Preconcentration of triazine herbicides from water by an ion chromatography column and determination by gas chromatography-mass spectrometry

The efficiency of ion chromatography columns packed with styrene-divinylbenzene copolymer containing quaternary ammonium groups to preconcentrate triazine herbicides and their degradation products below μg/l levels has been established. Retention is studied for different types of water. Pure methanol was used in a one-step elution. Enrichment factors of at least 4000 are achieved. Determination was carried out by using gas chromatography-single-ion monitoring mass spectrometry. Recoveries for run-off agricultural water were between 67–100% and close to 100% for ground water. The maximum admissible concentration ion drinking water (0.1 μg/l) and the alert and alarm threshold values in surface water (1 and 3 μg/l, respectively) dictated by the European Union can be measured.     

A simple direct enzyme immunoassay of antibodies based on the differences in diffusion rates in a gel of a synthetic antigen and of its complex with antibodies

A simple direct enzyme immunoassay for semiquantitative detec tion of antibodies is suggested. It is based on the difference in diffusion rates in a gel for a synthetic low-mol-wt antigen and of its complexes with antibodies to be detected. Sensitivity and specificity of the devel oped assay are equal to an ELISA method. The assay has been tested with antibodies against HIV protein gp41 in rabbit serum. Possible applications and limitations of the method are discussed.     

Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor

A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL⁻¹), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.     

Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin

At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody V_H/V_L interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the V_H/V_L interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL⁻¹) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g⁻¹) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.     

Development of a highly sensitive inhibition immunoassay for microcystin-LR

The development of a rapid one-step antigen-immobilized inhibition ELISA for microcystin-LR is described. For microplate coating a microcystin-biotin conjugate was synthesized. Using the commercially available monoclonal antibody MC10E7 in our newly established assay, IC₅₀ values of 0.045 μg l⁻¹ have been achieved. The detection limit for microcystin-LR was 4 ng l⁻¹. Considering the guidelines proposed by the world health organization (WHO) for microcystin-LR in drinking water (1 μg l⁻¹) the sensitivity of our test is more than sufficient. The period of assay processing could successfully be shortened to about 3 h without any loss in sensitivity. The suitability of the newly developed assay was evaluated with microcystin-LR spiked environmental water samples. Recovery rates for microcystin-LR between 60 and 165% were obtained in the linear range of the test format. The antigen-immobilized test format provides a highly reproducible, easy, and fast to perform detection system for microcystin allowing an internal retrospective quality control of the assay.     

Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR-OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L⁻¹, the 50% inhibition concentration (IC₅₀) was 0.20 ± 0.02 μg L⁻¹ and the quantitative detection range was 0.062-0.65 μg L⁻¹. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples.     

An immunoassay based on lab-on-a-chip for simultaneous and sensitive detection of clenbuterol and ractopamine

Both clenbuterol (CLB) and ractopamine (RAC) are β-adrenergic agonists. After long-term excessive intake, there will be adverse reactions such as headache, chest tightness, limb numbness, and serious life-threatening. Simultaneous detection of CLB and RAC in related samples is of great importance for human health. In this work, we outline a microfluidics-based indirect competitive immunoassay (MICI) system that can sensitively detect residual CLB and RAC in pork, swine blood and swine urine. The rapid detection of multiple samples can be achieved in one chip, which greatly improves the detection efficiency. This method has good stability and reproducibility and the microfluidic chips are easy to manufacture. The linear ranges for CLB and RAC detection by MICI are 0.1–2.5 ng/mL and 0.1–5 ng/mL, and the limits of detection (LODs) are 0.094 ng/mL and 0.091 ng/mL, respectively. This straightforward and portable immunoassay system provides a good platform for rapid detection of harmful substances in food samples.     

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.     

Selective extraction of triazine herbicides from food samples based on a combination of a liquid membrane and molecularly imprinted polymers

A selective extraction technique based on the combination of liquid membrane (microporous membrane liquid–liquid extraction) and molecularly imprinted polymers (MIP) was applied to triazines herbicides in food samples. Simazine, atrazine and propazine were extracted from aqueous food samples through the hydrophobic porous membrane that was impregnated with toluene, which also formed part of the acceptor phase. In the acceptor phase, the compounds were re-extracted onto MIP particles. The extraction technique was optimised for the amount of molecularly imprinted polymers particles in the organic acceptor phase, extraction time, and type of organic acceptor solvent and desorption solvent. An extraction time of 90 min and 50 mg of MIP were found to be optimum parameters. Toluene as the acceptor phase was found to give higher triazines binding onto MIP particles compared to hexane and combinations of diethyl ether and hexane. 90% methanol in water was found to be the best desorption solvent compared to acetonitrile, methanol and water. The selectivity of the technique was demonstrated by extracting spiked lettuce and apple extracts where clean chromatograms were obtained compared to liquid membrane extraction alone or to the microporous membrane liquid–liquid extraction – non-imprinted polymer combination. The MIP showed a certain degree of group specificity and the extraction efficiency in lettuce extract was 79% (0.72) for simazine, 98% (1.55) for atrazine and 86% (3.08) for propazine.     

Preparation of Polyclonal Antibodies with Application for an Organophosphorus Pesticide Immunoassay

Haptens of dichlorvos and paraoxon were conjugated to the carrier proteins of bovine serum albumin. The obtained conjugates were characterized by infrared and ultraviolet–visible spectroscopy. The binding ratios of dichlorvos and paraoxon-to-carrier proteins were also evaluated. The number of hapten molecules per protein molecule of dichlorvos–cationized bovine serum albumin conjugate was higher than for paraoxon–bovine serum albumin conjugate. The sheep polyclonal antibodies were produced against the dichlorvos and paraoxon. New multipolyclonal antibodies were obtained and characterized following the immunization of a 1:1 mixture of the immunogens for the simultaneous determination of dichlorvos and paraoxon by the immunoassay. An indirect enzyme-linked immunosorbent assay was used to characterize the reactivity of the antibodies to hapten conjugates. The multiantibodies showed lower affinities than the separate antibodies, but their affinities were sufficient for an immunoassay for the simultaneous determination of the analytes. The detection limit and linear range for the determination of dichlorvos and paraoxon alone and together were determined. The recovery was characterized to determine dichlorvos and paraoxon fortified in model solutions and milk. These results demonstrate the potential of this immunoassay for the quantitative screening of dichlorvos and paraoxon.     

Development of a sequential injection chromatography (SIC) method for determination of simazine,atrazine, and propazine

This paper describes the development of a sequential injection chromatography (SIC) procedure for separation and quantification of the herbicides simazine, atrazine, and propazine exploring the low backpressure of a 2.5 cm long monolithic C₁₈ column. The separation of the three compounds was achieved in less than 90 s with resolution >1.5 using a mobile phase composed by ACN/1.25 mmol/L acetate buffer (pH 4.5) at the volumetric ratio of 35:65 and flow rate of 40 μL/s. Detection was made at 223 nm using a flow cell with 40 mm of optical path length. The LOD was 10 μg/L for the three triazines and the quantification limits were of 30 μg/L for simazine and propazine and 40 μg/L for atrazine. The sampling frequency is 27 samples per hour, consuming 1.1 mL of ACN per analysis. The proposed methodology was applied to spiked water samples and no statistically significant differences were observed in comparison to a conventional HPLC–UV method. The major metabolites of atrazine and other herbicides did not interfere in the analysis, being eluted from the column either together with the unretained peak, or at retention times well‐resolved from the studied compounds.     

Laser-induced fluorescence reader with a turbidimetric system for sandwich-type immunoassay using nanoparticles

A unique laser-induced fluorescence (LIF) reader equipped with a turbidimetric system was developed for a sandwich-type immunoassay using nanoparticles. The system was specifically designed to reduce experimental error caused by particle loss, aggregation and sinking, and to improve analytical performance through ratiometric measurement of the fluorescence with respect to the turbidimetric absorbance. For application to determine the concentration of salinomycin, magnetic nanoparticles (MNPs) and FITC-doped silica nanoparticles (colored balls) immobilized with antibody were synthesized for magnetic extraction and for tagging as a fluorescence probe, respectively. The detection limit of about 39 pg mL⁻¹ was obtained, which was an improvement of about 2-fold compared to that obtained without employment of the turbidimetric system. Calibration linearity and sensitivity were also improved, with increase from 0.8601 to 0.9905 in the R²-coefficient and by 1.92-fold for the curve slope, respectively. The developed LIF reader has the potential to be used for fluorescence measurements using various nanomaterials, such as quantum dots.     

Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl

One-step membrane-based competitive colloidal gold-based immunoassays in flow-through and lateral-flow formats for the rapid detection of carbaryl were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and carbaryl hapten-OVA conjugate (test line). Anti-carbaryl antibody labeled with colloidal gold particles was firstly incubated with carbaryl. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for flow-through and lateral flow of 50 and l00 μg/L, respectively. The assay time for both tests was less than 5 min, suitable for rapid testing on-site.     

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 °C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Ag and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000 ng mL⁻¹, with detection limits 8 (thermal initiation) and 12 ng mL⁻¹ (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode.     

Selective extraction of triazine herbicides based on a combination of membrane assisted solvent extraction and molecularly imprinted solid phase extraction

A selective extraction technique based on the combination of membrane assisted solvent extraction and molecularly imprinted solid phase extraction for triazine herbicides in food samples was developed. Simazine, atrazine, prometon, terbumeton, terbuthylazine and prometryn were extracted from aqueous food samples into a hydrophobic polypropylene membrane bag containing 1000μL of toluene as the acceptor phase along with 100mg of MIP particles. In the acceptor phase, the compounds were re-extracted onto MIP particles. The extraction technique was optimised for the type of organic acceptor solvent, amount of molecularly imprinted polymers particles in the organic acceptor phase, extraction time and addition of salt. Toluene as the acceptor phase was found to give higher triazine binding onto MIP particles compared to hexane and cyclohexane. Extraction time of 120min and 100mg of MIP were found to be optimum parameters. Addition of salt increased the extraction efficiency for more polar triazines. The selectivity of the technique was demonstrated by extracting spiked cow pea and corn extracts where clean chromatograms were obtained compared to only membrane assisted solvent extraction or only molecularly imprinted solid phase extraction. The study revealed that this combination may be a simple way of selectively extracting compounds in complex samples.     

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V_H and V_L) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V_H and V_L genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 ± 0.03 and 0.02 ± 0.004 ng mL⁻¹, respectively, and the linear response range extended from 0.05 to 1.45 ng mL⁻¹. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). The results showed a good correlation between the data of dc-CLEIA and HPLC–MS (R² > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety.     

免疫毛细管电泳-激光诱导荧光分析DNA加合物的方法学研究

DNA加合物是一类重要的生物标志物,可应用于人体致癌物暴露监测、癌症风险评价和人群易感性研究。DNA加合物作为生物标志物的应用需要安全、灵敏、快速的先进分析技术。我们利用免疫毛细管电泳-激光诱导荧光分析,发展了高灵敏的DNA加合物分析方法和技术。本文主要介绍了相关的仪器研制及方法学研究。方法学研究涉及DNA加合物荧光探针的合成和表征、抗体与DNA加合物的相互作用及其结合计量学、抗原-抗体复合物的稳定化和DNA驱动电泳聚焦技术。     

A continuous flow system combined with a sensing fluorimetric transductor for the determination of β-naphthol

A single automatic method for continuous flow determination of β-naphthol based on the enhancement of its native fluorescence once the analyte was transitorily retained on-line on a solid support (QAE A-25 resin) is reported. So, a flow-through optosensor was developed using a flow-injection analysis system with solid phase fluorimetric transduction. KCl (0.15 mol l⁻¹) at pH 12.0 was used as carrier solution. To obtain the optimum fluorescence signal the wavelengths chosen were 245 nm (excitation) and 420 nm (emission). The response of the sensor was directly proportional to the sample volume injected in the studied range 40-1500 μl. Approximately one higher order of magnitude is achieved in sensitivity when 1500 μl are used with respect to the use of 40 μl of sample. The sensor was calibrated for three different injection volumes: 40, 600 and 1500 μl, responding linearly in the measuring range of 2-60, 0.5-15 and 0.2-5 μg l⁻¹ with detection limits of 0.5, 0.09 and 0.05 μg l⁻¹, respectively. The relative standard deviation for ten independent determination is 0.6% (40 μl), 0.9% (600 μl) and 2.3% (1500 μl). A recovery study was performed onto three different spiked water samples at concentration levels from 1 to 2.5 μg l⁻¹ and the recovery percentage from the experimental data ranged between 101±2 and 105±5.     

Photodegradation study for assessing the environmental fate of some triazine-, urea- and thiolcarbamate-type herbicides

Various biotic or abiotic factors influence the fate of pesticides in the natural environment, of which ultraviolet (UV) component in the sunlight is one of the most powerful forces. Studies on the photodegradation of pesticides have not only significance from the point of view of environmental science, but also very important in researching and developing new, safer, and effective pesticides. Previously we have studied the photodegradation of triazine- and urea-type herbicides in details. This paper is intended to outline the photodegradation pattern of some commonly used N-containing herbicides that belong to the groups of the mentioned triazines (atrazine, cyanazine, terbuthylazine, terbutryn) and ureas (chloroxuron, methabenzthiazuron, diuron, fenuron), as well as thiolcarbamates (butylate, cycloate, EPTC, molinate, vernolate), in order to gain information about their possible fate and derivatives in the environment.The most significant processes of photodegradation of triazines are the partial or complete loss of side-chains, or rather the substitution of the heteroatom-containing side-chain to hydroxyl-group. The chemical characteristics of the side chains determines basically the speed of the degradation. It can be concluded that the C-S bond breaks down much easier than C-Cl bond, and the chlorine atom remaining on the triazine-ring promotes the loss of alkyl-chains to a higher extent than that the hydroxy-group. In the case of ureas, the chemical properties of groups in both the N and N′ position can influence the degradation process. It could be stated the proximity of large aryl-substituent and methyl-group in the N position is unfavorable and instabilizing, promoting the further degradation of compound, at the same time, loss of these groups is the preferred degradation route. In the case of thiolcarbamates, the most frequent processes are the α- and β-oxidation of alkyl-groups connecting to the nitrogen atom. In the most cases, the N-formyl and N-dealkylated products were identified in the degradation mixture. The thioalkyl-group showed fair stability under the circumstances of photodegradation. Its partly or completely degraded products cannot be detected, and represent only very small part of the mixture.