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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.

Bioindicators are of fundamental importance in environmental monitoring of contaminants produced and spread by men’s activities. Honeybees are cheap and3 flexible test organisms for passive biomonitoring and bioaccumulation of a large variety of pollutants. Bees can collect on their bodies or include in beehive products the chemicals diffused in the air, on plant surfaces, and dissolved in water allowing an effective screening of possible hazards for human and ecosystem health. We carried out monitoring activities on the environmental distribution of agrochemicals, heavy metals, radionuclides, and phytopathogenic organisms. We placed the “monitoring stations” in selected areas. These consist of a couple of beehives equipped with baskets to collect the dead bees, which are enumerated to evaluate the mortality and analyzed to identify the pollutants. Palynological analyses were combined with analytical results and data processing. Currently, monitoring plans are ongoing in different municipalities. The increasing interest in environmental protection has led us to develop the “BIOAPI-OER” (Open access Educational Resource) project, a short online, free access course on the theme of bees as environmental bioindicators. It targets different target groups such as scholars, Ph.D. students, beekeepers, and professionals interested in environmental biomonitoring.

Introduction Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. Methods Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. Results Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. Conclusions Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. Implications IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

The global range of the environmental quality issues we all face necessitates integrated action from all of the countries that suffer from a particular environmental issue. We must be able to obtain data on the presence of pollutants and their effects on biota from any territory, regardless of its environmental, infrastructural, social, and economic conditions. Biomonitoring utilizes organisms and natural materials to obtain this information. In particular, the honeybee is a ubiquitous, easy-to-breed organism with great mobility. Its body, which is covered with hairs, picks up materials and particulates that it encounters in the environment. Therefore, bees are highly effective accumulators of materials from the soil, vegetation, air, and water. These characteristics mean that the honeybee is both a bioindicator and a passive bioaccumulator organism, making it an ideal agent for easily monitoring vast areas inexpensively, even in regions where infrastructure is scarce. In this short review, we summarize the main targets of the honeybee-based monitoring campaigns that have been carried out to date, highlighting the results obtained in assessments of organic and inorganic pollution performed by coupling more modern technologies with this long-standing practice. It is hoped that this review will make scientists more aware of the incredible potential of these delicate organisms to provide data that could prove useful in the management of environmental issues.

Effective resin disinfection is mandatory to ensure the microbiological quality of water treated by domestic softeners. The wet and sometimes warm environment inside the softener is ideal for bacteria growth. Our research was focused on the evaluation of the microbial quality of water from softeners sanitized by chlorine solutions or by electrolytic systems. We employed the heterotrophic plate count and specific tests to monitor the presence of opportunistic and pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, enterococci, and coliforms). Completely new devices were equipped with a commercially available electrolytic system or with a newly patented one or sanitized by automatic or manual addition of chlorine solutions. In all cases, the contamination was reduced, not completely avoided. In particular, the patented electrolytic system significantly reduced bacterial proliferation in strongly contaminated devices. Our data confirm the difficulties encountered to solve the problem of microbiological quality of softened water and offer encouraging information on new possible solutions. This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.

Due to the lack of European Massive Open Online Courses (MOOCs) in the field of the Toxicology and the major differences in the styles of teaching and learning of this important subject in amongst various European life science-oriented institutions, the European Erasmus+ project “Learning Toxicology through Open Educational Resources (TOX-OER)” was developed and implemented. Considering the complexity and heterogeneity of the toxicology field, the TOX-OER project main objective was to develop and share toxicology-related knowledge and skills among students/ earners from seven countries, which promote the internationalization of Higher Education Institutions in Europe but also in countries from other continents. The project was coordinated by Universidad de Salamanca (Spain), and the partners were: Space Research and Technology Institute (Bulgaria), Univerzita Karlova V Praze (Czech Republic), South-Eastern Finland University of Applied Sciences (Finland), Università di Bologna (Italy), Universidade do Porto (Portugal) and Universitatea Transilvania din Brasov (Romania). One of the goals/ objectives of the project is the dissemination, popularizing the information and reaching potentially interested people who can benefit from the offered courses. The aim of this paper is to present and analyze part of the TOX-OER’s outcomes developed by the project’s partnership, especially the modules/ topics related to the drugs, gaseous and persistent organic pollutants, as the principal groups of xenobiotics.

Hemp cultivation is living a period of renewed interest worldwide after long years of opposition and abandonment. The European Union (EU) allows and subsidizes the growing of fiber and oilseed cultivars of Cannabis sativa L. with respect to the THC content limit of 0.2%. The EU method for the quantitative determination of Δ9-tetrahydrocannabinol (THC) content in hemp varieties provides to apply a tolerance of 0.03 g of THC per 100 g of sample concerning compliance assessment to that limit. However, the method does not report any precision data, especially useful as a function of THC content to evaluate measurement uncertainty and therefore to establish the conformity of hemp at different THC legal limits. Measurement uncertainty of the method by both bottom-up and top-down approach, besides repeatability and reproducibility, was investigated and estimated in the THC concentration range 0.2–1.0%, which includes the different legal limits set out for hemp around the world. We proposed Decision Rules for conformity of hemp showing that a non-compliant declaration beyond reasonable doubt should be stated when the THC content, as a mean result on a duplicate analysis, exceeds the limit by at least 11–15%, depending on THC limit. We highlighted other issues concerning practical aspects of hemp analysis, from sampling to evaluation of results, as well as the need to carry out collaborative studies on the EU method. Graphical abstract

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.

In this work, PM10 samples previously subjected to thorough chemical speciation and receptor modelling, have been investigated for their bio-toxicity using an inhibition test based on bacterial luminescence modulation when in contact with airborne particulate samples. The variation of light emission intensity from a luminescent bacteria strain, the Photobacterium phosphoreum, is proposed as an efficient proxy for the quantification of bio-toxic effects induced by airborne particulate matter. PM10 samples characterized by definite levels of pollutants from the pertaining air shed were found to induce a decrease in the bacterial bioluminescence intensity, expressed as percentage of Inhibition Ratio (IR%). This behaviour suggests the decay of this energy-consuming activity because of a toxic effect. Cluster analysis on chemical composition and IR% data provides evidence of a statistically significant association between the adverse effects on living cells and the range of specific chemical species in PM10.

This article is an update version of G. Gübitz, M.G. Schmid, IMMUNOASSAYS, TECHNIQUES | Luminescence Immunoassays, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole, Encyclopedia of Analytical Science (Second Edition), Elsevier, 2005, Pages 352–360, ISBN 9780123693976.Immunoassays (IAs) are analytical methods based on the specific recognition between the antigen (Ag), which molecular weight can vary in a wide range, and the variable regions of antibody (Ab), a Y shaped macromolecule composed by four polypeptide chains. IAs are applicable without analytes enrichment, purification, or pretreatment having great advantages over standard methods. Immunoassays relate to medicine, food quality control, forensic veterinary, and environmental analysis. A detectable label conjugated on one or both of the immunoreagents (IRs) reveals the recognition between Ag and Ab. The evolution of IAs corresponded to the evolution of the labeling technology and the optical detection is the simplest way to evaluate the analytical signal.

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 (

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.

Conventional detection of toxic contaminants on surfaces, in food, and in the environment takes time. Current analytical approaches to chemical detection can be of limited utility due to long detection times, high costs, and the need for a laboratory and trained personnel. A non-specific but easy, rapid, and inexpensive screening test can be useful to quickly classify a specimen as toxic or non toxic, so prompt appropriate measures can be taken, exactly where required. The bioluminescent bacteria-based tests meet all these characteristics. Bioluminescence methods are extremely attractive because of their high sensitivity, speed, ease of implementation, and statistical significance. They are usually sensitive enough to detect the majority of pollutants toxic to humans and mammals. This tutorial provides practical guidelines for isolating, cultivating, and exploiting marine bioluminescent bacteria as a simple and versatile analytical tool. Although mostly applied for aqueous phase sample and organic extracts, the test can also be conducted directly on soil and sediment samples so as to reflect the true toxicity due to the bioavailability fraction. Because tests can be performed with freeze-dried cell preparations, they could make a major contribution to field screening activity. They can be easily conducted in a mobile environmental laboratory and may be adaptable to miniaturized field instruments and field test kits.

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.

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.

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%.

This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000–2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

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.

The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the “bioactive” metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

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.

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.

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.

In this study we describe an efficient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the firefly luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our findings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.

A sensitive, rapid and simple bioluminescent (BL) assay using bioluminescent bacteria to detect the toxic activity of several chemicals is described. This assay is based on the measurement of inhibition of light production of a bioluminescent bacterial strain, isolated from seawater, in the presence of different toxins like heavy metals, organic chemicals, such as benzene, toluene, ethylbenzene, xylene (BTEX) and a wide range of pesticides in environmental samples. The improvement with respect to other commercial and non-commercial bioluminescent assays consists of the possibility to work at room temperature without the need to thermostat, thus allowing the use of simpler and low cost instruments, or to improve the assay using a microplate format, which makes it possible to analyse several samples also continuously for several hours. Using lyophilised bacteria, the assay is performed in less than an hour, without any bacterial cultivation, which makes the test suitable for rapid and sensitive evaluation of chemical pollutants in environmental samples.

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.

Competitive ELISA estimation based on application of polyclonal mouse antibodies to somatic antigen O:9, 12 was developed. The optimization of the protocol is reported. The optimal concentration of immobilized somatic antigen O:9, 12 was found to be 4.9 2 104 cells ml−1; optimal concentration of mouse IgG was 6.25 w g ml−1; and the optimal concentration of peroxidase labelled antibody to mouse IgG was 8 w g ml−1. The tested antibody exhibited neither cross reactions with chosen strains (serotypes) of salmonellas group 04 (B), 07 (C1), 08 (C2-C3), nor with members of Enterobacteriaceae: Escherichia coli, Klebsiella pneumonia, Citrobacter freundii and non-fermenting bacterium Pseudomonas fluorescens. Application of chemiluminiscent substrates increased the sensitivity of S. enteritidis detection up to three times. Competitive ELISA tested on model samples produced results comparable with standard cultivation techniques for Salmonella spp.

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.

An enzyme immunoassay was used to determine benzo[a ]pyrene (BaP) in smoked meat products and other samples of food and environmental origin. The method used has a detection limit (3 σ) of 0.1 μg kg−1 and a coefficient of variation less than 10%. The main aim of the study was to compare the possible influence of different smoking processes and packaging material on the amount of BaP deposited on smoked meat product, mainly different sausages. The lowest amount of BaP was found when smoke produced by steam in the indirect method smoking-chamber was used. A slightly protective effect of polyamide casing was noted. © 1999 Society of Chemical Industry

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.

The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when single-stranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co(2+) and Br(-) on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co(2+) and Br(-) quenchers. The Stern-Volmer constants (K (SV)) for Co(2+) and Br(-) quenching effect on the studied alkaloids were considerably reduced (10-50%) after incubation of the compounds in the presence of DNA with regard to the K (SV) values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (K (ass) = 2.2 × 10(2) - 1.3 × 10(3)), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.

Bioremediation has proven successful in numerous applications to petroleum hydrocarbons or chlorinated aromatic hydrocarbons contaminated soils. There is increasing interest for application of biotoxicity tests for ecological assessment and for supporting management decisions for remediation. Luminescent assays, light-emitting bacteria in particular, can be a suitable tool for environmental analysis, and in vivo luminescence is a rapid and precise indicator of the toxic effects of xenobiotic on micro-organisms. In this study three different strains of marine bioluminescent bacteria have been employed to follow the changes in biotoxicity occurring during the laboratory scale bioremediation of water and soil samples contaminated by hydrocarbons and collected at an industrial area. The degradation was made by hydrocarbons degrading bacteria, both of commercial sources and isolated from polluted water and soils. The samples were treated for 45 days. The toxicity of the samples, before and after the bioremediation was determined directly on water samples or on the extracts of soil samples. The yield of extraction of different solvents (acetone, dioxane, ethanol, and dichloromethane) was evaluated by the bioluminescent test. The measurements were carried out using a microplate format both for short time of contact (60 minutes, acute toxicity) and for longer time intervals (24 hours, chronic toxicity). The results have been expressed as percentage of inhibition with respect to the blank emission (100% emission). Original and treated samples have been analysed by gas chromatography to assess the hydrocarbons (C>12 and Poly Chlorinated Biphenyls, PCB) content. The autochthonous bacteria isolated from polluted samples resulted less effective, due to the short time for selection, in remediation activity with respect to the commercial ones, but their capacity to degrade long chain hydrocarbons was satisfactory. The presented laboratory study can be applied also in case of on-field conditions.

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.