Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA–bovine thyroglobulin conjugate and OA–N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 μl min⁻¹; flow rate, 25 μl min⁻¹. An assay action limit of 126 ng g⁻¹ was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g⁻¹, which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC–MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r ² = 0.991). Figure Biacore     

Comparison of two solid-phase microextraction methods for the quantitative analysis of VOCs in indoor air

Competitive adsorption on adsorptive solid-phase microextraction (SPME) fibres implies careful determination of operating conditions for reliable quantitative analysis of VOCs in indoor air. With this objective, two analytical approaches, involving non-equilibrium and equilibrium extraction, were compared. The average detection limit obtained for GC-MS analysis of nine VOCs by the equilibrium method is 0.2 μg m⁻³, compared with 1.9 μg m⁻³ with the non-equilibrium method. The effect of the relative humidity of the air on the calibration plots was studied, and shown to affect acetone adsorption only. Hence, the concentrations that can be accurately determined are up to 9 μmol m⁻³. The methods were then applied to indoor air containing different concentrations of VOCs. The non-equilibrium method, involving short extraction time, can be used for detection of pollution peaks whereas equilibrium extraction is preferable for measurement of sub-μg m⁻³ ground concentration levels.      

Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Environmental monitoring of phenolic pollutants in water by cloud point extraction prior to micellar electrokinetic chromatography

Many aromatic compounds can be found in the environment as a result of anthropogenic activities and some of them are highly toxic. The need to determine low concentrations of pollutants requires analytical methods with high sensitivity, selectivity, and resolution for application to soil, sediment, water, and other environmental samples. Complex sample preparation involving analyte isolation and enrichment is generally necessary before the final analysis. The present paper outlines a novel, simple, low-cost, and environmentally friendly method for the simultaneous determination of p-nitrophenol (PNP), p-aminophenol (PAP), and hydroquinone (HQ) by micellar electrokinetic capillary chromatography after preconcentration by cloud point extraction. Enrichment factors of 180 to 200 were achieved. The limits of detection of the analytes for the preconcentration of 50-ml sample volume were 0.10 μg L⁻¹ for PNP, 0.20 μg L⁻¹ for PAP, and 0.16 μg L⁻¹ for HQ. The optimized procedure was applied to the determination of phenolic pollutants in natural waters from San Luis, Argentina. Figure Schematic representation of the cloud point extraction process.     

Filtration-based tyramide amplification technique—a new simple approach for rapid detection of aflatoxin B<Subscript>1</Subscript>

The catalyzed reporter deposition (CARD) method of signal amplification, also called “tyramide signal amplification”, has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B₁ (AFB₁) in a variety of foodstuffs with a detection limit of 12.5 μg kg⁻¹. The assay procedure involves sequential addition of standards or sample, AFB₁–horseradish peroxidase (HRP) conjugate, B-T, avidin–HRP, and substrate solution over anti-AFB₁ antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB₁ at concentrations from 25 to 100 mg kg⁻¹ were between 95 and 105%. AFB₁ results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC. Figure The principle of filtration-based tyramide filtration technique     

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA _HP of 3.27 × 10⁻³ μmol⁻¹ L), followed by α-tocopherol (SA _HP of 2.36 × 10⁻³ μmol⁻¹ L) and quercetin (SA _HP of 0.31 × 10⁻³ μmol⁻¹ L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees     

Study on an on-line molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography for separation and determination of trace estrone in environment

Estrone is one of the important potential endocrine-disrupting compounds, and the sensitive and reliable analytical methods for the determination of estrone are required for the assurance of human health. In this paper, using estrone as template molecule, 3-aminopropyltriethoxysilane as function monomer, and tetraethoxysilicane as cross-linker, a highly selective molecularly imprinted microsphere was synthesized by surface molecular imprinting technique combined with a sol–gel process. The imprinted material was characterized by the Fourier transform infrared and static adsorption experiments, and the results showed that it exhibited good recognition and selective ability for estrone. A novel method for separation and determination of trace estrone in environmental sample was developed using on-line molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography. With a sample loading flow rate of 2.6 mL min⁻¹ for a 9.6-min extraction, the enrichment factor obtained by the slopes of the linear portion in comparison with the direct injection of 10 μL standard sample solution was 1,045. The detection limit (S/N = 3) was 5.7 ng L⁻¹, and the relative standard deviations for nine replicate extractions of 5.0 μg L⁻¹ estrone was less than 10.0%. This method was evaluated for quantitative determination of estrone in well and lake water samples spiked at two levels (0.5 and 1.0 μg L⁻¹) with recoveries ranging from 86% to 95%.      

An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube–cross-linked chitosan composite

A simple method has been devised for immobilization of acetylcholinesterase (AChE)—covalent bonding to a multiwall carbon nanotube (MWNT)–cross-linked chitosan composite (CMC)—and a sensitive amperometric sensor for rapid detection of acetylthiocholine (ATCl) has been based on this. Fourier-transform infrared spectroscopy proved that the native structure of the immobilized enzyme was preserved on this chemically clean and homogeneous composite film, because of the excellent biocompatibility and non-toxicity of chitosan. Glutaraldehyde was used as cross-linker to covalently bond the AChE, and efficiently prevented leakage of the enzyme from the film. Because of the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl, with a value of 132 μmol L⁻¹, forming thiocholine, which was then oxidized to produce a detectable and rapid response. Under optimum conditions the amperometric current increased linearly with the increasing concentration of ATCl in the range 2.0–400 μmol L⁻¹, with a detection limit of 0.10 μmol L⁻¹. Fabrication reproducibility of the sensor was good and the stability was acceptable. The sensor is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis. Abstract     

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe<Superscript>−</Superscript> ions in aqueous solution

Water-soluble cadmium sulfide (CdS) quantum dots (QDs) capped by mercaptoacetic acid were synthesized by aqueous-phase arrested precipitation, and characterized by transmission electron microscopy, spectrofluorometry, and UV-Vis spectrophotometry. The prepared luminescent water-soluble CdS QDs were evaluated as fluorescence probes for the detection of highly reactive hydrogen selenide ions (HSe⁻ ions). The quenching of the fluorescence emission of CdS QDs with the addition of HSe⁻ ions is due to the elimination of the S²⁻ vacancies which are luminescence centers. Quantitative analysis based on chemical interaction between HSe⁻ ions and the surface of CdS QDs is very simple, easy to develop, and has demonstrated very high sensitivity and selectivity features. The effect of foreign ions (common anions and biologically relevant cations) on the fluorescence of the CdS QDs was examined to evaluate the selectivity. Only Cu²⁺ and S²⁻ ions exhibit significant effects on the fluorescence of CdS QDs. With the developed method, we are able to determine the concentration of HSe⁻ ions in the range from 0.10 to 4.80 μmol L⁻¹, and the limit of detection is 0.087 μmol L⁻¹. The proposed method was successfully applied to monitor the obtained HSe⁻ ions from the reaction of glutathione with selenite. To the best of our knowledge, this is the first report on fluorescence analysis of HSe⁻ ions in aqueous solution. Figure CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe- ions in aqueous solution     

Determination of diquat by flow injection–chemiluminescence

A simple, economic, sensitive and rapid method for the determination of the pesticide diquat was described. This new method was based on the coupling of flow injection analysis methodology and direct chemiluminescent detection; to the authors’ knowledge, this approach had not been used up to now with this pesticide. It was based on its oxidation with ferricyanide in alkaline medium; significant improvements in the analytical signal were achieved by using high temperatures and quinine as sensitiser. Its high throughput (144 h⁻¹), together with its low limit of detection (2 ng mL⁻¹), achieved without need of preconcentration steps, permitted the reliable quantification of diquat over the linear range of (0.01–0.6) μg mL⁻¹ in samples from different origins (river, tap, mineral and ground waters), even in the presence of a 40-fold concentration of paraquat, a pesticide commonly present in the commercial formulations of diquat. Figure Quartz luminometer cell     

Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection

To identify the explosive used in a terrorist attack, or to obtain an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral-flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC₅₀ values in the ng mL⁻¹ range, and CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an “improvised explosive device”. Three extraction procedures were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (LFIA), developed by using the same immunoreagents, reached a detection limit of 1 μg mL⁻¹ when tested on the same samples analysed by CL-ELISA.      

Forensic analysis of dyed textile fibers

A rapid, specific, and sensitive method has been developed using molecularly imprinted polymers (MIPs) as solid-phase extraction sorbents for extraction of trace tetracycline antibiotics (TCs) in foodstuffs. MIPs were prepared by precipitation polymerization using tetracycline as the template. Under the optimal condition, the imprinting factors for MIPs were 4.1 (oxytetracycline), 7.0 (tetracycline), 7.4 (chlortetracycline), 7.7 (doxycycline), respectively. Furthermore, the performance of MIPs as solid-phase extraction sorbents was evaluated and high extraction efficiency of molecularly imprinted solid-phase extraction (MISPE) procedure was demonstrated. Compared with commercial sorbents, MISPE gave a better cleanup efficiency than C18 cartridge and a higher recovery than Oasis HLB cartridge. Finally, the method of liquid chromatography–tandem mass spectrometry coupled with molecular-imprinted solid-phase extraction was validated in real samples including lobster, duck, honey, and egg. The spiked recoveries of TCs ranged from 94.51% to 103.0%. The limits of detection were in the range of 0.1–0.3 μg kg⁻¹. Chromatograms obtained by direct injection of the spiked egg extracts (5 × 10^-3 mmol L⁻¹) and purification with MISPE     

DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection     

Highly sensitive spectrofluorimetric determination of trace amounts of lecithin

A new spectrofluorimetric method was developed for the determination of trace amounts of lecithin using the ciprofloxacin (CIP)–terbium (Tb³⁺) ion complex as a fluorescent probe. In a buffer solution at pH=5.60, lecithin can remarkably reduce the fluorescence intensity of the CIP–Tb³⁺ complex at λ=545 nm. The reduced fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. Optimum conditions for the determination of lecithin were also investigated. The linear range and detection limit for the determination of lecithin were 1.0×10⁻⁶–3.0×10⁻⁵ mol L⁻¹ and 3.44×10⁻⁷ mol L⁻¹, respectively. This method is simple, practical, and relatively free of interference from coexisting substances. Furthermore, it has been successfully applied to assess lecithin in serum samples.      

Instrumental modification intended to save time,and volumes of sample and reagent solutions,in the atomic fluorescence spectrometric determination of mercury

Use of small membrane pumps, instead of peristaltic pumps, to introduce sample and reagent solutions into the spectrometer has several advantages in atomic fluorescence spectrometric determination of mercury. This simple modification results in a substantial saving in the time required for the measurements and so 90% of reagent solution volumes and 95% of sample solution volumes are saved, with a consequent decrease in the volume of waste generated. The sampling frequency is almost tripled, with no deterioration in sensitivity, which is similar to that obtained by use of peristaltic pumps. The relative standard deviation for ten consecutive measurements of a 1 μg L⁻¹ mercury solution was approximately 2%. Figure Small membrane pumps for the atomic fluorescene spectro metric determination of mercury     

Flow-injection solid surface lanthanide-sensitized luminescence sensor for determination of <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 μL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ _ex = 290 nm and λ _em = 546 nm. The method shows a linear range from 0.2 to 6.0 μg mL⁻¹ with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h⁻¹. A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment. Figure Typical emission bands of Tb(III) in a solid-phase PABA–Tb(III) luminescence spectrum     

Determination of Etofenprox in environmental samples by HPLC after anionic surfactant micelle-mediated extraction (coacervation extraction)

A method is described for determination of residues of the insecticide Etofenprox in environmental samples. Anionic surfactant micelle-mediated extraction (coacervation extraction) was evaluated for isolation of Etofenprox before HPLC. The optimum conditions used for extraction included: 0.09 g sodium dodecanesulfonate (SDoS), 3.1 mL (3.3, for concentrations below 0.04 mg L⁻¹) 12 mol L⁻¹ HCl, 5 min vortex stirring, 5 min centrifugation at 4000 rpm, 2 h equilibration time. The limits of quantification (LOQ) and detection (LOD) were 0.01 and 0.004 mg L⁻¹, respectively, and recoveries obtained from five real samples ranged from 94.33±2.48 to 100.13±2.71%. The precision of the method was good; relative standard deviations (RSD) were less than 7%.