Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Tylosin in Milk and Water Samples

A specific polyclonal antibody was prepared based on a new hapten with stable structure. Based on this, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for determination of tylosin residues in food and environmental samples. The experimental conditions were optimized as follows: the coating antigen and antibody were respectively diluted by 6000 times and 2000 times, and the competitive reaction time was 40 min. Under the optimized conditions, the method showed a detection limit of 0.07 ng mL^?1, an IC₅₀ of 1.39 ng mL^?1 and a linear range of 0.17–11.0 ng mL^?1. The recoveries of spiked raw milk and water samples were ranged from 78.4% to 105.6%, with the RSDs of less than 15%. Good correlation between icELISA and HPLC method was obtained for spiked samples (R² = 0.97). This method was suitable for the determination of tylosin residues in milk and water samples.     

Determination of Florfenicol in Freshwater, Sediments and Bryophyte Fontinalis antipyretica by HPLC with Fluorescence Detection

Novel procedures for the determination of florfenicol in freshwater, sediments and bryophyte Fontinalis antipyretica, using reversed-phase high-performance liquid chromatography are described. Liquid chromatography was performed on a 5 µm PuroSpher RP-18E^® column using methanol and 0.05 M phosphate buffer (18/82 v/v, pH 7.3) as mobile phase (0.8 ml min^?1) and fluorescence detection (excitation wavelength 265 nm and emission wavelength 295 nm). Florfenicol was determined in centrifuged freshwater samples. Florfenicol was extracted from sediments and bryophytes samples by using a solid-liquid extraction step followed by a solid phase extraction step. Linearity was confirmed over the concentration range 25–1000 ng mL^?1 water and 50-1000 ng g^?1 sediment or bryophyte. Limits of detection and quantitation were 8 and 25 ng mL^?1 water and 17 and 50 ng g^?1 sediment or bryophyte respectively. Mean extraction recoveries of florfenicol from sediments and bryophyte were from 85.9 to 109.1%.     

Rabbit Monoclonal Antibody-Based Lateral Flow Immunoassay Platform for Sensitive Quantitation of Four Sulfonamide Residues in Milk and Swine Urine

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP), and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which was based on antigen-antibody properties, the hapten conjugate N¹-[4-(carboxymethyl)-2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detection reagent. With quantitative assessment aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD, STZ, SP, and SMX were 0.91 ng mL^?1, 0.10ng mL^?1,0.12ng mL^?1, and 2.13ng mL^?1, and the half-maximum inhibition concentrations (IC₅₀) were 5.19 ng mL^?1, 1.25 ng mL^?1, 0.66 ng mL^?1, and 24.14 ng mL^?1, respectively. The recoveries at three spiked levels (5, 20, 50 ng mL^?1for SD, STZ, and SP; 20, 50, 100 ng mL^?1 for SMX) were in the range of 78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More importantly, the detection performance of the established platform was consistent with that of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the potential for fast, sensitive and relatively accurate quantification of four sulfonamide residues in practical uses.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

Synchronized separation,concentration and determination of trace chloramphenicol,thiamphenicol and florfenicol in food by using polyoxyethylene cetyl ether-salt aqueous two-phase system coupled with high-performance liquid chromatography

As a novel and green pretreatment technique to trace samples, polyoxyethylene cetyl ether (POELE20)–(NH₄)₂SO₄ aqueous two-phase extraction system was coupled with high-performance liquid chromatography to analyse synchronously chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) in chicken and pork samples. It was found that the extraction efficiency (E%) and enrichment factor (F) of the three antibiotics were influenced by the types of salts, the concentration of salt, the concentration of POELE20, system temperature and pH. The final optimal condition was as following: the phase-forming salt is (NH₄)₂SO₄, the concentration of (NH₄)₂SO₄ is 0.141 g mL^?1, the concentration of POELE20 is 0.03 g mL^?1, the temperature is 298.15 K, and the system pH is 4.5. This POELE20–(NH₄)₂SO₄ ATPS was applied to separate and enrich three antibiotics in real sample under the optimal conditions, and it was found that the recovery was 97.20–102.00 % with a RSD of 0.61–4.85 %. The limit of detection for CAP, TAP and FF were 0.10, 0.50 and 0.50 μg kg^?1, and the limit of quantitation for CAP, TAP and FF was 0.15, 1.50 and 1.50 μg kg^?1. Seven times the experiments were used to verify the repeatability and veracity of this method, and the RSD for the intra-day and inter-day were 1.13–3.22 and 1.74–4.72 %.     

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for several (fluoro)quinolones in milk

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a visual colloidal gold-based immunochromatographic assay (CGIA) for simultaneous determination of ofloxacin, marbofloxacin, and fleroxacin residues in milk using polyclonal antibodies. The half-maximum inhibition concentrations (IC₅₀) of ofloxacin, marbofloxacin, fleroxacin, and limits of detection (LODs; calculated as IC₁₅ values) are between 0.20 and 0.53?ng mL^?1, and between 0.02 and 0.05?ng mL^?1, respectively. The average recoveries range from of 78% to 113%, and the coefficients of variation of intra- and inter-assays are between 2 and 11%, and 3 to 19%, respectively. The LODs for ofloxacin, marbofloxacin, fleroxacin in milk are between 3.5 and 8.9?ng mL^?1. The visual minimum detection limit of the optimized CGIA is 2?ng mL^?1 for milk samples. The detection process can be completed within 10?min. The strips can be stored at 4?°C for 8?weeks without significant loss of activity. The results of the analysis of spiked samples showed that the CGIA can be applied to preliminary, fast, and on-site screening of milk samples. The ELISA and CGIA allow for a rapid, sensitive, and low-cost determination of (fluoro)quinolones residues in milk samples.

Trace Determination of Germanium by Continuous Flow Hydride Generation Laser-Induced Fluorescence Spectrometry

A continuous flow hydride generation laser-induced fluorescence (HG-LIF) spectrometry technique has been investigated for performing trace determination of germanium (Ge). Hydride generation of Ge is performed using reagent concentrations of 2?M H₃PO₄ and 0.5% NaBH₄ and fluorescence detection is performed using tunable dye laser radiation at 253.323?nm for Ge excitation with fluorescence measured at 303.907?nm. The HG-LIF approach provides a linear response for Ge in the concentration range from 1.0 to 50?ng mL^?1 and a limit of detection of 0.1?ng mL^?1. Replicate measurements at 10?ng mL^?1 have a relative standard deviation of 0.1% (n?=?8). Measurements of Ge in different sample matrices have demonstrated the effectiveness of thiourea and ascorbic acid as masking agents that compensate for samples containing interfering ions. The determinations of the Ge content in reference water samples, fly ash samples, and supplement capsules demonstrate that the HG-LIF approach has feasibility for measuring Ge in different sample matrices at environmentally relevant concentrations.     

Simultaneous chiral separation and determination of carvedilol and 5′-hydroxyphenyl carvedilol enantiomers from human urine by high performance liquid chromatography coupled with fluorescent detection

A sensitive and specific high performance liquid chromatography coupled with fluorescent detection (HPLC-FL) and tandem mass spectrometry detection (HPLC-MS/MS) methods for separation and determination of carvedilol (CAR) enantiomers and 5′-hydroxyphenyl carvedilol (5′-HCAR) enantiomers has been developed and validated. The analysed compounds were extracted from human urine by solid phase extraction. Good enantioseparation of the studied enantiomers was achieved on CHIRALCEL® OD-RH column using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile in a gradient elution. The mass spectrometric data were acquired using the multiple reaction monitoring mode by positive electrospray ionisation. The method was validated over the concentration range from 25.0 ng mL^?1 to 200 ng mL^?1 for the analysed compounds. The limit of quantification varied from 14.2 ng mL^?1 to 24.2 ng mL^?1. Both the repeatability and inter-day precisions were below 10.0%, and the accuracy varied from ?13.2% to 3.77%. The extraction recoveries ranged from 79.2% to 108%. The present paper reports the method for the simultaneous determination of CAR enantiomers and their metabolite enantiomers (5′-HCAR) in human urine samples. This newly developed method was successfully used to analyse the aforementioned analytes in human urine samples obtained from patients suffering from cardiovascular disease.      

Simultaneous determination of residues of chloramphenicol,thiamphenicol, florfenicol,and florfenicol amine in farmed aquatic species by liquid chromatography/mass spectrometry

A liquid chromatographic (LC)/mass spectrometric (MS) method was developed for determining the residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species. The phenicols are extracted with acetone, the extracts are partitioned with dichloromethane, the aqueous layer is removed, and the organic layer is evaporated to dryness. The residue is dissolved in dilute acid and defatted with hexane, and the aqueous layer is prepared for analysis by LC. The phenicols are determined by reversed-phase LC by using a Hypersil C18-BD column with a water-acetonitrile gradient and MS detection using selected-ion recording. Calibration curves were linear for all analytes between 0.015 and 0.425 ng injected. The relative standard deviations for measurements by the proposed method were < 10% for all of the analytes studied, with recoveries ranging from 71% for florfenicol amine to 107% for florfenicol in salmon tissue spiked at the 2 ng/g level. Detection limits of 0.1 ng/g for florfenicol and chloramphenicol, 0.3 ng/g for thiamphenicol, and 1.0 ng/g for florfenicol amine are easily obtainable. The operational errors, interferences, and recoveries for spiked samples compare favorably with those obtained by established LC methodology. The proposed method is simple, rapid, and specific for monitoring residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species.     

Development of Immunoaffinity Sample-Purification for GC–MS Analysis of Ractopamine in Swine Tissue

A method combining immunoaffinity chromatography with gas chromatography–mass spectrometry (GC–MS) has been established for determination of ractopamine residues in swine liver and urine. After clean-up on an immunoaffinity chromatography column, GC–MS analysis revealed recovery from blank swine liver and urine fortified at 2.5–20 ng g^?1 (ng mL^?1 for urine), respectively, was 68.2–78.6 and 76.2–83.1%. The limits of detection and quantification were 0.5 ng g^?1 (or ng mL^?1) and 2.0 ng g^?1 (or ng mL^?1), respectively. The procedure was used for analysis of ractopamine residues in samples of swine liver and urine in which the levels were unknown. The amounts detected were 9–216 ng g^?1 (ng mL^?1).     

Determination of Mercury in Urine and Seawater by Flow Injection Vapor Generation Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

A simple and inexpensive laboratory-built vapor generator was used with inductively coupled plasma mass spectrometry (ICP-MS) for the determination of mercury in urine and seawater samples. The applications of vapor generation ICP-MS alleviated the non-spectroscopic interferences and the sensitivity problem of mercury determination encountered when the conventional pneumatic nebulizer was used for sample introduction. The concentration of mercury was determined by isotope dilution method. The isotope ratio of mercury was calculated from the peak areas of each injection peak. The repeatability of the peak areas and isotope ratio determinations of seven consecutive injections of 1 ng mL^?1 Hg solution were 2.3% and 2.2%, respectively. This method has a detection limit of 0.07 ng mL^?1 for mercury. This method was applied to determine mercury in a CASS-3 nearshore seawater reference sample, NASS-4 open ocean seawater reference sample, NIST SRM 2670 freeze-dried urine reference sample and several urine and seawater samples collected from National Sun Yat-Sen University. The results for the reference samples agreed satisfactorily with the reference values. Results for other samples analyzed by the isotope dilution method and the method of standard additions agreed satisfactorily. Precision was better than 10% for most of the determinations.     

Preparation and characterization of ultrasensitive and specific polyclonal antiserum against ciprofloxacin based on cationized bovine serum albumin

An ultrasensitive polyclonal antiserum against ciprofloxacin (CPFX) was developed by introducing cationized bovine serum albumin (BSA) as the carrier. Conjugates used as immunogens were synthesized using CPFX coupled to BSA cationized with ethylenediamine and hexamethylenediamine, namely CPFX-eBSA and CPFX-hBSA. The results showed that the antisera immunized with CPFX-hBSA exhibit an improved sensitivity and specificity compared with those immunized with CPFX-eBSA. Under the conditions of optimal heterologous enzyme-linked immunosorbent assay, the optimal antiserum yielded an IC₅₀ of 0.097 ng mL^?1, which proves it to be more sensitive than any CPFX antibody. Cross-reactivities with norfloxacin, enrofloxacin, and ofloxacin were < 3.0 %. No cross-reactivities with penicillin and gentamicin were found. The limit of detection was 0.01 ng mL^?1, which is far below the maximum residue level established by the EU regulation, suggesting the great potential of the presented antiserum for quantitative assays of CPFX in samples.     

Highly sensitive deoxynivalenol immunosensor based on a glassy carbon electrode modified with a fullerene/ferrocene/ionic liquid composite

We describe a sensitive electrochemical immunosensor for the detection of deoxynivalenol (DON). It is based on a glassy carbon electrode modified with a composite made from fullerene (C₆₀), ferrocene and the ionic liquid. The components were immobilized on the surface of the electrode using chitosan cross-linked with epichlorohydrin. Then, the antibody to DON was covalently conjugated to the surface which then was blocked with serum albumin. The performance of the immunosensor was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. It offers good repeatability (RSD?=?1.2%), selectivity, a stability of more than 180?days, an impedimetric response to DON in the range of 1?pgmL^?1 to 0.3?ng?mL^?1, and a detection limit (at S/N?=?3) of 0.3?pgmL^?1. The limit of detection is better than that of GC, HPLC, GC-MS, HPLC-MS and LC-MS-MS. The effects of omitting C₆₀ or the ionic liquid were also examined. The results indicate that the sensitivity of the biosensor is 2-fold better if C₆₀ and ionic liquids are used. This demonstrates that C₆₀ facilitates electron transfer on the surface of the modified electrode due to its unique electrochemical properties, while the ionic liquid provides a biocompatible microenvironment for the antibody. This results in increased sensitivity and stability. The method was satisfactorily applied to the determination of DON in food samples.

Chromatography of Uranium on High-Performance Ion Exchangers

Abstract

The potential of high-performance liquid chromatography (HPLC) for the determination of U(VI) in ground waters and urine has been examined under a variety of HPLC experimental conditions. Conventional cross-linked and bonded-phase ion exchangers, both cation and anion, were studied with aqueous mobile phases containing tartrate, citrate, or α-hydroxyisobutyrate. The best chromatography was obtained on bonded-phase cation exchangers with an α-hydroxyisobutyrate eluent. The metal ions were detected either by visible spectrophotometry after a post-column reaction with a complexing reagent, or with a polarographic detector. Dectection after post-column reaction gave the best sensitivity; the detection limit (2 × baseline noise was 6 ng or 60 ng.ml^?1 for 100 μl samples. In-line trace enrichment was used to decrease detection limits and linear calibration curves were observed in the ranges studied; 0.5 to 50 ng.mL^?1 for ground waters and 25 to 400 ng.mL^?1 for artificial urine.     

Development of colloidal gold-based immumochromatographic assay for the rapid detection of medroxyprogesterone acetate residues in biological materials

A competitive colloidal gold-based immunoassay in lateral-flow format for the rapid detection of medroxyprogesterone acetate (MPA) in biological materials was developed. A nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was first incubated with MPA. The limit of detection for lateral flow was 5?ng?g^?1 for detecting an MPA standard solution, and the limit of detection was 10?ng?mL^?1 for detecting the MPA spiked in pig urine and 10?ng?g^?1 for spiked in pig liver. The results were confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and indicated that there was a good agreement between both methods (R ²?=?0.976). The assay time for the test was less than 5?min, suitable for rapid testing on site.     

Construction of Modified Carbon Paste Electrode for Highly Sensitive Simultaneous Electrochemical Determination of Trace Amounts of Copper (II) and Cadmium (II)

A chemically modified electrode was constructed for rapid, simple, accurate, selective and highly sensitive simultaneous determination of Cu(II) and Cd(II) using square wave anodic stripping voltammetry. The electrode was prepared by incorporation of SiO₂ nanoparticles, coated with a newly synthesized Schiff base, in carbon paste electrode. The limit of detection was found to be 0.28 ng mL^?1 and 0.54 ng mL^?1 for Cu(II) and Cd(II), respectively. The proposed chemically modified electrode was used for the determination of copper and cadmium in several foodstuffs and water samples.     

Solid phase extraction of trace amounts of Pb(II) in opium, heroin, lipstick, plants and water samples using modified magnetite nanoparticles prior to its atomic absorption determination

A new, simple, fast and reliable solid-phase extraction method has been developed for separation/preconcentration of trace amounts of Pb(II) using dithizone/sodium dodecyl sulfate-immobilized on alumina-coated magnetite nanoparticles, and its determination by flame atomic absorption spectrometry (FAAS) and graphite furnace atomic absorption spectrometry (GFAAS) after eluting with 4.0?mol?L^?1 HNO₃. Optimal experimental conditions including pH, sample volume, eluent concentration and volume, and co-existing ions have been studied and established. Under the optimal experimental conditions, the preconcentration factor, detection limit, linear range and relative standard deviation of Pb(II) using FAAS technique were 280 (for 560?mL of sample solution), 0.28?ng?mL^?1, 1.4?C70?ng?mL^?1 and 4.6% (for 10?ng?mL^?1, n?=?10), respectively. These analytical parameters using GFAAS technique were 300 (for 600?mL of sample solution), 0.002?ng?mL^?1, 0.006?C13.2?ng?mL^?1 and 3.1% (for 5?ng?mL^?1, n?=?10), respectively. The presented procedure was successfully applied for determination of Pb(II) content in opium, heroin, lipstick, plants and water samples.     

Application of dispersive liquid–liquid microextraction and reversed phase-high performance liquid chromatography for the determination of two fungicides in environmental water samples

Dispersive liquid–liquid microextraction (DLLME) has been developed for the extraction and preconcentration of diethofencarb (DF) and pyrimethanil (PM) in environmental water. In the method, a suitable mixture of extraction solvent (50 µL carbon tetrachloride) and dispersive solvent (0.75 mL acetonitrile) are injected into the aqueous samples (5.00 mL) and the cloudy solution is observed. After centrifugation, the enriched analytes in the sediment phase were determined by HPLC-VWD. Different influencing factors, such as the kind and volume of extraction and dispersive solvent, extraction time and salt effect were investigated. Under the optimum conditions, the enrichment factors for DF and PM were both 108 and the limit of detection were 0.021 ng mL^?1 and 0.015 ng mL^?1, respectively. The linear ranges were 0.08–400 ng mL^?1 for DF and 0.04–200 ng mL^?1 for PM. The relative standard deviation (RSDs) were both almost at 6.0% (n = 6). The relative recoveries from samples of environmental water were from the range of 87.0 to 107.2%. Compared with other methods, DLLME is a very simple, rapid, sensitive (low limit of detection) and economical (only 5 mL volume of sample) method.