Centri-voltammetry for biosensing systems: biocentri-voltammetric xanthine detection

Centri-voltammetry is a method that combines centrifugation and voltammetry. This method, developed by our group in 2003, yielded promising results when applied to trace analysis of metal ios. We demonstrate here the first application of centri-voltammetry to biosensing systems. A xanthine biosensor was constructed by immobilizing xanthine oxidase on a planar platinum electrode which then was placed at the bottom of a centri-voltammetric cell. The experimental parameters were optimized to give two linear ranges. The first is from 0.1 to 1???M, and the second from 5 to 50???M. The RSD is 3.4 (n?=?5). The biosensor was applied to the determination of xanthine in wine and in urine. Calculated recoveries are 101?±?0.61% (n?=?3) for wine samples, and 102?±?0.556% (n?=?3) for urine samples.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg^?1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg^?1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg^?1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n?=?6), mean intra-day precision 3.2 % (n?=?3 within a plate; n?=?2 repeated within a day), mean inter-day precision 3.7 % (n?=?3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n?=?2), and mean recovery 106.9 % (n?=?5 different concentrations; n?=?4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO₂ chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Facile synthesis of a porous network-like silver film for electrocatalytic detection of nitrate

We have developed a method for in-situ construction of a porous network-like silver film on the surface of a glassy carbon electrode (GCE). It is based on a galvanic replacement reaction where a layer of copper nanoparticles is first electrodeposited as a sacrificial template. The silver film formed possesses a porous network-like structure and consists of an assembly of numerous nanoparticles with an average size of 200 nm. The electrode displays excellent electrocatalytic activity, good stability, and fast response (within 2 s) toward the reduction of nitrate at a working potential of ?0.9 V. The catalytic currents linearly increase with the nitrate concentrations in the range of 0.08–6.52 mM, with a detection limit of 3.5 μM (S/N?=?3) and a repeatability of 3.4 % (n?=?5).

Biosensor for aluminium(III) based on its inhibition of α-chymotrypsin immobilized on a screen-printed carbon electrode modified with gold nanoparticles

We report on an amperometric assay for Al(III) ions that is based on the inhibition of the enzyme α-chymotrypsin. Screen-printed carbon electrodes modified with gold nanoparticles were used as solid supports for the immobilization of the enzyme. The amperometric response of the synthetic enzyme substrate substrate N-benzoyl-L-tyrosine ethyl ester is affected by Al(III) ions, and this leads to a decrease in the amperometric oxidation current. The assay has a detection limit of 3.3?μM of Al(III). The repeatability and reproducibility of the method are 6.9% (n?=?3) and 6.4% (n?=?5), respectively. Main interferents include Mo(VI), W(VI) and Fe(III) ions. The method was successfully applied to the determination of Al(III) in tap water.

Extraction of phthalate esters from water and beverages using a graphene-based magnetic nanocomposite prior to their determination by HPLC

We have developed a highly sensitive microextraction method for the preconcentration of some phthalate esters such as diethyl phthalate, di-n-propylphthalate, di-n-butyl-phthalate, dicyclohexyl-phthalate, and diethyl-hexyl phthalate prior to their determination by HPLC. It is based on a magnetic graphene nanocomposite as an effective adsorbent. The effects of the amount of the extractant composite employed, extraction time, pH values, salt concentration and desorption conditions were investigated. Under the optimum conditions, the enrichment factors range from 1574 to 2880. Response is linear in the concentration range from 0.1 to 50?ng?mL^?1. The limits of detection (at S/N?=?3) were between 0.01 and 0.04?ng?mL^?1. The method was successfully applied to the determination of five phthalate esters in water and beverage samples.

Gas-phase chemiluminescent determination of inorganic selenium in water samples following flow injection hydride generation and cryotrapping

We describe a method for the determination of inorganic selenium in water samples via gas-phase chemiluminescence (GPCL). Se(IV) was first derivatized with 4-nitro-o-phenylenediamine to form 5-nitropiazselenol. The latter was decomposed by persulfate through photocatalytic oxidation to give Se(VI), which was reduced to Se(IV). Selenium hydride was generated from Se(IV) through reduction with sodium borohydride and then preconcentrated using cryotrapping. The cryotrapped hydride was evaporated and carried to a reaction chamber by a stream of helium, where it produced GPCL as a result of ozonation. The method exhibits a wide linear calibration range (from 0.5?μg?L^?1 to 1.0?mg?L^?1) with a detection limit of 0.12?μg?L^?1 (for n?=?11), and a relative standard deviation of 3.90?% (at n?=?11) at 5.0?μg?L^?1 level of selenium. The method was applied to the determination of inorganic selenium in water samples and gave satisfactory results.

Colorimetric determination of 8-hydroxy–2′-deoxyguanosine using label-free aptamer and unmodified gold nanoparticles

We report on a sensitive and specific method for the visual and colorimetric detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in human urine by using the 8-OHdG-aptamer (Apt) as the recognition element. The aptamer was adsorbed on the surface of gold nanoparticles which enhances their stability. On addition of 8-OHdG, the conformation of the aptamer changes to form a G-quadruplex structure, thereby losing the ability to protect the nanoparticles and causing a color change from red to blue. The conformational changes were also studied by circular dichroism. The response is linearly dependent on the concentration of 8-OHdG in the range from 5.6 to 282 nM, the limit of detection is 1.7 nM, the relative standard deviation ranges from 1.1 to 4.2 % (for n?=?6), and the recoveries range from 95.9 to 104.9 %. The method paves the way for visual analysis of samples without the use of costly instruments.

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.

Carbon-based magnetic nanocomposites in solid phase dispersion for the preconcentration some of lanthanides, followed by their quantitation via ICP-OES

We report on a method for the extraction of the lanthanide ions La(III), Sm(III), Nd(III) and Pr(III) using a carbon-ferrite magnetic nanocomposite as a new adsorbent, and their determination via flow injection ICP-OES. The lanthanide ions were converted into their complexes with 4-(2-pyridylazo)resorcinol, and these were adsorbed onto the nanocomposite. Fractional factorial design and central composite design were applied to optimize the extraction efficiencies to result in preconcentration factors in the range of 141–246. Linear calibration plots were obtained, the limits of detection (at S/N?=?3) are between 0.5 and 10 μg?L^?1, and the intra-day precisions (n?=?3) range from 3.1 to 12.8 %. The method was successfully applied to a certified reference material.

Preconcentration and separation of ultra-trace palladium ion using pyridine-functionalized magnetic nanoparticles

We present a study on the application of magnetic nanoparticles (MNPs) prepared from Fe₃O₄ and functionalized with pyridine as an adsorbent for the solid-phase extraction of trace quantities of Pd(II) ion. The pyridine group was immobilized on the surface of the MNPs by covalent bonding of isonicotinamide. The modified MNPs can be readily separated from an aqueous solution by applying an external magnetic field. Effects of pH, the amount of functionalized MNPs, extraction time, type and quantity of eluent, desorption time, break-through volume and interfering ions on the extraction efficiency were optimized. The amount of Pd(II) was then determined using FAAS. Under the optimized conditions, the detection limit and preconcentration factor are 0.15?μg?L^-1 and 196, respectively, and the relative standard deviation (at 20?μgL^?1; for n?=?10) is 3.7?%. The method had a linear analytical range from 1 to 80?μg?L^-1 and was applied to determine Pd(II) in spiked tape water and soil.

Voltammetric sensing of thallium at a carbon paste electrode modified with a crown ether

We describe a new method for differential-pulse anodic stripping voltammetric determination of thallium(I) using a carbon paste electrode modified with dicyclohexyl-18-crown-6. The effect of supporting electrolyte (type and pH), accumulation and reduction potential, and of time and amount of modifier were investigated by differential pulse anodic stripping voltammetry. A method was then worked out for the determination of thallium at low levels. Under optimized conditions, the response to Tl(I) is linear in the range from 3.0 to 250 ng mL^?1. The detection limit is 0.86 ng mL^?1. The sensor displays good repeatability (with a relative standard deviation of ±2.70 % for n?=?7) and was applied to the determination of Tl(I) in water, hair samples, and certified reference materials.

A cloud point extraction for spectrophotometric determination of ultra- trace antimony without chelating agent in environmental and biological samples

We report on a simple, sensitive and reliable method for the cloud point extraction of antimony (Sb) and its subsequent spectrophotometric detection. It is based on the color reaction of Sb (III) with iodide in acidic medium and subsequent micelle-mediated extraction of tetraiodoantimonate using a non-ionic surfactant in the absence of any chelating agent. The effects of reaction and extraction parameters were optimized. The calibration plot is linear in the range of 0.80–95?ng?mL^?1 of antimony in the sample solution, with a regression coefficient (r) of 0.9994 (for n?=?9). The detection limit (at SNR?=?3) is 0.23?ng?mL^?1, and the relative standard deviations at 10 and 70?ng?mL^?1 of antimony are 3.32 and 1.85?% (at n?=?8), respectively. The method compared favorably to other methods and was applied to determine antimony in seawater, anti-leishmania drug (glucantime), and human serum.

Bioactivity fingerprint analysis of cyclooxygenase-2 ligands from radix Aconiti by ultrafiltration–UPLC–MSn

A novel fingerprinting method, bioactivity fingerprint analysis, based on an ultrafiltration–ultraperformance liquid chromatography–multistage tandem mass spectrometry (UPLC–MS ⁿ ) method is proposed for the quality control of herbal medicines from the bioactivity viewpoint concerning the efficacy of herbal medicines. The bioactivity fingerprints reflecting the anti-inflammatory activities of radix Aconiti and radix Aconiti preparata were established. With use of ultrafiltration UPLC–MS ⁿ , 11 cyclooxygenase-2 ligands from radix Aconiti preparata and 14 cyclooxygenase-2 ligands from radix Aconiti were found after incubation with cyclooxygenase-2. Twelve of the cyclooxygenase-2 ligands were identified by the ultraperformance UPLC–MS ⁿ method. The enrichment factor of each peak in the bioactivity fingerprint was calculated and was demonstrated to be characteristic, which makes bioactivity fingerprint analysis for the quality control of herbal medicines possible from the viewpoint of their bioactivities.

Extraction of tributyltin by magnetic molecularly imprinted polymers

We have prepared core-shell magnetic molecularly imprinted polymer nanoparticles for recognition and extraction of tributyl tin (TBT). The use of particles strongly improves the imprinting effect and leads to fast adsorption kinetics and high adsorption capacities. The functional monomer acrylamide was grafted to the surface of Fe₃O₄ nanospheres in two steps, and MIP layers were then formed on the surface by creating a MIP layer on the surface consisting of poly(ethyleneglycol dimethacrylate) with a TBT template. The particles were characterized in terms of morphological, magnetic, adsorption, and recognition properties. We then have developed a method for the extraction of TBT from spiked mussel (Mytilidae), and its determination by liquid chromatography-tandem mass spectrometry. The method has a limit of detection of 1.0 ng?g^?1 (n?=?5) of TBT, with a linear response between 5.0 and 1,000 ng?g^?1. The proposed method was successfully applied to the determination of trace TBT in marine food samples with recoveries in the range of 78.3–95.6 %.

Ionic liquid-based hollow fiber supported liquid membrane extraction combined with thermospray flame furnace AAS for the determination of cadmium

We have developed a simple and effective method for hollow fiber liquid-phase microextraction of cadmium. It is based on the use of a room temperature ionic liquid (RTIL) and was coupled to thermospray flame furnace AAS. The RTIL was placed in the pores of a polypropylene hollow fiber (acting as a liquid membrane) and also used as the acceptor solution. Ammonium pyrrolidinedithiocarbamate (APDC) was used as the chelating agent. The effects of the concentration of APDC, the pH of samples, stirring rates, extraction time, and potential interferences were optimized to result in a detection limit of 9?ng?L^?1 and an enrichment factor of 90. The relative standard deviation is 4.7% (at 0.5?ng?mL^?1, for n?=?5). The method was successfully applied to the determination of cadmium.

Double stable isotope ultra performance liquid chromatographic-tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase, the crucial enzyme responsible for synthesis of epinephrine

Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-l-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n?=?10) and 10.9 to 13.6 % (n?=?10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.

Lipidomic profiling of plasma in patients with chronic hepatitis C infection

Chronic hepatitis C virus (HCV) infection is a global health issue. Although its progression is reported to be closely associated with lipids, the way in which the plasma lipidome changes during the development of chronic HCV infection in humans is currently unknown. Using an improved quantitative high-throughput lipidomic platform, we profiled 284 lipids in human plasma samples obtained from healthy controls (n?=?11) and patients with chronic HCV infection (n?=?113). The intrahepatic inflammation grade (IG) of liver tissue was determined by biopsy. Two types of mass spectrometers were integrated into a single lipidomic platform with a wide dynamic range. Compared with previous methods, the performance of this method was significantly improved in terms of both the number of target sphingolipids identified and the specificity of the high-resolution mass spectrometer. As a result, 44 sphingolipids, one diacylglycerol, 43 triglycerides, 24 glycerophosphocholines, and 5 glycerophospho-ethanolamines were successfully identified and quantified. The lipid profiles of individuals with chronic HCV infection were significantly different from those of healthy individuals. Several lipids showed significant differences between mild and severe intrahepatic inflammation grades, indicating that they could be utilized as novel noninvasive indicators of intrahepatic IG. Using multivariate analysis, healthy controls could be discriminated from HCV patients based on their plasma lipidome; however, patients with different IGs were not well discriminated. Based on these results, we speculate that variations in lipid composition arise as a result of HCV infection, and are caused by HCV-related digestive system disorders rather than progression of the disease.

The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu²⁺ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Multiwalled carbon nanotubes modified with 2-aminobenzothiazole modified for uniquely selective solid-phase extraction and determination of Pb(II) ion in water samples

A solid phase extraction method is presented for the selective preconcentration and/or separation of trace Pb(II) on multiwalled carbon nanotubes modified with 2-aminobenzothiazole. Inductively coupled plasma optical emission spectrometry was used for detection. The effects of pH, shaking time, sample flow rate and volume, elution condition and interfering ions were examined using batch and column procedures. An enrichment factor of 100 was accomplished. Common other ions do not interfere in both the separation and determination. The maximum adsorption capacity of the sorbent at optimum conditions is 60.3?mg?g^?1 of Pb(II), the detection limit (3??) is 0.27?ng?mL^?1, and the relative standard deviation is 1.6% (n?=?8). The method was validated using a certified reference material, and has been applied to the determination of trace Pb(II) in water samples with satisfactory results.