Analytical approach for characterization of cadmium-induced thiol peptides—a case study using <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Phytochelatins (PC) were described earlier to play a role in metal detoxification in Chlamydomonas reinhardtii but were not clearly identified. The focus of this case study was to identify PC synthesized by C. reinhardtii exposed to Cd. Only low intracellular concentrations of cadmium (85 nmol g⁻¹ fresh weight) were sufficient to cause significant changes in thiol peptide pools. Thus, results showed a progressive decline of the glutathione content, accompanied by an induction of phytochelatins. Not only canonic phytochelatins but for the first time also the iso-phytochelatins CysPC _n and PC₂Ala were identified in this unicellular green alga using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Additionally, CysPC _n desGly, PC _n desGly, CysPC _n Glu, and PC₂Glu were found throughout MS analysis. Also, low abundant PCs could be detected due to the high sample preconcentration combined with little sample amounts (0.3 μL min⁻¹) necessary for electrospray. Identified PCs had a maximum number of 5 γ-glutamyl cysteine (γ-GluCys) units. Thiol peptides of higher molecular masses suggesting PC _n with n > 5 could be identified as intermolecular oxidation products of smaller PCs. Thiols may easily be oxidized. Therefore, PCs were reduced prior to MS analysis. Dithiothreitol and tris(2-carboxyethyl) phosphine were compared concerning their reduction effort.      

Centri-voltammetric determination of glutathione

Centri-voltammetry is a method for concentrating an analyte on an electrode with the aid of a centrifuge. It can be performed in the presence or the absence of a solid carrier/support. This is followed by a voltammetric (analytical) scan. Specifically, we describe here an application of the technique to the determination of glutathione (GSH). Silica gel is used as the carrier precipitate to which gold nanoparticles were added in order to improve accumulation as a result of their strong affinity for thiols. Voltammetry was performed with a carbon paste electrode modified with multi-wall carbon nanotubes. The response to GSH is linear in the 25 and 800 μM concentration range (the correlation coefficient being 0.9915) and the relative standard deviation is 3.40 % (at 250 μM of GSH and n?=?6). The procedure was successfully applied to the determination of GSH in wine and in synthetic plasma using the standard addition method. The recoveries are 100.8 % and 100.0 %, respectively.

Simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes using oscillatory-flow multiplex PCR

An oscillatory-flow multiplex PCR method in a capillary microfluidic channel has been developed for the simultaneous determination of pre-purified DNA of multiple foodborne bacterial pathogens. The PCR solution passes three temperature zones in an oscillatory manner. The thermal stability and sample evaporation of the microfluidic device were investigated. Under controlled conditions, a highly efficient multiplex PCR was accomplished as demonstrated for the simultaneous amplifications of 278 bp, 168 bp, and 106 bp DNA fragments within 35 min after 35 cycles for simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. This is much shorter than that of a conventional PCR machine. The detection limits of bacterial genome DNA for the three species are about 399, 314, and 626 copies per μL, respectively. This is comparable to those obtained with the conventional multiplex PCR. Consequently, the oscillatory-flow multiplex PCR technology holds good potential for rapid amplification and detection of nucleic acids of microbial foodborne pathogens.

Colloidal gold based immunochromatographic strip for the simple and sensitive determination of aflatoxin B1 and B2 in corn and rice

We have developed a simple and fast immunochromatographic test strip for the simultaneous quantitation of aflatoxin B₁ and aflatoxin B₂ in corn and rice. The strip contains three pads (sample, conjugate, and absorbing pad) and uses the respective polyclonal antibodies immobilized on gold nanoparticles. Matrix interferences were minimized by application of fugacity theory. Clean-up of samples and pre-treatment of strip pads is not required. The visual detection limit is 0.1 ng mL^?1, and the process can be completed within 5 min. Out of 113 natural samples, 16 rice and 27 corn samples (38% in total) were aflatoxin positive and the test results were confirmed by HPLC. The strip shows, however, high cross reactivity to aflatoxins G₁, G₂, and M₁. We consider this strip to possess wide applicability because of its ease of use, sensitivity, stability, and low cost.

On-Plate Self-Desalting and Matrix-Free LDI MS Analysis of Peptides With a Surface Patterned Sample Support

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH₄HCO₃ (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

Preconcentration of trace cadmium ion using magnetic graphene nanoparticles as an efficient adsorbent

Graphene-based magnetic nanoparticles (G-Fe₃O₄) were prepared and used as an effective adsorbent for the solid-phase extraction of trace quantities of cadmium from water and vegetable samples. The method avoids some of the time-consuming steps associated with traditional solid phase extraction. The excellent sorption property of the G-Fe₃O₄ system is attributed to π - π stacking interaction and hydrophobic interactions between graphene and the Cd-PAN complex. The effects of pH, the amount of G–Fe₃O₄, extraction time, type and volume of eluent, desorption time and interfering ions on the extraction efficiency were optimized. The preconcentration factor is 200. Cd(II) was then quantified by flame atomic absorption spectrometry with a detection limit of 0.32 ng mL^?1. The relative standard deviation (at 50 ng mL^?1; for n?=?10) is 2.45 %. The method has a linear analytical range from 1.1 to 150 ng mL^?1, and the recoveries in case of real samples are in the range between 93.1 % and 102.3 %.

Development of monolith-based stir bar sorptive extraction and liquid chromatography tandem mass spectrometry method for sensitive determination of ten sulfonamides in pork and chicken samples

A highly sensitive method was developed for the simultaneous determination of ten sulfonamides in pork and chicken samples by monolith-based stir bar sorptive extraction (SBSE) coupled to high-performance liquid chromatography tandem mass spectrometry. The samples were freeze-dried and extracted by acetonitrile, then enriched and further extracted by SBSE which was based on poly(vinylphthalimide-co-N,N-methylenebisacrylamide) monolith (SBSE-VPMB) as coating. To achieve optimum extraction performance of SBSE for sulfonamides, several parameters, including pH value and ionic strength in the sample matrix and extraction and desorption time, were investigated in detail. Under the optimal conditions, the limits of detection (S/N?=?3) for target sulfonamides were 1.2–6.1 ng/kg in pork and 2.0–14.6 ng/kg in chicken, respectively. Real samples spiked at the concentration of 0.5 and 5.0 μg/kg showed recoveries above 55 % and relative standard deviations below 12 %. At the same time, the extraction performances of target sulfonamides on SBSE-VPMB were compared with other SBSE based on porous monolith and commercial SBSE.

Determination of formaldehyde based on the enhancement of the chemiluminescence produced by CdTe quantum dots and hydrogen peroxide

Water-soluble cadmium telluride quantum dots (CdTe QDs) capped with glutathione (GSH) display chemiluminescence (CL) emission on reaction with hydrogen peroxide (H₂O₂) in strongly alkaline medium. It is found that the CL is strongly enhanced on addition of formaldehyde in aqueous solution. A flow injection system was developed, and it is shown that there is good linearity between CL intensity and the concentration of formaldehyde in the 0.06–3.0 μg L^?1 range. The limit of detection is as low as 10 ng L^?1. The method was successfully applied to the determination of formaldehyde in indoor air after adsorption into an aqueous phase. The recoveries for the real samples range from 97 % to 102.5 %, and the relative standard deviation is <3.8 % for intra- and inter-assay precision.

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5?×?4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min^?1 and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100?×?4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5 % triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min^?1 and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL^?1.

Microextraction by packed sorbents combined with surface-enhanced Raman spectroscopy for determination of musk ketone in river water

Microextraction by packed sorbents (MEPS) combined with Surface-enhanced Raman spectroscopy (SERS) was investigated, and applied to the determination of musk ketone (MK) in river water samples. The full MEPS–SERS method includes analyte enrichment by MEPS preconcentration with C₁₈ sorbent followed by SERS detection supported by silver nanoparticles. An eluent drop containing the analyte is deposited directly from the MEPS syringe on a CaF₂ glass plate. When the drop has dried, a specific volume of silver nanoparticles solution is added on it before each SERS measurement. Several experimental variables were studied in depth; under the optimum experimental conditions MK can be extracted from a 500 μL sample with recoveries in the range 47–63 %. The limit of detection was 0.02 mg L^?1 and the relative standard deviation 15.2 % (n?=?4). Although not investigated in this work, the proposed method might be suitable for in-situ monitoring, because of the portability of the Raman spectrometer used.

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

Polythiophene-coated Fe3O4 nanoparticles as a selective adsorbent for magnetic solid-phase extraction of silver(I), gold(III), copper(II) and palladium(II)

We have developed a fast method for sensitive extraction and determination of the metal ions silver(I), gold(III), copper(II) and palladium(II). Fe₃O₄ magnetic nanoparticles were modified with polythiophene and used for extraction the metal ions without a chelating agent. Following extraction, the ions were determined by flow injection inductively coupled plasma optical emission spectrometry. The influence of sample pH, type and volume of eluent, amount of adsorbent, sample volume and time of adsorption and desorption were optimized. Under the optimum conditions, the calibration plots are linear in the 0.75 to 100 μg L^?1 concentration range (R²?>?0.998), limits of detection in the range from 0.2 to 2.0 μg L^?1, and enhancement factors in the range from 70 to 129. Precisions, expressed as relative standard deviations, are lower than 4.2 %. The applicability of the method was demonstrated by the successful analysis of tap water, mineral water, and river water.

Determination of phthalate esters in edible oils by use of QuEChERS coupled with ionic-liquid-based dispersive liquid–liquid microextraction before high-performance liquid chromatography

A selective and low organic-solvent-consuming method of sample preparation combined with high-performance liquid chromatography with diode-array detection is introduced for analysis of phthalic acid esters in edible oils. Sample treatment involves initial liquid–liquid partitioning with acetonitrile, then QuEChERS cleanup by dispersive solid-phase extraction with primary secondary amine as sorbent. Preconcentration of the analytes is performed by ionic-liquid-based dispersive liquid–liquid microextraction, with the cleaned-up extract as disperser solvent and 1-hexyl-3-methylimidazolium hexafluorophosphate as extraction solvent. Under the optimized conditions, correlation coefficients (r) were 0.998–0.999 and standard errors (S _y/x ) were 2.67–3.37?×?10³ for calibration curves in the range 50–1000 ng g^?1. Detection limits, at a signal-to-noise ratio of 3, ranged from 6 to 9 ng g^?1. Intra-day and inter-day repeatability, expressed as relative standard deviation, were in the ranges 1.0–6.9 % and 2.4–9.4 %, respectively. Recovery varied between 84 % and 106 %. The developed method was successfully used for analysis of the analytes in 28 edible oils. The dibutyl phthalate content of four of the 28 samples (14 %) exceeded the specific migration limit established by domestic and international regulations.

Preconcentration of ultra-trace amounts of iron and antimony using ion pair solid phase extraction with modified multi-walled carbon nanotubes

Ion pair solid phase extraction was applied to the simultaneous preconcentration of iron and antimony. The ion pairs consisting of FeCl₄ ^? or SbCl₄ ^? anions and the benzyldimethyltetradecyl ammonium cation were formed on the surface of multi-walled carbon nanotubes, then eluted with nitric acid, and the elements finally quantified by ETAAS. The adsorption capacities of the impregnated MWCNTs are 9.2 mg g^?1 for iron and 27.5 mg g^?1 for antimony. The following analytical figures of merit were determined for iron and antimony, respectively: Enrichment factors of 210 and 230, assay precisions of ±5.3 % and ±4.8 %, linear calibration plots from 0.7 to 9.4 and 13.0 to 190 ng L^?1, and detection limits of 0.17 and 3.5 ng L^?1. The method was applied to the determination of iron and antimony in human hair, synthetic sample, and to the certified reference materials gold ore (MA-1b) and trace elements in water (SRM 1643d).

Nanostructured platform for the detection of Neisseria gonorrhoeae using electrochemical impedance spectroscopy and differential pulse voltammetry

We report on a nanocomposite based genosensor for the detection of Neisseria gonorrhoeae, a bacterium causing the sexually transmitted disease gonorrhoea. Amino-labeled probe DNA was covalently immobilized on electrochemically prepared polyaniline and iron oxide (PANI-Fe₃O₄) nanocomposite film on an indium tin oxide (ITO) electrode. Scanning electron microscopy, transmission electron microscopy, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) techniques have been employed to characterize surface of the modified electrode. The genosensor has detection limits of 1?×?10^-15 M and 1?×?10^-17 M, respectively, using the EIS and DPV techniques. This biosensor can discriminate a complementary sequence from a single-base mismatch and from non-complementary DNA, and has been utilized for detection of DNA extracted from N. gonorrhoeae culture, and from patient samples with N. gonorrhoeae. It is found to exhibit good specificity for N. gonorrhoeae species and shows no response towards non-gonorrhoeae type of Neisseria species (NgNs) and other gram-negative bacterias (GNBs). The affinity constant for hybridization calculated using the Langmuir adsorption isotherm model is found to be 3.39?×?10⁸ M^-1.

A magnetic metal-organic framework as a new sorbent for solid-phase extraction of copper(II), and its determination by electrothermal AAS

We report on the synthesis of Fe₃O₄-functionalized metal-organic framework (m-MOF) composite from Zn(II) and 2-aminoterephthalic acid by a hydrothermal reaction. The magnetic composite is iso-reticular and was characterized by FTIR, X-ray diffraction, SEM, magnetization, and TGA. The m-MOF was then applied as a sorbent for the solid-phase extraction of trace levels of copper ions with subsequent quantification by electrothermal AAS. The amount of sorbent applied, the pH of the sample solution, extraction time, eluent concentration and volume, and desorption time were optimized. Under the optimum conditions, the enrichment factor is 50, and the sorption capacity of the material is 2.4 mg g^?1. The calibration plot is linear over the 0.1 to 10 μg L^?1 Cu(II) concentration range, the relative standard deviation is 0.4 % at a level of 0.1 μg L^?1 (for n?=?10), and the detection limit is as low as 73 ng L^?1. We consider this magnetic MOF composite to be a promising and highly efficient material for the preconcentration of metal ions.

Cetyltrimethylammonium-coated magnetic nanoparticles for the extraction of bromate,followed by its spectrophotometric determination

We report on a combination of magnetic solid-phase extraction and spectrophotometric determination of bromate. Cetyltrimethylammonium ion was adsorbed on the surface of phenyl-functionalized silica-coated Fe₃O₄ nanoparticles (Ph-SiO₂@Fe₃O₄), and these materials served as the sorbent. The effects of surfactant and amount of sorbent, the composition of the desorption solution, the extraction time and temperature were optimized. Under optimized conditions, an enrichment factor of 12 was achieved, and the relative standard deviation is 2.9 % (for n?=?5). The calibration plot covers the 1–50 ng mL^?1 range with reasonable linearity (r ²?>?0.998); and the limit of detection is 0.5 ng mL^?1. The method is not interfered by ionic compounds commonly found in environmental water samples. It was successfully applied to the determination of bromate in spiked water samples.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4?±?11.5 and 10.1?±?4.0 nmol/mg protein, respectively.

Enzyme-linked immunosorbent assay and immunochromatographic strip for rapid detection of atrazine in water samples

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic (ICG) strip for the determination of the herbicide atrazine in water samples. The ELISA had a half-maximum inhibition concentration (IC₅₀) of 0.12 ng mL^?1 and a limit of detection (LOD, calculated as the IC₁₅ value) of 0.01 ng mL^?1. The average of recoveries for all spiked water samples was 96.5%. There was a good correlation between the data determined by this ELISA and those obtained by high performance liquid chromatography (HPLC) (r ² ?=?0.996). The visual LOD of the ICG strip assay was 2 ng mL^?1. The assay process only took 10 min, and no sample pretreatment was required. Its high specificity, sensitivity and fast detection made the strip well suited for on-site screening of atrazine in water samples. Both the ELISA and the ICG strip assay are useful for rapid analysis of a large number of water samples at low cost.

Development of a microwave-assisted-extraction-based method for the determination of aflatoxins B1, G1, B2, and G2 in grains and grain products

This article describes the use of microwave-assisted extraction (MAE) as a pretreatment technique for the determination of aflatoxins B₁, G₁, B₂, and G₂ in grains and grain products. The optimal operation parameters, including extraction solvent, temperature, and time, were identified to be acetonitrile as the extraction solvent at 80 °C with 15 min of MAE. The extracts were cleaned up using solid-phase extraction followed by derivatization with trifluoroacetic acid and were determined by liquid chromatography–fluorescence detection. A Sep-Pak cartridge was chosen over Oasis HLB and Bond Elut cartridges. By the use of aflatoxin M₁ as an internal standard, relative recoveries of the aflatoxins ranged from 90.7 to 105.7 % for corn and from 88.1 to 103.4 % for wheat, with relative standard deviations between 2.5 and 8.7 %. A total of 36 samples from local markets were analyzed, and aflatoxin B₁ was found to be the predominant toxin, with concentrations ranging from 0.42 to 3.41 μg/kg.