The management and exploitation of naturally light-emitting bacteria as a flexible analytical tool: A tutorial.

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.

Trace analysis of pollutants by use of honeybees, immunoassays, and chemiluminescence detection.

Specific and sensitive analysis to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and food is urgently required because of the increasing attention devoted to the environment and health protection. Our research group has been involved in monitoring the presence and distribution of agrochemicals by monitoring beehives distributed throughout the area studied. Honeybees have been used both as biosensors, because the pesticides affect their viability, and as "contaminant collectors" for 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 luminescence detection. The range of chemiluminescent immunoassays developed was extended to include the determination of completely different pollutants, for example explosives, volatile organic compounds (including benzene, toluene, ethylbenzene, xylenes), and components of plastics, for example bisphenol A. An easier and portable format, a lateral flow immunoassay (LFIA) was added to the ELISA format to increase application flexibility in these assays. Aspects of the 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 assay for analysis of peroxide-based explosives.

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.

Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection.

To identify the explosive used in a terrorist attack, or to obtain 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 particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of 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 and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC(50) values in the ng mL(-1) range, and 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 were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (LFIA), developed by using the same immunoreagents, reached a detection limit of 1 microg mL(-1) when tested on the same samples analysed by CL-ELISA.

Column switching liquid chromatography and post-column photochemically fluorescence detection to determine imidacloprid and 6-chloronicotinic acid in honeybees.

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%.

Trace metals in arcid clam Scapharca inaequivalvis: effects of molluscan extracts on bioluminescent bacteria.

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.

Bioluminescent bacteria assay of veterinary drugs in excreta of food-producing animals.

The residues of pharmacological treatments on food-producing animals, present in the manure dispersed on agricultural land, can impact environmental and human health through toxic, genotoxic, and drug-resistance development effects. Biotoxicity assays can easily reveal the presence of noxious substances and those based on bioluminescent bacteria (BLB) are particularly simple and rapid. A BLB assay was developed as microplate format by using various strains of Vibrio sp. and was employed to evaluate their response to pure antibiotic solutions and to residues extracted from excreta of antibiotic treated pigs and turkeys. The residues were quantified by HPLC analysis. The BLB assay can be proposed as an easy-to-perform screening tool to assess the presence of residues due to undeclared current, or recently ended, pharmacological treatments, as well as to evaluate their permanence in manure.

Application of matrix solid phase dispersion to the determination of imidacloprid, carbaryl, aldicarb, and their main metabolites in honeybees by liquid chromatography-mass spectrometry detection.

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 0.004mgkg(-1) for imidacloprid to 0.09mgkg(-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.

Occurrence and distribution of pesticides in the province of Bologna, Italy, using honeybees as bioindicators.

Samples of honeybees (Apis mellifera, n = 92) from 14 beehive monitoring stations located in 3 townships in the province of Bologna were analyzed from April to October 2000. The concentration of 32 organophosphorus pesticides and 5 carbamates was determined through liquid-liquid extraction followed by gas chromatography with a nitrogen-phosphorus detector and liquid chromatography coupled to mass spectrometry using atmospheric pressure chemical ionization in positive and negative ion modes. The most contaminated samples were from Granarolo Emilia where cereals (wheat, sorghum, and corn), sugar beets, and potatoes are the main agriculture products. Thirty-five pesticides were detected, with organophosphorus being the most abundant ones. Malathion was detected in 58% of the samples (mean level 0.360 mg/kg) followed by fenithrothion in 53% of the samples (mean level 0.544 mg/kg) and pirimiphos methyl in 48% of the samples (mean level 0.006 mg/kg). Temporal trends showed that the maximum detection frequency occurred in late spring and was associated with the use of treatment products and less rainfall. The obtained results demonstrated the feasibility of using honeybees for assessing pesticide exposure in agriculture settings.

Automated and manual luminescent assay of antioxidant capacity: analytical features by comparison.

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.

Analysis of organophosphorus pesticides in honeybee by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.

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.

Determination of organophosphorus pesticides in honeybees after solid-phase microextraction.

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.

Multiresidual method for the gas chromatographic analysis of pesticides in honeybees cleaned by gel permeation chromatography.

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.

Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29.

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.

Determination of superoxide dismutase in erythrocytes by a chemiluminescent assay.

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 3x10(-2)+/-7x10(-3) U/ml of SOD at 2sigma 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.

Chemiluminescent determination of xanthine oxidase activity in milk.

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 degrees C followed by a 1:10 dilution with UHT ('XOD-free') milk. The assay was carried out at 25 degrees 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%.

Development of a chemiluminescence competitive PCR for the detection and quantification of parvovirus B19 DNA using a microplate luminometer.

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

A chemiluminescent flow sensing device for determination of choline and phospholipase D activity in biological samples.

A chemiluminescent flow-sensing device for the determination of phospholipase D (PLD) activity and/or choline (Ch) in biological samples using choline oxidase (ChO) and horseradish peroxidase (HRP) immobilized on Eupergit C (polymer beads of methacrylamide, N-methylene-bis-methacrylamide, and allyl-glycidyl-ether) was developed. The best results were obtained with immobilized ChO and HRP at a polymer beads wet weight ratio of 16:1. The optimized parameters of the developed sensing device were 56 microM luminol in working solution; sample volume, 60 microliters; flow rate, 0.3 ml/min; and sample throughput, 15/h. The detection limit (3 SD) using a luminescent enhancer was 1.2 microM for Ch, corresponding to 0.167 mIU of PLD activity per milliliter. Without enhancer the values were 3.0 microM and 0.417 mIU, respectively. The Ch recovery varied between 80.4 and 109%. The biological samples quenched the luminescent light to different extents, and this matrix effect was readily overcome by measuring the luminescent signal of added Ch standard. The flow biosensor was used for the determination of PLD in samples of different origin, including rape seeds during maturation.

Chemiluminescent immunoperoxidase assay for the dot blot hybridization detection of Parvovirus B19 DNA using a low light imaging device.

A new synthesized 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 sigma 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.

Chemiluminescent low-light imaging of biospecific reactions on macro- and microsamples using a videocamera-based luminograph.

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.