Determination of 2,4-dichlorophenoxyacetic acid in cereals by fluorescence polarization immunoassay

A fluorescence polarization immunoassay (FPIA) procedure is developed for the determination of 2,4-dichlorophenoxyacetic acid in cereal grains using a Sentry-200 portable device. We synthesized tracers, that is, antigens labeled with fluorescein derivatives, based on two fluorescent compounds and two chlorophenoxyacids. The tracer synthesized from 2,4-dichlorophenoxyacetic acid and 4-aminomethylfluorescein is found to be optimal for the FPIA of 2,4-dichlorophenoxyacetic acid. The limit of detection for 2,4-dichlorophenoxyacetic acid in flour was 40 ng/g; the analytical range is from 80 to 1000 ng/g. The percentage of recovery was 85 ± 7% at the relative standard deviation (RSD) 2–10%.     

Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid

We present a novel immunoassay format utilizing the catalytic properties of gold nanoparticles in the luminol-silver nitrate-gold nanoparticle based chemiluminescence (CL) system for the detection of widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Highly sensitive anti-2,4-D antibody was produced and conjugated with gold nanoparticles of various sizes. In the present assay format, employing a competitive inhibition approach, a well-characterized hapten-protein conjugate (2,4-D-BSA) was used to coat the microtiter plates. The analyte (2,4-D) was pre-incubated with anti-2,4-D antibody labeled with gold nanoparticles and added to each well of the microtiter plate. The gold label triggered the reaction between luminol and silver nitrate generating a luminescence signal at 425 nm. Under the optimized conditions, the CL based immunoassay showed the detection limit of 2,4-D in standard water samples around 3 ng mL(-1). The CL based immunoassay format, based on gold nanoparticles as a catalyst, could be used as a fast screening methodology (<30 min) for pesticide detection.     

测定人体尿液中苄基丙二酸的免疫分析新方法研究

将苄基丙二酸(BA)与牛血清白蛋白通过活化酯法偶联制备人工免疫原,然后免疫新西兰大白兔制得抗苄基丙二酸多克隆抗体;使用该抗体建立了测定人体尿液中的苄基丙二酸的间接竞争酶联免疫吸附方法(ic-ELISA),方法线性范围为1.0×10-2～1.0×103ng/mL,最低检测浓度为0.01 ng/mL,板内差异为5.5%,板间差异在3.8%～7.8%的范围内;用于实际尿样中苄基丙二酸的测定,回收率为87.8%～115.1%。     

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO₂ groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC₅₀ values for atrazine and 2,4-D equal to 0.8 ng mL⁻¹ and 7 ng mL⁻¹, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL⁻¹ respectively in the optimum working range between 0.01 and 1000 ng mL⁻¹ with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.     

荧光免疫法测定生物样品中的氯胺酮

制备了氯胺酮的多克隆抗体,以间接酶联免疫吸附法为基础,异硫氰酸荧光素为荧光探针,建立了检测氯胺酮的荧光免疫法。在优化的反应条件下,方法的线性范围为1～1000μg/L,检出限为0.48μg/L。不同浓度的氯胺酮在人体血清和尿液样品中的加标回收率在99%～107%。方法适用于生物样品中氯胺酮的检测。     

Determination of 2,4-dichlorophenoxyacetic acid and silvex in water with high-performance liquid chromatography

The determination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2,4,5-trichlorophenoxy) propionic acid (Silvex) in water at the μg l^-1 level is based on liquid/ liquid extraction and high-performance liquid chromatography. Sample preparation for water samples is simplified. The ranges of linear response are 50 ng to 60 μg for 2,4-D and 30 ng to 60 μg for Silvex. The average recoveries of 2,4-D at the 10 μg l^-1 and 1 μg l^-1 levels are 91% and 120%, respectively, while the average recoveries of Silvex at the 10 μg l^-1 and 1 μg l^-1 levels are 85% and 110%, respectively.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Development of a non-competitive immunoassay for monitoring DDT, its metabolites and analogues in water samples

This report highlights the characteristics of a general method of performing non-competitive immunoassays for low-molecular-mass analytes, which was developed and applied to 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) determination in aqueous samples. The method is based on the separation of the analyte-bound antibody from the excess of the free antibody by a chromatographic step, followed by the dissociation of the complex and the capture of the previously bound antibody on a solid phase. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 8 ng l⁻¹) obtained by the above method enabled us to decidedly improve the sensitivity of the corresponding enzyme-linked immunosorbant assay (ELISA) and of all reported immunoassays for DDT.In addition, by applying this new format, even if a very selective antibody was used, a broad selectivity was observed, which allowed DDT + DDD + DDE to be determined instead of only p,p′-DDT as in the ELISA performed with the same antibody. In addition, real water samples were validated in a percentage recovery test. Very good recovery rates were obtained, highlighting the validity of the proposed method to accurately determine the total DDT content in water.     

Long-wavelength homogeneous enzyme immunoassay for the determination of amikacin in water samples

A simple and rapid homogeneous enzyme immunoassay involving the use of the malic dehydrogenase enzyme and a long-wavelength fluorophor, the oxazine Cresyl Violet, is proposed for the determination of the antibiotic amikacin in water samples. An enzymatic tracer has been synthesized by covalent binding of amikacin to malic dehydrogenase via a carbodiimide derivative. Free tracer catalyses the reaction between Cresyl Violet and malic acid giving rise to a decrease in the fluorescence of the fluorophor. Kinetic curves for this reaction have been monitored at λ_ex 585 and λ_em 624 nm using the stopped-flow mixing technique, being the initial rate measured in only 2-3 s. The dynamic range of the method is 1-15 ng mL⁻¹ and the detection limit is 0.3 ng mL⁻¹, using aqueous standard solutions or water samples. The precision, obtained at 1 and 5 ng mL⁻¹ and expressed as relative standard deviation, was 6.0 and 9.6%, respectively. The method has been applied to the analysis of drinking, river and wastewater samples. The sample pre-treatment involved a solid-phase extraction step for the clean-up of the samples. A recovery study was carried out to validate the method, being the values obtained in the range 80-114%, with a mean value of 96.7%.     

Development of a simple extraction procedure for chlorpyrifos determination in food samples by immunoassay

The suitability of immunoassay methodology for rapid and accurate determination of chlorpyrifos in vegetables was tested. The optimised ELISA detection limit was 0.32 ng/ml, with a working range from 0.69 to 6.21 ng/ml and an immunoassay test-mid point (IC₅₀) of 2.08 ng/ml. A rapid sample preparation procedure considering different parameters such as the amount of sample, volume of extractant, extraction time and dilution factor was optimised. The developed direct extraction (DE) and multiresidue (ME) standard procedures were performed in different fortified fresh and processed vegetable samples (tomato, bonnet pepper, bean, pea, asparagus, broccoli, watermelon, melon, lettuce, cucumber, celery and red pepper). Recoveries were in all cases in the whole range 85.2-108.9% for both DE and ME extracts. Also, the comparison of the results obtained by both immunochemical and chromatographic methods for spiked fruits and vegetables were good with a correlation coefficient (r) of 0.97.     

Gold nanoparticles for enhanced chemiluminescence and determination of 2,4-dichlorophenol in environmental water samples

Gold nanoparticles (AuNPs) of different sizes were synthesized by the citrate reduction method. It was found that AuNPs could enhance the chemiluminescence (CL) of the luminal-NaOH system and 2,4-dichlorophenol (2,4-DCP) could inhibit AuNPs-luminal-NaOH CL signals in alkaline solution. CL spectra, UV-visible spectra, and transmission electron microscopy (TEM) were used to investigate the CL mechanism. On the basis of the inhibition, a flow-injection CL method has been established for determination of 2,4-DCP in water samples. Under the optimized conditions, the linear range for determination of the 2,4-DCP was 0.1 × 10(-6) to 20 × 10(-6) g mL(-1), LOD (limit of detection, S/N = 3) was 1.36 × 10(-8) g mL(-1), with RSD of 1.8% (n = 11). This method has been successfully used for analysis of 2,4-DCP in environmental water samples.     

Development of a fast capillary electrophoresis method for determination of creatinine in urine samples

The aim of this work was to develop a fast method using capillary electrophoresis for the determination of creatinine in human urine samples. The pH and constituents of the background electrolyte were selected by inspection of effective mobility of creatinine and candidate urine interferents versus pH curves. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated by the Peakmaster software and considered in the optimization of the background electrolyte, composed by 10 mmol L(-1) tris(hydroxymethyl)aminomethane and 20 mmol L(-1) 2-hydroxyisobutyric acid (HIBA) at pH 3.93. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 microm I.D.), with short-end injection configuration and direct UV detection at 215 nm. The migration time of creatinine was only 22s. A few figures of merit of the method are as follows: good linearity in the concentration interval of 5-70 mg L(-1) (R(2)>0.99), limit of detection of 0.5 mg L(-1), inter-day precision better than 2.7% (n=9) and recovery in the range 99.0-103.7% at three concentration levels (50, 100 and 150 mg L(-1)). Urine samples were prepared by deproteination with acetonitrile (1:3 sample:acetonitrile, v/v), centrifugation and dilution of a deproteinated aliquot with 12.5 mmol L(-1) HIBA (1:4, v/v). Creatinine concentrations between 489 and 1063 mg L(-1) were obtained in the urine of four healthy volunteers.     

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg⁻¹, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg⁻¹, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.     

Development of a microparticle-based on-site immunoassay for the detection of atrazine in soil and water samples

Atrazine is widely used as a herbicide in agriculture and has been identified as a major groundwater contaminant in the US. Because of the possible hazard associated with its usage, there is a need for an efficient and economic screening method for on-site field testing of atrazine and other s-triazine herbicides in soil and water. We have developed a rapid, on-site test for the detection of atrazine based on the principle of microparticle agglutination inhibition immunoassay. The test detects 50 microg kg(-1) (0.050 ppm) atrazine in soil samples with direct extraction and 1.0 microg L(-1) atrazine in water samples when coupled with solid phase extraction.     

Application of NaClO-treated multiwalled carbon nanotubes as solid phase extraction sorbents for preconcentration of trace 2,4-dichlorophenoxyacetic acid in aqueous samples

Multiwalled carbon nanotubes functionalized by oxidation of original multiwalled carbon nanotubes with NaClO were prepared and their application as solid phase extraction sorbent for 2,4-dichlorophenoxyacetic acid (2,4-D) was investigated systemically, and a new method was developed for the determination of trace 2,4-D in water samples based on extraction and preconcentration of 2,4-D with solid phase extraction columns packed with NaClO-treated multiwalled carbon nanotubes prior to its determination by HPLC. The optimum experimental parameters for preconcentration of 2,4-D, including the column activating conditions, the amount of the sorbent, pH of the sample, elution composition, and elution volume, were investigated. The results indicated 2,4-D could be quantitatively retained by 100 mg NaClO-treated multiwalled carbon nanotubes at pH 5, and then eluted completely with 10 mL 3:1 (v/v) methanol-ammonium acetate solution (0.3 mol/L). The detection limit of this method for 2,4-D was 0.15 μg/L, and the relative standard deviation was 2.3% for fortified tap water samples and 2.5% for fortified riverine water sample at the 10 μg/L level. The method was validated using fortified tap water and riverine water samples with known amount of 2,4-D at the 0.4, 10, and 30 μg/L levels, respectively.     

Preconcentration of 2,4-dichlorophenoxyacetic acid on molecularly imprinted polymers and its subsequent determination by high performance liquid chromatography

The potency of molecularly imprinted polymers (MIP) for 2,4-dichlorophenoxyacetic acid (2,4-D) in the dynamic sorption preconcentration (solid phase extraction) of 2,4-D and three other structurally related species (2,4-dichlorophenol, 2-chlorophenol, and dikamba) from aqueous solutions is assessed. The optimal conditions for preconcentration are found: 0.01 M HCl, 25–100 mL of solution, flow rate of solution 0.7 mL/min, column (15 × 2.7 mL), and sorbent weight 0.04 g. The analytes are desorbed with 1 mL of methanol and detected in the eluate by reversed-phase HPLC with spectrophotometric detection at 225 nm. The detection limit for 2,4-D makes 0.4 μg/mL without preconcentration and 0.01 μg/mL with preconcentration from a volume of 100 mL. The procedure is applied to the analysis of model mixtures based on fresh river water.     

Adsorption and preconcentration of 2,4-dichlorophenoxyacetic acid on a chemically modified silica gel surface

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), chemically anchored to a silica gel surface, was used to adsorb and preconcentrate the same herbicide from aqueous solutions at room temperature. From a series of adsorption isotherms adjusted to a modified Langmuir equation, the maximum number of moles adsorbed was calculated as 4.67 x 10(-5) mol g(-1), with the highest retention capacity at pH 5. This modified silica gel was used in a column for preconcentrating trace levels of 2,4-D. The preconcentrated herbicide can be directly eluted with methanol with a recovery efficiency higher than 97%. The concentration factor was 8.33.     

类Fenton试剂-罗丹明B-HCl体系动力学荧光法测定环境水样中 2,4-二氯酚

在酸性介质中,痕量2,4-二氯酚对类Fenton试剂与罗丹明B的反应具有明显阻抑作用,且其阻抑作用大小与2,4-二氯酚的浓度有关。据此建立了一种动力学荧光法测定痕量2,4-二氯酚的新方法。考察了各种因素对测定方法灵敏度的影响。在最佳条件下,方法的线性范围为0.04~0.8μg/mL,检出限为0.012μg/mL。将该方法用于7种环境水样和6种合成样品中2,4-二氯酚的测定,加标回收率为93.5%~108.0%,相对误差为-5.45%~4.60%。     

Gel-based immunoassay for non-instrumental detection of pyrene in water samples

A new qualitative immunologically based tube test for non-instrumental detection of pyrene (PYR) in water samples was developed. The method combines the pre-concentration of analyte by immunoextraction and its detection by immunoassay using Sepharose 4B-immobilized IgG-fraction of a polyclonal anti-PYR antiserum (immunoaffinity gel) and 1-pyrenebutyric acid-horseradish peroxidase conjugate (PYR-BA-HRP). The immunoaffinity gel was placed in a standard 1-ml SPE column through which a 10-ml aliquot of water sample spiked with 10% acetonitrile was passed. Following, free antibody binding sites were detected by application of PYR-BA-HRP. Four minutes after addition of the chromogenic substrate the results were visually evaluated by occurring or stayed away blue colour development for negative and positive samples, respectively. Total time for assay was about 15 min for six samples. Under optimized conditions a cut-off level for pyrene of 0.04 ng ml(-1) was found. At this defined concentration, a set of spiked samples (n=175) was analyzed and very low rates of false negatives (1.2%) and false positives (4.6%) determined which fulfils the requirement set by Commission Decision 2002/657/EC for a screening method. No interference by other PAH compounds like naphthalene, fluoranthene, phenanthrene, anthracene, and benzo[a]pyrene at a concentration of 20 ng ml(-1), i.e., 500-fold excess compared to the defined cut-off level was observed. Different water types like surface water, tap water, bottled water, and melted snow were analyzed for PYR contamination by the proposed method and results confirmed by HPLC-FLD.