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Publications in this journal
Article: Kinetic-exclusion analysis-based immunosensors versus enzyme-linked immunosorbent assays for measurement of cancer markers in biological specimens.[show abstract] [hide abstract]
ABSTRACT: Kinetic exclusion analysis (KinExA)-based immunosensors and enzyme-linked immunosorbent assays (ELISA) have been developed and validated for measurement of five different cancer markers in biological specimens. These markers were: 2'-deoxycytidine (dCyd), 8-hydroxy-2'-deoxyguanosine (8HdG), carbohydrate antigen (CA15-3), α-fetoprotein (AFP), and β-subunit of human chorionic gonadotropin (β-HCG). The KinExA-based assays were conducted on the KinExA™ 3200 instrument. The ELISA assays employed the competitive immunoassay format for dCyd and 8HdG, however they employed the direct sandwich-type format for CA15-3, AFP, and β-HCG. Each assay was validated in terms of its limit of detection, working range, precision profile, and accuracy. The analytical performances of the KinExA-based sensors were found to be superior to the ELISA for the five markers. The data demonstrated that the format of the assay may influence its performance characteristics (sensitivity, precision, etc.), even when exactly the same reagents are employed. The superior performance of the KinExA format is most likely due to: (1) the high surface area of beads containing the immobilized capture in the flow cell of the instrument, (2) the high flow rate of the reagents passing through the beads, which minimizes the diffusion limitations at the reaction surface, and (3) the limited time that the antibody is in contact with the capture reagent. The KinExA-based assays exhibited three noteworthy properties compared with ELISA: (1) avoiding the problems of mass transport limitations, and mobility effects, (2) KinExA analysis with automated sampling increase the assay convenience; and (3) providing high sensitivity with a lower limit of detection and better precision than ELISA. The proposed KinExA-based immunosensors are anticipated to have a great value in measurement of the cancer markers where more confident results are needed.Talanta 07/2013; 111C:13-9.
Article: Separation and determination of degradation products of acid orange 7 by capillary electrophoresis/capacitively coupled contactless conductivity detector.[show abstract] [hide abstract]
ABSTRACT: Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detector (C(4)D) was developed to separate azo-dyestuff acid orange 7 (AO7) and its six degradation products. The analyzed products were sulfamic acid, oxalic acid, benzenesulfonic acid, 4-hydroxybenzene sulfonic acid, phthalic acid, and 4-aminobenzene sulfonic acid. In developing the method, types and concentrations of running buffers, injecting voltage and time, and applied voltage were tested to obtain optimum conditions to analyze target compounds. The separation was successfully achieved within 10min using a fused-silica capillary under the following conditions: 20mmolL(-1) acetate acid buffer, electrokinetic injection of -12kV×10s, and applied voltage of -13kV. The developed method was applied to analyze degradation products in situ during the reaction of AO7 with Fenton reagent (Fe(II)+H2O2 at pH 4.0).Talanta 07/2013; 111C:54-61.
Article: Method for the purification of polybrominated diphenyl ethers in sediment for compound-specific isotope analysis.[show abstract] [hide abstract]
ABSTRACT: A purification method for lower polybrominated diphenyl ethers (PBDEs, from tri- to hexa-BDE) in sediment for compound-specific isotope analysis (CSIA) was developed in this study. The compounds were extracted using a Soxhlet apparatus with addition of activated alumina and Florisil in the paper tube during the Soxhlet extraction provided for less complex extracts. Then, the extract was isolated from polar compounds using a multi-layer silica gel column, separated into different fractions using alumina/silica (Al/Si) gel columns and finally purified using a Florisil column. The mean recoveries of the major PBDE congeners in the spiked samples ranged from 76.2% to 82.4%. The purity of the samples was verified by GC-MS in full scan mode. The stable isotopic integrity of the spiked samples after the purification was tested by comparing the stable carbon isotope ratios (δ(13)C) of the processed and the unprocessed standard materials. The differences in the δ(13)C values for each compound between the processed and unprocessed standards were less than 0.5‰, with the exception of BDE100 (0.54‰). Finally, the purification and isotope analysis method was successfully applied to measure the δ(13)C of PBDEs in sediments. This application of the method indicated that CSIA seems to be a promising method for providing intrinsic characteristics for further environmental fate studies of PBDEs.Talanta 07/2013; 111C:93-7.
Article: Multi-residue method for determination of selected neonicotinoid insecticides in honey using optimized dispersive liquid-liquid microextraction combined with liquid chromatography-tandem mass spectrometry.[show abstract] [hide abstract]
ABSTRACT: The objective of this study was to develop analytical method based on optimized dispersive liquid-liquid microextraction (DLLME) as a pretreatment procedure combined with reversed phase liquid chromatographic separation on C18 column and isocratic elution for simultaneous MS/MS determination of selected neonicotinoid insecticides in honey. The LC-MS/MS parameters were optimized to unequivocally provide good chromatographic separation, low detection (LOD, 0.5-1.0μgkg(-1)) and quantification (LOQ, 1.5-2.5μgkg(-1)) limits for acetamiprid, clothianidin, thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different types (chloroform, dichloromethane) and volumes of extraction (0.5-3.0mL) and dispersive (acetonitrile; 0.0-1.0mL) solvent and by mathematical modeling it was possible to establish the optimal sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the concentration range of LOQ-100.0μgkg(-1) were used to compensate the matrix effect and to fulfill the requirements of SANCO/12495/2011 for the accuracy (R 74.3-113.9%) and precision (expressed in terms of repeatability (RSD 2.74-11.8%) and within-laboratory reproducibility (RSDs 6.64-16.2%)) of the proposed method. The rapid (retention times 1.5-9.9min), sensitive and low solvent consumption procedure described in this work provides reliable, simultaneous, and quantitative method applicable for the routine laboratory analysis of seven neonicotinoid residues in real honey samples.Talanta 07/2013; 111C:125-33.
Article: Trace analysis of mono-, di-, tri-substituted polyfluoroalkyl phosphates and perfluorinated phosphonic acids in sewage sludge by high performance liquid chromatography tandem mass spectrometry.[show abstract] [hide abstract]
ABSTRACT: A new method using ultrasonic extraction and solid phase extraction (SPE) clean-up pretreatments was developed for the analysis of mono-, di- and tri-substituted polyfluoroalkyl phosphates (abbreviated as mono-PAPs, di-PAPs and tri-PAPs) and perfluorinated phosphonic acids (PFPAs) in sludge from wastewater treatment plants (WWTPs). For the ultrasonic extraction of three mono-PAPs, three di-PAPs and three PFPAs in sludge samples, a mixture of tetrahydrofuran/acetic acid (1:1, v/v) was found to be the most suitable extraction solvent. The subsequently optimized clean-up and enrichment procedures were carried out with weak anion exchange (WAX) cartridges in-line coupled with graphitized carbon black (ENVI-Carb) tubes. Two tri-PAPs were ultrasonically extracted by acetonitrile/tetrahydrofuran (1:1, v/v) and cleaned by mixed-mode anion exchange (MAX) in-line coupled with ENVI-Carb cartridges. The analytes were analyzed by optimized high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method either in negative or positive ionization mode. The method quantification limits (MQLs) of the 11 analytes in sludge ranged from 0.6 to 5.1ng/g, meanwhile the total recoveries of the pretreatment varied from 24% (6:2 mono-PAP) to 107% (PFDPA). The method was successfully applied to analyze 16 sewage sludge samples collected from seven provinces in China, and two mono-PAPs were identified with concentrations ranging from <MQLs to 10.7ng/g.Talanta 07/2013; 111C:170-7.
Article: Determining quality of caviar from Caspian Sea based on Raman spectroscopy and using artificial neural networks.[show abstract] [hide abstract]
ABSTRACT: The purpose of this study was to evaluate the feasibility of Raman spectroscopy for predicting purity of caviars. The 93 wild caviar samples of three different types, namely; Beluga, Asetra and Sevruga were analysed by Raman spectroscopy in the range 1995cm(-1) to 545cm(-1). Also, 60 samples from combinations of every two types were examined. The chemical origin of the samples was identified by reference measurements on pure samples. Linear chemometric methods like Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were used for data visualisation and classification which permitted clear distinction between different caviars. Non-linear methods like Artificial Neural Networks (ANN) were used to classify caviar samples. Two different networks were tested in the classification: Probabilistic Neural Network with Radial-Basis Function (PNN) and Multilayer Feed Forward Networks with Back Propagation (BP-NN). In both cases, scores of principal components (PCs) were chosen as input nodes for the input layer in PC-ANN models in order to reduce the redundancy of data and time of training. Leave One Out (LOO) cross validation was applied in order to check the performance of the networks. Results of PCA indicated that, features like type and purity can be used to discriminate different caviar samples. These findings were also supported by LDA with efficiency between 83.77% and 100%. These results were confirmed with the results obtained by developed PC-ANN models, able to classify pure caviar samples with 93.55% and 71.00% accuracy in BP network and PNN, respectively. In comparison, LDA, PNN and BP-NN models for predicting caviar types have 90.3%, 73.1% and 91.4% accuracy. Partial least squares regression (PLSR) models were built under cross validation and tested with different independent data sets, yielding determination coefficients (R(2)) of 0.86, 0.83, 0.92 and 0.91 with root mean square error (RMSE) of validation of 0.32, 0.11, 0.03 and 0.09 for fatty acids of 16.0, 20.5, 22.6 and fat, respectively.Talanta 07/2013; 111C:98-104.
Article: Potential of time series-hyperspectral imaging (TS-HSI) for non-invasive determination of microbial spoilage of salmon flesh.[show abstract] [hide abstract]
ABSTRACT: This study investigated the potential of using time series-hyperspectral imaging (TS-HSI) in visible and near infrared region (400-1700nm) for rapid and non-invasive determination of surface total viable count (TVC) of salmon flesh during spoilage process. Hyperspectral cubes were acquired at different spoilage stages for salmon chops and their spectral data were extracted. The reference TVC values of the same samples were measured using standard plate count method and then calibrated with their corresponding spectral data based on two calibration methods of partial least square regression (PLSR) and least-squares support vector machines (LS-SVM), respectively. Competitive adaptive reweighted sampling (CARS) was conducted to identify the most important wavelengths/variables that had the greatest influence on the TVC prediction throughout the whole wavelength range. As a result, eight variables representing the wavelengths of 495nm, 535nm, 550nm, 585nm, 625nm, 660nm, 785nm, and 915nm were selected, which were used to reduce the high dimensionality of the hyperspectral data. On the basis of the selected variables, the models of PLSR and LS-SVM were established and their performances were compared. The CARS-PLSR model established using Spectral Set I (400-1000nm) was considered to be the best for the TVC determination of salmon flesh. The model led to a coefficient of determination (rP(2)) of 0.985 and residual predictive deviation (RPD) of 5.127. At last, the best model was used to predict the TVC values of each pixel within the ROI of salmon chops for visualizing the TVC distribution of salmon flesh. The research demonstrated that TS-HSI technique has a potential for rapid and non-destructive determination of bacterial spoilage in salmon flesh during the spoilage process.Talanta 07/2013; 111C:39-46.
Article: Screen-printed biosensor based on the inhibition of the acetylcholinesterase activity for the determination of codeine.[show abstract] [hide abstract]
ABSTRACT: The current paper presents the chronoamperometric determination of codeine using screen-printed carbon electrodes that incorporate tetrathiafulvalene in the matrix of the working electrode, as mediator, and cross-linked acetylcholinesterase. Applying a potential of +250mV, a 1mM solution of acetylthiocholine in electrolyte solution pH 7 gives an oxidation signal due to the dimerization of its metabolite after the reaction with the enzyme. This electrochemical signal is decreased by consecutive additions of a solution of codeine, which allows the performance of curves of calibration for the validation of this electrochemical method, giving a reproducibility of 3.31% (n=6) and a capability of detection of 20µM. This type of inhibition has been studied by means of a Lineweaver-Burk plot. Additionally, the developed biosensor was used for the determination of the quantity of codeine in pharmaceutical commercial tablets and urine samples.Talanta 07/2013; 111C:8-12.
Article: Determination of the titratable acidity and the pH of wine based on potentiometric flow injection analysis.[show abstract] [hide abstract]
ABSTRACT: A FIA system using a pH-sensitive detector based on a graphite/quinhydrone/silicone composite electrode was applied to determine sequentially the titratable acidity and the pH of wine, as well as the sum of calcium and magnesium ions. For all measurements the same FIA configuration was used employing different carrier solutions. The results for the determination of acidity and pH are in good agreement with those obtained by classical potentiometric titrations and by pH measurements using a conventional glass electrode. The standard deviation was less than 1.5% for both kinds of measurements and the sample volume was 150μL. The method allows about 40 determinations of titratable acidity per hour and 30 pH measurements per hour. The titration method can be adjusted to the legal requirements in USA and Europe.Talanta 07/2013; 111C:134-9.
[show abstract] [hide abstract]
ABSTRACT: A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.Talanta 07/2013; 111C:206-14.
Article: A comparative study between different alternatives to prepare gaseous standards for calibrating UV-Ion Mobility Spectrometers.[show abstract] [hide abstract]
ABSTRACT: An UV-Ion Mobility Spectrometer is a simple, rapid, inexpensive instrument widely used in environmental analysis among other fields. The advantageous features of its underlying technology can be of great help towards developing reliable, economical methods for determining gaseous compounds from gaseous, liquid and solid samples. Developing an effective method using UV-Ion Mobility Spectrometry (UV-IMS) to determine volatile analytes entails using appropriate gaseous standards for calibrating the spectrometer. In this work, two home-made sample introduction systems (SISs) and a commercial gas generator were used to obtain such gaseous standards. The first home-made SIS used was a static head-space to measure compounds present in liquid samples and the other home-made system was an exponential dilution set-up to measure compounds present in gaseous samples. Gaseous compounds generated by each method were determined on-line by UV-IMS. Target analytes chosen for this comparative study were ethanol, acetone, benzene, toluene, ethylbenzene and xylene isomers. The different alternatives were acceptable in terms of sensitivity, precision and selectivity.Talanta 07/2013; 111C:111-8.
Article: Microfluidic cytometer based on dual photodiode detection for cell size and deformability analysis.[show abstract] [hide abstract]
ABSTRACT: Cellular mechanical properties play an important role in disease diagnosis. Distinguishing cells based on their mechanical properties provides a potential method for label-free diagnosis. In this work, a convenient and low-cost microfluidic cytometer was developed to study cell mechanical properties and cell size based on the change of transmission intensity, using a low-cost commercial laser as a light source and two photodiodes as detectors. The cells pass through a narrow microchannel with a width smaller than the cell dimension, integrated in a polydimethylsiloxane chip, below which the laser is focused. The transit time of individual cells is measured by the time difference detected by two photodiodes. This device was used to study the difference in cell mechanical properties between HL60 cells treated with and without Cytochalasin D. Furthermore, it was also applied to distinguish cells with different diameters, HL60 cells and red blood cells, by measuring the transmission intensity.Talanta 07/2013; 111C:178-82.
Article: Detection and discrimination of alpha-fetoprotein with a label-free electrochemical impedance spectroscopy biosensor array based on lectin functionalized carbon nanotubes.[show abstract] [hide abstract]
ABSTRACT: A label-free electrochemical impedance spectroscopy (EIS) biosensor for the sensitive determination and discrimination of alpha-fetoprotein (AFP) was developed by employing wheat-germ agglutinin (WGA) lectin as molecular recognition element. The EIS biosensor was fabricated by adsorbing carboxyl-functionalized single-wall carbon nanotubes (SWNTs) onto a screen-printed carbon electrode (SPCE) and subsequently covalently coupling WGA onto the surface of the SWNTs-modified electrode. Upon binding of AFP to the biosensor, the electron transfer resistance was increased and the increase in the electron transfer resistance was linearly proportional to the logarithm of the concentration of AFP in the range from 1 to 100ng/L with a detection limit of 0.1ng/L. It was found that the employment of SWNTs as immobilization platform could reduce the background and enhance the EIS response. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of AFP N-glycan and discriminate AFP between healthy and cancer patients serum samples. This work demonstrates that the employment of carbon nanotubes as immobilization platform and lectin as molecular recognition element in biosensor array is a promising approach for the determination and discrimination of glycoproteins for cancer diagnosis. The strategy proposed in this work could further be used for high-throughput, label-free profiling of the glycan expression of cancer-related glycoproteins and to develop methods for cancer diagnosis in the early stages.Talanta 07/2013; 111C:62-8.
Article: A novel amperometric sensor for ascorbic acid based on poly(Nile blue A) and functionalised multi-walled carbon nanotube modified electrodes.[show abstract] [hide abstract]
ABSTRACT: A new type of modified electrode sensor for ascorbic acid has been prepared by deposition of multi-walled carbon nanotubes (MWCNT) and poly(Nile blue A) on the surface of glassy carbon electrodes. Nile blue A was electropolymerised either beneath (directly on glassy carbon) or onto the MWCNT layer by potential cycling in phosphate buffer solution at pH 6.0. Characterisation of the modified electrodes was carried out by cyclic voltammetry and electrochemical impedance spectroscopy. Quantitative determination of ascorbate was achieved by cyclic voltammetry and fixed potential amperometry in phosphate buffer solution at pH 5.3. The modified electrodes exhibited good sensitivity, wide linear range, a detection limit of 1.6μM and good stability, showing that they can be used as sensors for ascorbic acid. There is no interference from compounds commonly found in clinical and pharmaceutical samples and the determination of ascorbic acid in commercial tablet samples was successfully performed.Talanta 07/2013; 111C:76-84.
Article: Optimisation and application of accelerated solvent extraction and flash chromatography for quantification of PCBs in tree barks and XAD-2 passive samplers using GC-ECD with dual columns.[show abstract] [hide abstract]
ABSTRACT: An analytical method for the quantification of Polychlorinated biphenyls (PCBs) concentrations in XAD-2 passive air samplers (PAS) and tree barks collected close to the Rhine River between France and Germany was developed. This method used Accelerated Solvent Extraction (ASE) followed by a purification step by flash chromatography using a 4gr cartridge (3g of silica gel and 1g of 44% acidified silica) and analysis by GC-ECD with dual columns. Quantification (QL) and detection (DL) limits varied 0.5 from and 5.7ngPAS(-1) and from 0.5 and 3.0ngPAS(-1) respectively. For tree barks, quantification and detection limits were calculated for each congener on washed tree bark sample with a signal to noise ratio of 3:1 and 10:1 (corresponding to a LQ in the range of 1-4ng per bark sample by congeners). Uncertainties on each congener concentration were calculated to be in the range of 3-20% XAD-2 passive samplers were field calibrated by using Hi-vol. Samplers. Sampling rates of 4.2, 11.5, 1.6, and 7.9m(3)PAS(-1)d(-1) for tri-, tetra-, penta- hexa-PCBs, respectively were obtained and are comparable to those already obtained with PUF-PAS for gas phase only (gas/particle distribution was 90/10). Method was applied to real atmospheric samples collected by XAD-2 passive samplers and tree barks in the east of France.Talanta 07/2013; 111C:140-6.
Article: Development of a new chromium reducing antioxidant capacity (CHROMAC) assay for plants and fruits.[show abstract] [hide abstract]
ABSTRACT: A chromium reducing antioxidant capacity (CHROMAC) assay was presented to measure antioxidant capacity of selected plants and fruits and compared its performance with other commonly used antioxidant capacity methods of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cupric reducing antioxidant capacity (CUPRAC). The assay is based on the spectrophotometric measurement of colored a chelate complex of Cr(III) and diphenylcarbazone formed by the reaction of Cr(VI) and 1,5-diphenylcarbazid in acidic medium. Phenolic compounds react with excessive amounts of Cr(VI) at low pH values, causing reduction of Cr(VI) to Cr(III) and conversion of phenols to oxidized products. The assay comprises of the antioxidant with a chromium(VI) solution, a 1,5-diphenylcarbazid in acidic medium and subsequent measurement of the developed absorbance at 540nm after 50min. The color development is stable for phenolic compounds in plant and fruit. The selectivity of the assay for phenolic compounds was improved by adjusting pH to 2.8 and reduction potential between 0.2 and 0.9V. The developed assay was successfully applied to the measurement of antioxidant capacity of three plants and one fruit (Prunus divaricata Ledeb.subsp. divaricata) samples and comparable results were obtained by ABTS and CUPRAC assays.Talanta 07/2013; 111C:119-24.
Article: Eletrochemical reduction of patulin and 5-hydroxymethylfurfural in both neutral and acid non-aqueous media. Their electroanalytical determination in apple juices.[show abstract] [hide abstract]
ABSTRACT: The electro-reduction of patulin mycotoxin and 5-hydroxymethylfurfural at glassy carbon electrodes in acetonitrile +0.1molL(-1) tetrabutylammonium perchlorate, in both the absence and the presence of different aliquots of trifluoroacetic acid is reported. 5-hydroxymethylfurfural is the most common interference in the determination of patulin in products derived from apples. The electrochemical techniques were cyclic and square wave voltammetries, and controlled potential bulk electrolysis. The number of electrons exchanged in the patulin electro-reduction of n=1 could be inferred from controlled potential bulk electrolysis measurements. Ultraviolet-visible and infrared spectroscopies were used to identify patulin electro-reduction product/s. A value of (2.1±0.1)×10(-5)cm(2)s(-1) for the patulin diffusion coefficient was calculated from convoluted cyclic voltammograms. A method based on square wave voltammetry was developed for the quantitative determination of patulin in both fresh, and commercial apple juices in the presence of 5-hydroxymethylfurfural. Calibration curves obtained from solutions of the commercial reagent, and commercial apple juices were linear in the range from 3.0×10(-7) to 2.2×10(-5)molL(-1). The lowest concentration measured experimentally for a signal to noise ratio of 3:1 was 3×10(-7)molL(-1) (45ppb) and a recovery percent of 84% was determined for commercial apple juices. This electroanalytical methodology appears as a good screening method for the determination of patulin in apple juices.Talanta 07/2013; 111C:85-92.
Article: Development of a reagentless electrochemiluminescent electrode for flow injection analysis using copolymerised luminol/aniline on nano-TiO2 functionalised indium-tin oxide glass.[show abstract] [hide abstract]
ABSTRACT: In this study, a nano-structured copolymer of luminol/aniline (PLA) was deposited onto nano-TiO2-functionalised indium tin oxide (ITO)-coated glass by electrochemical polymerisation using cyclic voltammetry (CV). The resulting reagentless electrochemiluminescent (ECL) electrode (ECLode) can be used for flow injection analysis (FIA). The properties of the ECLode were characterised by CV, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The ECLode has high background ECL emission as well as excellent stability and reproducibility, and yielding sensitive response towards target analytes. The ECL emissions of the ECLode were 50 times higher than PLA/ITO, and 500 times higher than polyluminol (PL)/ITO. The ECLode showed sensitive responses to reactive oxygen species (ROSs), permitting its application for determination of antioxidants by quenching. Under optimised conditions, an absolute detection limit of 69.9pg was obtained for resveratrol, comparable to the highest levels of sensitivity achieved by other methods. Thus, the gross antioxidant content of red wine was determined, with satisfactory recoveries between 87.6% and 108.3%. These results suggest a bright future for the use of the ECLode for single-channel FIA due to its high sensitivity, accuracy and reproducibility.Talanta 07/2013; 111C:156-62.
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