidoreductase and bacterial luciferase co-immobilized on a nylon coil placed in front of a photomultiplier, and alcohol dehydrogenase separately immobilized on a second nylon coil, is described for the assay of ethanol in serum and saliva. The flow is air-segmented and 5–50-μl samples are required. The sample throughput is 25–30 h −1 with no carryover. The detection limit is 1 μmol l −1 ethanol (50 pmol injected) and the response is linear between 50 and 2500 pmol of ethanol. The relative standard deviation is 3–10% for both within-day and between-day assays, and serum and saliva can be analyzed directly. The immobilized enzymes are satisfactorily stable and up to 900 samples can be analyzed with one enzyme reactor.

A carbon dioxide electrode has been used for kinetic studies of decarboxylase activity. The methodology used requires only a sensitive pH meter operating in the millivolt mode with the electrode. The CO2 release may be followed continuously. Compared to other assay procedures the method outlined offers the advantages of rapidity, continuity, specificity, and sufficient precision. Techniques for the calibration of the electrode response are discussed. The applicability of this assay was appraised by determining the Km of lysine decarboxylase. The value obtained showed good agreement with that reported in literature. The procedure might be useful for the study of enzyme-inhibitor interactions.

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).

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.