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.

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.

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.

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%.

Bioluminescent flow sensor for L-phenylalanine determination in serum.

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.

Microdialysis and luminescent probe: analytical and clinical aspects.

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

Bioluminescent flow sensor for the determination of L-(+)-lactate.

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

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence.

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H2O2 (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.

Bioluminescent flow sensors: L-lactate dehydrogenase activity determination in serum.

The catalytic activity of serum L-lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L-lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37 degrees C and from 3 to 2000 U/l at 25 degrees C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.

Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.

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

Radioactivity in breast milk and placentas during the year after Chernobyl.

After the April 1986 nuclear reactor accident at Chernobyl in the Union of Soviet Socialist Republics, samples of human placenta and breast milk were tested for 1 year to determine the levels of radioactivity. The radionuclide iodine 131 was never beyond the detection limit of our gamma detector for both matrices. As to cesium isotopes 134 and 137, the highest levels detected in breast milk (6 Bq.L-1) and placenta (15.8 Bq.kg-1) were recorded in March 1987. Study data for breast milk and placenta are in agreement with the values calculated by means of double-compartment food-milk and food-placenta models. With regard to placental content, the cesium contribution to the average dose during the year after the Chernobyl accident was calculated to be 40 to 60 microSv.

Continuous-flow determination of primary bile acids, by bioluminescence, with use of nylon-immobilized bacterial enzymes.

We describe a continuous-flow bioluminescence method for measuring primary bile acid in serum, with nylon as the solid support. 7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1. 159), a bacterial luciferase (EC 1.14.14.3), and NAD+:FMN oxido-reductase (EC 1.6.8.1) are covalently co-immobilized on a nylon coil (1 m X 1.0 mm i.d.). The assay is highly specific for 7 alpha-hydroxy bile acids. Other bile acids and steroids do not interfere. The continuous-flow light-emitting system, in which the reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer, is versatile and simple. The flow is air-segmented, and serum samples (5-50 microL) can be injected directly. Concentration and response are linearly related from 10 to 2500 pmol per assay tube. The precision of the method is satisfactory (CV 5-10%), both inter- and intra-assay. We validated the technique by comparing results with those by RIA, enzyme immunoassay, and "high-performance" liquid chromatography. More than 20 samples an hour can be analyzed, with no carryover. The nylon-immobilized enzymes are stable for more than two months, and greater than 500 samples can be analyzed with use of a few milligrams of enzymes. Normal values for bile acid content of serum ranged from 1 to 2.5 mumol/L, in agreement with those obtained by other methods.