Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish

Dioxin-like polychlorinated biphenyls (DL-PCBs) often make up the majority of the toxic equivalent (TEQ) contribution of dioxins found in fish samples. For the purpose of making risk assessments, it is therefore important to develop screening methods for determining TEQ concentrations of DL-PCBs in retail fish. We have developed a rapid biosensor immunoassay (BIA) for DL-PCBs that uses a surface plasmon resonance sensor (Biacore 3000). The BIA is highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118) that is generally the most abundant DL-PCB isomer found in fish. The fish extracts were first cleaned up on a multilayer silica gel column followed by an alumina column, then subjected to the assay. The quantitative limit of the assay was 1 ng PCB 118 per gram of tested sample. Dilution and recovery tests using purified fish extracts suggested that the matrix effect was minimized in the assay by diluting the analyzed samples. The assay results for retail fish samples (n=7) agreed well with those obtained by an enzyme-linked immunoassay (ELISA) using the same monoclonal antibody: ELISA has been already validated for determining DL-PCBs in fish samples, so BIA performs well in this analysis. Finally, BIA results for the TEQ concentrations of DL-PCBs in retail fish samples (n=10) correlated well with those obtained by high-resolution gas chromatography coupled to high-resolution mass spectrometry (r=0.89). Our method is therefore useful for screening retail fish to determine the TEQ concentrations of DL-PCBs.     

Dual biosensor immunoassay-directed identification of fluoroquinolones in chicken muscle by liquid chromatography electrospray time-of-flight mass spectrometry

Fluoroquinolones (FQs) are synthetic antibiotics of broad-spectrum antibacterial activity widely used to treat infections in farmed fish, turkeys, pigs, calves and poultry. Monitoring these substances residues is therefore regulated by law. For the detection of FQs, we studied the feasibility of coupling the simultaneous screening of several FQs, using a dual surface plasmon resonance (SPR) biosensor immunoassay (BIA), in parallel, with an analytical chemical methodology for their identification. Six FQs were simultaneously screened at or below their maximum residue level (MRL) in chicken muscle using a multi-FQ BIA for norfloxacin, ciprofloxacin, enrofloxacin, difloxacin and sarafloxacin, and a specific BIA for flumequine. The two BIAs were serially coupled in a multi-channel SPR biosensor featuring a dual BIA in a competitive inhibition format. The samples non-compliant during the screening with the dual BIA were further concentrated and fractionated with gradient liquid chromatography (LC). The effluent was splitted toward two 96-well fraction collectors resulting in two identical 96-well plates. One was re-screened with the dual BIA to identify the immunoactive fractions and direct the identification efforts toward the relevant fractions in the second well-plate with high resolution LC-electrospray time-of-flight mass spectrometry (ESI-TOFMS). The system not only allows the possibility to screen and identify known FQs, but also to discover unknown chemicals of similar structure which show activity in the dual BIA.     

An Electrochemical Acetylcholinesterase Biosensor Based on Fluorene(bisthiophene) Comprising Polymer for Paraoxon Detection

In this study, a novel conductive polymer comprising biosensor based on poly-2,2′-(9,9-dioctyl-9 h-fluorene-2,7-diyl)bistiophene (Poly(BT)) and acetylcholinesterase (AChE) was reported for the determination of paraoxon. This practical biosensor allowed to catalyze electrochemical oxidation of acetylthiocholine (+0.6 V vs. Ag reference). The detection range for acetylcholine chloride (AThCl) with Poly(BT)/AChE was found to be 0.025–4 mM. In pesticide analysis, wide linear ranges from 0.5 to 1 μg/L and 1 to 14 μg/L, and a low detection limit of 0.033 μg/L were estimated. Under optimum operating conditions, the developed biosensor was used for pesticide detection in milk and tap water samples, effectively.     

Determination of pyrimethamine in animal tissue and egg by liquid chromatography with fluorescence detection

A sensitive and selective liquid chromatographic (LC) assay was developed to determine the concentration of pyrimethamine in animal tissue and egg by fluorescent derivative. Animal samples were extracted with acetonitrile, centrifuged, and purified by hexane. Fluorescent derivatization was performed by reacting pyrimethamine with chloroacetaldehyde and subjected to LC with fluorescence detection (excitation wavelength 300 nm, emission wavelength 420 nm). The limit of detection was 10 ng/g (10 ppb) and the standard calibration curve was linear in the range of 1-100 ppb (0.01-1 ng/10 microL). Recoveries from samples fortified at levels of 0.1 and 1 ppm (microg/g) were 61.0-77.4 and 65.5-81.2%, respectively. The method was applied to the monitoring of marketed samples. Pyrimethamine was not determined in any of the 70 samples: 20 swine muscle; 20 chicken muscle; 10 chicken liver; and 20 egg.     

Determination of endocrine-disrupting phenols in water samples by a new manual shaking-enhanced, ultrasound-assisted emulsification microextraction method

Manual shaking-enhanced, ultrasound-assisted emulsification microextraction (MS-USAEME) combined with ultraperformance liquid chromatography (UPLC) with UV detection has been developed for the determination of five endocrine-disrupting phenols (EDPs) in seawater samples and detergent samples: 4-tert-butylphenol (4-t-BP), 4-cumylphenol (4-CP), 4-tert-octylphenol (4-t-OP), 2,4-di-tert-butylphenol (2,4-di-t-BP) and 4-nonylphenol (4-NP). Optimum conditions were found to be: 25 μL 1-bromohexadecane as extraction solvent, 5 mL of aqueous sample and 1 g of NaCl to control the ionic strength; manual shaking for 10 s; ultrasonication for 1 min; centrifugation for 3 min at 5000 rpm (speed). For MS-USAEME, manual shaking for 10 s is essential for effective extraction when the ultrasonic extraction time is as brief as 1 min. The small volume of aqueous sample enhances the effect of manual shaking significantly. For seawater samples, the limit of detection (LOD) was 0.5-2.8 ng mL(-1), the limit of quantification (LOQ) was 1.8-9.3 ng mL(-1) with the relative standard deviation (RSD) in the range 4.2-10.3%. For detergent samples, the LOD was 0.4-2.4 ng mL(-1), LOQ was 1.6-8.2 ng mL(-1) and RSD 4.7-10.0%. The relative recovery was 96-109% for seawater samples and 81-106% for the detergent samples.     

Determination of ochratoxin A at part-per-trillion level in Italian salami by immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detection

A fast high-performance liquid chromatography method has been devised for the determination of ochratoxin A (OTA) in Italian salami in the low part-per-trillion (pg/g) level. The samples were extracted with ethyl acetate and purified by immunoaffinity column (IAC). The IAC eluate could be directly injected or previously concentrated 10-fold. Recovery at 0.5 and 1 ng/g was 77 +/- 4%. The between-day coefficient of variation measured over 5 days on samples spiked at 1 ng/g was 8%. The developed method required a relatively small volume of non-halogenated organic solvent and the whole procedure was simpler and faster compared to other existing procedures. The limit of detection was 0.06 ng/g that could be even lowered using a preconcentration step. A total of 30 salami samples were analysed using this procedure; the most contaminated sample was found to have OTA concentration at 0.4 ng/g level.     

Determination of chloramphenicol residue in fish and shrimp tissues by gas chromatography with a microcell electron capture detector

A gas chromatography method with microcell electron capture detection was developed for the determination of chloramphenicol residue in fish and shrimp muscle tissues. The tissue samples were extracted with ethyl acetate, defatted with hexane, and derivatized with Sylon BFT [N,O-bis (trimethylsilyl) trifluoroacetamide-trimethylchlorosilane (99 + 1)]. The limit of detection was 0.04 ng/g and the limit of quantitation 0.1 ng/g. Average recoveries were 70.8-90.8% for fish and 69.9-86.3% for shrimp, respectively. The method was validated for the determination of practical samples.     

Quantification of the xenoestrogens 4-tert.-octylphenol and bisphenol A in water and in fish tissue based on microwave assisted extraction, solid-phase extraction and liquid chromatography-mass spectrometry

Extraction methods were developed for quantification of the xenoestrogens 4-tert.-octylphenol (tOP) and bisphenol A (BPA) in water and in liver and muscle tissue from the rainbow trout (Oncorhynchus mykiss). The extraction of tOP and BPA from tissue samples was carried out using microwave-assisted solvent extraction (MASE) followed by solid-phase extraction (SPE). Water samples were extracted using only SPE. For the quantification of tOP and BPA, liquid chromatography mass spectrometry (LC-MS) equipped with an atmospheric pressure chemical ionisation interface (APCI) was applied. The combined methods for tissue extraction allow the use of small sample amounts of liver or muscle (typically 1 g), low volumes of solvent (20 ml), and short extraction times (25 min). Limits of quantification of tOP in tissue samples were found to be approximately 10 ng/g in muscle and 50 ng/g in liver (both based on 1 g of fresh tissue). The corresponding values for BPA were approximately 50 ng/g in both muscle and liver tissue. In water, the limit of quantification for tOP and BPA was approximately 0.1 microg/l (based on 100 ml sample size).     

Determination of clenbuterol residues in bovine urine by optical immunobiosensor assay

Clenbuterol (CBL) is an orally active beta2-adrenoceptor agonist which has been used in veterinary medicine as a broncodilator and an agent of uterine relaxation. It has however become better known as a drug used illegally to promote growth in farm animals. A rapid and sensitive biosensor assay was developed to detect CBL residues in bovine urine. The method involved a simple extraction procedure using tert-butyl methyl ether followed by analysis on the biosensor with results obtained against a buffer calibration curve. The assay allowed up to 88 samples to be analyzed per working day, with each cycle on the biosensor taking approximately 7 min to complete. The limit of detection (LOD) was determined as 0.27 ng/mL using 20 EU reference blank urine samples. The intra-assay Sr ranged from 4.7-7.6% for 3 control samples while the interassay Sr ranged from 9.2-12.7%. The recovery was found to be approximately 95%. A series of incurred urine samples were assayed and the results compared by Enzyme immunoassay (EIA), radio-immunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS) analysis. Urine samples taken from local abattoirs were also analyzed by the biosensor method and by EIA analysis. The antibody used in the biosensor test exhibited high cross reactivity with at least 7 other beta-agonists allowing detection of these compounds at less than 1 ng/mL in bovine urine.     

Determination of beryllium in solid samples by flame atomic absorption spectrometry after preconcentration on activated carbon.

It is generally supposed that the preconcentration procedure is used for the determination of metal concentrations under the sensitivity of the measurement method. This study showed that preconcentration is also need for the beryllium (Be) concentration over the sensitivity of atomic absorption spectrometry. For this purpose, a simple and selective method for the determination of Be in solid samples is modified. The method is based on the preconcentration of the complexes of beryllium-acetylacetone plus morin, oxine, PAN and PAR on activated carbon at different pH values. The adsorbed beryllium was eluted with aqua regia and measured by flame atomic absorption spectrometry (FAAS). Recoveries of up to 85% were achieved. For removing chemical interferences and applying the method to Be determination in solid samples, the masking studies and reproducibility were examined. The detection limit was found to be 0.12 ng mL(-1). The relative sandard deviations (RSD) were found to be 8% for 60 mL of 10.0 ng mL(-1) using 10 replicate enrichment procedures. Beryllium concentrations in the studied solid samples were found in the range of 0.28 - 3.95 mg kg(-1).     

Biosensor assay of sulfadiazine and sulfamethazine residues in pork

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.     

Gold nanoparticles/carbon nanotubes/ionic liquid microsized paste electrode for the determination of cortisol and androsterone hormones

A novel nanocomposite electrode material constituted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and n-octylpyridinium hexafluorophosphate (OPPF₆) ionic liquid was prepared and checked for the development of electrochemical (bio)sensing devices. AuNPs/MWCNTs/OPPF₆ paste electrodes with micrometer dimensions (500 μm, i.d.) were constructed and applied to the determination of cortisol and androsterone hormones. Regarding cortisol determination, the microsized paste electrode was used to detect 1-naphtol generated upon addition of 1-naphthyl phosphate as enzyme substrate in the competitive immunoassay between alkaline phosphatase-labelled cortisol and cortisol. Squarewave voltammetry allowed determining the hormone within the 0.1- to 10-ng/mL linear range (r?=?0.990) with a detection limit of 15 pg/mL and a EC₅₀ value of 0.46?±?0.06 ng/mL cortisol. The method was applied to the determination of cortisol in urine and serum samples containing a certified cortisol content. Moreover, a microsized enzyme biosensor prepared by bulk modification of the AuNPs/MWCNTs/OPPF₆ electrode with the enzyme 3α-hydroxysteroid dehydrogenase was used for the determination of androsterone through the amperometric detection of reduced nicotinamide adenine dinucleotide. A calibration plot with a linear range between 0.1 and 120 μg/mL (r?=?0.993) and a limit of detection of 89 ng/mL were obtained. The biosensor was applied to the analysis of human serum spiked with androsterone at the 250 ng/mL concentration level.     

A rapid surface plasmon resonance (SPR) biosensor immunoassay for screening of somatotropins in injection preparations

The use of growth hormones (recombinant somatotropins (rSTs)) is approved in several countries, e.g. the USA, Brazil and Australia to enhance growth or lactating performances of livestock. Their use in the EU is banned, however, due to the widespread application, the illegal use within the EU cannot be excluded. To screen for rSTs in injection preparations, a biosensor immunoassay (BIA) using surface plasmon resonance (SPR) technology was developed. Compared to existing analysis methods for rSTs, like radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), this technique provides a rapid (7 min) alternative. A direct BIA was compared to an indirect (inhibition) BIA and the performances of several antibodies against (r)STs were compared in the indirect BIA. In the final inhibition assay, using rabbit anti-bovine rST, extracts from several injection preparations were shown to contain bovine rST (rbST). The limit of detection for rbST in the assay is 0.008 microg mL(-1) which is far below the expected concentrations in injection preparations. Although the cross-reactivities for STs of other species were low, screening of injection preparations for porcine, equine and human ST was feasible through the analysis of less diluted extracts. Tryptic digestion followed by nano-electrospray liquid chromatography-ion trap tandem mass spectrometry (nano-LC-MS/MS) was used to identify STs.     

High-performance liquid chromatographic separation of caffeine, theophylline, theobromine and paraxanthine in rat brain and serum.

Caffeine and its metabolites theophylline, theobromine and paraxanthine have been determined in rat brain and serum samples by high-performance liquid chromatography with ultraviolet detection. The recovery, 85-103%, allowed quantification by external standard methods. The variability was found to be less than 3 and 7% for intra-day and inter-day assays, respectively. The detection limit, 1.57 ng of methylxanthines on column, allowed the determination of 62.5 ng/g or ml in biological material. Rats treated with 30 mg/kg caffeine (subcutaneously) were sacrificed at different times (1, 6, 12 and 24 h). Higher concentrations of methylxanthines (specially paraxanthine) were observed in the striatum than in the rest of the brain, and it was also observed that the clearance of methylxanthines was faster in serum than in brain structures.     

Protein-templated gold nanoclusters as fluorescence probes for the detection of methotrexate

In this contribution, bovine serum albumin stabilized gold nanoclusters as novel fluorescent probes were successfully utilized for the detection of methotrexate for the first time. Our prepared gold nanoclusters exhibited strong emission with peak maximum at 633.5 nm. However, the addition of methotrexate induced the strong fluorescence intensity of the gold nanoclusters to decrease. The decrease in fluorescence intensity of the gold nanoclusters caused by methotrexate allowed the sensitive detection of methotrexate in the range of 0.0016 μg mL(-1) to 24 μg mL(-1). The detection limit for methotrexate is 0.9 ng mL(-1) at a signal-to-noise ratio of 3. The present sensor for methotrexate detection possessed a low detection limit and wide linear range. In addition, the real samples were analyzed with satisfactory results.     

Facile and controllable preparation of glucose biosensor based on Prussian blue nanoparticles hybrid composites

A glucose biosensor based on polyvinylpyrrolidone (PVP) protected Prussian blue nanoparticles (PBNPs)-polyaniline/multi-walled carbon nanotubes hybrid composites was fabricated by electrochemical method. A novel route for PBNPs preparation was applied in the fabrication with the help of PVP, and from scanning electron microscope images, Prussian blue particles on the electrode were found nanoscaled. The biosensor exhibits fast current response (<6 s) and a linearity in the range from 6.7x10(-6) to 1.9x10(-3) M with a high sensitivity of 6.28 microA mM(-1) and a detection limit of 6x10(-7) M (S/N=3) for the detection of glucose. The apparent activation energy of enzyme-catalyzed reaction and the apparent Michaelis-Menten constant are 23.9 kJ mol(-1) and 1.9 mM respectively, which suggests a high affinity of the enzyme-substrate. This easy and controllable construction method of glucose biosensor combines the characteristics of the components of the hybrid composites, which favors the fast and sensitive detection of glucose with improved analytical capabilities. In addition, the biosensor was examined in human serum samples for glucose determination with a recovery between 95.0 and 104.5%.     

Simultaneous determination of emamectin and ivermectin residues in Atlantic salmon muscle by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method for determining residues of the antiparasitic drugs emamectin (EMA) and ivermectin (IVR) in fish tissues has been developed. EMA and IVR residues are extracted with acetonitrile and cleaned up on a C18 solid-phase extraction column. Extracts are derivatized with 1-methylimidazole and trifluoroacetic anhydride and the components are determined by LC on a C18 reversed-phase column with fluorescence detection (excitation: 365 nm, emission: 470 nm). The mobile phase is 94% acetonitrile-water run isocratically. Calibration curves were linear between 1 and 32 ng injected for both EMA and IVR. The limit of detection for both analytes was 0.5 ng/g, with a limit of quantitation of 1.5 ng/g. Recoveries of EMA and IVR added to salmon muscle averaged 96 +/- 9% and 86 +/- 6%, respectively, at levels between 5 and 80 ng/g. The percent relative standard deviation for the described method was less than 7% over the range of concentrations studied. The operational errors, interferences, and recoveries for fortified samples compare favorably with an established IVR method. The recommended method is simple, rapid, and specific for monitoring residues of EMA and IVR in Atlantic salmon muscle.     

Efficient and sensitive screening and confirmation of residues of selected polyether ionophore antibiotics in liver and eggs by liquid chromatography-electrospray tandem mass spectrometry.

A method using liquid chromatography tandem mass spectrometry (LC-MS-MS) with electrospray (ES) for the determination of traces of narasin, monensin and salinomycin in chicken liver and eggs was developed, validated and used for routine surveillance. The essence of this paper is to demonstrate that one single method can serve very well for two entirely different purposes, i.e., screening and confirmation. Highly reliable confirmation of the identity at low concentrations was demonstrated when residues of narasin were detected and quantified (0.2 to 11 ng g(-1)) in 50% of the Swedish eggs analysed in 1999. Four daughter ions were detected with ion ratios meeting suggested confirmation criteria for the European Union, even at 0.2 ng g(-1). The method was found to be highly cost-effective since both screening and confirmation of 98 liver samples were performed in only two analytical runs (the Swedish national surveillance scheme of 1999, report level 5 ng g(-1)). The high performance of the method for the different applications was possible due to a combination of the power of ES-LC-MS-MS, a procedure involving screening of pooled samples, and method optimisation of the work-up (automated solid phase extraction), LC and MS parameters. Validation data for narasin (0.5 to 20 ng g(-1)) in eggs are presented (accuracy 94 to 108%, relative standard deviation 4 to 10%, limit of detection 0.026 ng g(-1)). The time for an LC-MS-MS run was 4 min, corresponding to 48 s per sample in a pool.     

Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.     

Ionic liquid-functionalized graphene for fabricating an amperometric acetylcholinesterase biosensor

This work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated by modifying a glassy carbon electrode with acetylcholinesterase (AChE) immobilized on ionic liquid-functionalized graphene (IL-G). The functionalized graphene sheets had good dispersibility and long-term stability in various solvents. The as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.77 mM. Furthermore, based on the inhibition by OPs of the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.0025 to 0.48 and 0.48 to 1.42 μg mL(-1) with a detection limit of 0.8 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for OPs detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.