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ABSTRACT: An enzymatic fluorimetric method for the determination of polyphenol compounds in beverages is described, which is based on the temporal inhibition caused by these compounds on the oxidation of the long wavelength fluorophor indocyanine green (λ(ex) 764 nm, λ(em) 806 nm), in the presence of the enzyme laccase and positively charged gold nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its fluorescence, but it is delayed by the polyphenol, obtaining a time period directly proportional to its concentration, which has been used as the analytical parameter. The behaviour of several benzenediols and benzenetriols in the system and the modification of the activity of the enzyme by its interaction with AuNPs have been studied. The system has been optimized using gallic acid as a polyphenol model, but the dynamic ranges of the calibration graphs and the detection limits for several of the polyphenols assayed were obtained (μmol L(-1)): gallic acid (0.13-5, 0.04), catechol (0.08-5, 0.01), hydroquinone (0.05-2, 0.01), hydroxyhydroquinone (0.09-5, 0.03), pyrogallol (0.17-5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of the method, expressed as RSD% and checked at two concentration levels of each analyte, ranged between 1.8 and 5.6%. The method has been applied to the determination of polyphenol content in several foodstuff samples and the results compared with those obtained with the standard Folin-Ciocalteu method.
Analytica chimica acta 02/2012; 713:1-6. · 4.31 Impact Factor
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ABSTRACT: The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidants used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5s, using an integration time of 0.1s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were (μmolL⁻¹): gallic acid (0.04-0.59, 0.01), propyl gallate (0.04-1.41, 0.01), octyl gallate (0.03-0.35, 0.08), dodecyl gallate (0.02-0.30, 0.007), butylated hydroxyanisol (0.07-0.39, 0.009), butylated hydroxytoluene (0.04-0.32, 0.01), ascorbic acid (0.11-1.72, 0.03) and sodium citrate (0.07-1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%. The results obtained were validated using two reference methods.
Analytica chimica acta 06/2011; 695(1-2):11-7. · 4.31 Impact Factor
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ABSTRACT: An overview of the usefulness of different nanoparticles to improve the features of high throughput separation and individual and multiplexed detection bioassays is presented. Although the development of microarray and microfluidic systems has expanded the capabilities of these high throughput assays, the combined use of NPs and these devices has provided them with new applications in drug discovery, proteomic and genomic studies, and clinical diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor and other nanoparticles in high throughput bioassays. Also, the versatility of the detection systems described shows that NPs are useful alternatives to fluorescent dyes, which are often used in these assays.
Combinatorial chemistry & high throughput screening 01/2010; 13(4):309-17. · 2.46 Impact Factor
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ABSTRACT: The relevance of tumor markers in clinical diagnosis of cancer has given rise to the development of new approaches based on the use of nanoparticles to improve the features of the immunoassays developed for their control. This article reviews the usefulness of different nanoparticles to develop direct, sandwich and competitive assays for the individual and multiplexed determination of these compounds.
Mini Reviews in Medicinal Chemistry 09/2009; 9(9):1064-74. · 2.53 Impact Factor
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ABSTRACT: A method for the evaluation of liposome size populations using sucrose density gradient centrifugation coupled with a continuous flow system is presented. Liposomes, prepared using different methods (rapid solvent evaporation, rehydration, and detergent removal) and modified by assaying several procedures (shaking, sonication and extrusion) were evaluated according to the type of liposome, size and polydispersity. The preparation of liposomes was carried out in the presence of the fluorophor cresyl violet. Extracts of the liposomes were homogenised and centrifuged at 20,073 x g at 4 degrees C for 30 min using sucrose density gradient centrifugation programmes, which provide efficient liposome separation in different sizes. The results of the separation procedure were tested by aspiration of the extracts into a continuous flow system in which the liposomes were disrupted by the continuous mixing with a Triton X-100 solution, prior to their translation to the detector. The luminescence provided by the liberation of the encapsulated fluorophor indicates the distribution of liposomes in each density gradient stage. Three zones were obtained: zone alpha, containing giant unilamellar and multivesicular vesicles, zone beta, with large and medium size liposomes, and zone gamma, which contained small size liposomes. The precision of the separation zones obtained, expressed as RSD%, was lower than 5.6% in all instances. The method provides a relative rapid way to evaluate the liposome polydispersity and size after using conventional methods of synthesis and mechanical modifications.
Analytica chimica acta 08/2009; 645(1-2):79-85. · 4.31 Impact Factor
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ABSTRACT: A simple and rapid method for the determination of DNA, involving the interaction between a surfactant, a long-wavelength fluorophor (LWF) and the nucleic acid, is presented. Different chemical systems based on the local effective charge of the surfactant/LWF system with DNA were tested, choosing cetyltrimethyl ammonium bromide (CTAB) and indocyanine green (ICG) for the development of the method. The fluorescence of ICG increases in the presence of CTAB, but it rapidly decreases in the presence of deoxyribonucleic acid. The initial reaction-rate (v(0)) and signal at a prefixed-time (DeltaIF(20)) are monitored at 780 and 802 nm as excitation and emission wavelengths, respectively, using stopped-flow mixing technique, which makes the method applicable to automate routine analysis. Each measurement was obtained in about 30 s, being the integration time 0.1 s. The dynamic range of the calibration graph was 10-1500 ng mL(-1), with a detection limit of 5 ng mL(-1). The precision of the method, expressed as relative standard deviation, ranged between 2.1% and 4.5%. After a sample treatment consisting on a conventional extraction, the method was applied to the determination of DNA in several samples from different biological materials.
Analytica chimica acta 02/2009; 632(1):109-14. · 4.31 Impact Factor
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ABSTRACT: The potential usefulness of terbium(III) as reagent for the luminescent determination of flumequine residues in food samples has been studied using both fluorescence (FL) and time-resolved (TR) modes and both batch (B) and integrated liquid chromatography (LC)/derivatisation approaches. The system was optimised in each instance to establish the analytical features of the four methods. The dynamic ranges of the calibration graphs, obtained with standard solutions of flumequine, were (ng mL(-1)): B-FL 0.18-600; B-TR 2.4-150; LC-FL 3.7-1000 and LC-TR 52-3000. The detection limits were also obtained giving the following values (ng mL(-1)): B-FL 0.055; B-TR 0.7; LC-FL 1.1 and LC-TR 15. The precision, expressed as the percentage of relative standard deviation, was equal or lower than 5.1% in all instances. The LC methods, which avoid the interference of other quinolone antibiotics, were applied to the analysis of chicken muscle and liver, and whole milk samples. The sample pre-treatment only consisted of a deproteinisation step. The validation procedure for the analysis of samples was carried out using EC recommendations, and the decision limit and detection capability were calculated. The recoveries obtained ranged from 95.0% to 103.8%.
Analytica chimica acta 10/2006; 578(2):220-6. · 4.31 Impact Factor
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ABSTRACT: A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.
Analytical and Bioanalytical Chemistry 12/2003; 377(6):1071-8. · 3.78 Impact Factor
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ABSTRACT: A method for the determination of monoclonal antibody anti-canine-IgG based on a continuous filtration/dissolution system is presented as prototype for further developments. The basis of the system is the continuous formation of a high-molecular immunocomplex, which is temporally retained on a microfilter located prior to the detector. The immunochemical method consists of the development of a sandwich type heterogeneous non-competitive reaction to yield a high molecular immunocomplex, as a result of the affinity interaction between streptavidin and biotincanine IgG and the immunoreaction between canine IgG and mAb anti-canine IgG, which occurs in solution. Goat anti-mouse IgG labelled with peroxidase is used as tracer. The extension of the immunoreaction is monitored fluorimetrically via the condensation product between 4-hydroxyphenylacetic acid and hydrogen peroxide in the presence of the peroxidase retained on the filter. The method provides a dynamic range from 10(-4) to 500 mug l(-1) with an IC(50) of 0.554 mug 1(-1) (for a biotin-IgG dilution of 1:250, chi(2)=0.6085, r(2)=0.9991, n=14) and a precision, expressed as R.S.D.%, lower than 4.7%. After modifications, the method here proposed can be extended for monitoring analytes of interest in the agrochemical, food and environmental areas, as far as permitted by the availability to produce the corresponding monoclonal antibody.
Talanta 11/2001; 55(4):821-9. · 3.79 Impact Factor
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ABSTRACT: The application of laser-induced breakdown spectrometry (LIBS) and chemometrics [namely principal component analysis (PCA) and cluster analysis (CA)] is presented for the characterization of screen-printed electrodes with in-depth resolution. An Nd : YAG laser operating at the fundamental wavelength of 1064 nm was focused on the sample and the plasma emission was collected by a fibre-optic bundle coupled to a spectrograph–charge-coupled device system. The experimental variables were optimized for a satisfactory spatial characterization. The different zones of the screen-printed electrode were studied (working electrode, reference electrode and electrical contacts) in order to identify both the composition and distribution of the layers deposited on the inert support for the construction of the electronic device. Carbon, gold, silver, platinum, palladium, titanium and aluminium were identified as major components. The use of pattern recognition techniques (PCA and CA) for the statistical treatment of the spectroscopy data obtained by LIBS proved to be a powerful tool for the rapid analysis of miniaturized multilayer electronic devices. Copyright © 2001 John Wiley & Sons, Ltd.
Surface and Interface Analysis 04/2001; 31(4):313 - 320. · 1.18 Impact Factor
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ABSTRACT: A flow injection fluorimetric method is proposed for the determination of chloroquine based on the photochemical derivatisation in an alkaline medium of the analyte and fluorescence generation after irradiation with a pulsed Nd:YAG laser operated at 355 nm. Chemical, hydrodynamic and laser variables were studied in order to obtain the best conditions for quantification. A linear range from 25 to 600 micrograms/L was achieved, with a correlation coefficient of 0.997 (n = 8), an RSD of 4.3% (n = 11) and a detection limit of 8 micrograms/L (3 sigma). The sample throughput was 10 h-1. The method was successfully applied to the determination of chloroquine in human plasma. The increase of sensitivity with respect to the method based on monitoring the intrinsic fluorescence of chloroquine itself was 1.7 times.
Fresenius Journal of Analytical Chemistry 04/2001; 369(5):438-41.
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ABSTRACT: An innovative continuous flow approach consisting of the triple integration of chemical hydrolysis, analytical pervaporation and enzyme inhibition-based reaction for the determination of metrifonate is presented. The method is based on chemical degradation of the pesticide, a separation step consisting of analytical pervaporation of its volatile metabolite and inhibition action of this on the acetylcholinesterase catalysis. The subsequent derivatisation reaction is a two-step reaction involving choline oxidase (ChOD) and horseradish peroxidase (POD) with fluorimetric detection (lambda (ex )=310 nm and lambda (em )=415 nm ) of the dimer formed by the action of hydrogen peroxide. The efficiency of the inhibitory effect was increased using an open-closed flow system. Applied to liquid samples, the method has a linear determination range of 0.0025-0.15 g l(-1)(n=8, r=0.9993) with a precision, expressed as RSD, of 3.2-6.7% and a sampling frequency of 3 h(-1). When applied to solid samples the method shows a linear determination range of 0.0026-0.13 g kg(-1) (n=5, r(2)=0.9981, RSD 2.7-7.7%) and a sampling frequency of 2h(-1). The approach has been applied to the determination of metrifonate in natural water and spiked soil samples with recoveries ranging between 94.3 and 107.8% for liquid samples and between 86.5 and 99.6% for solid samples.
Talanta 02/2001; 53(5):961-70. · 3.79 Impact Factor
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ABSTRACT: A commercially available supercritical fluid extractor provided with carbon dioxide was coupled to a dual-beam thermal lens spectrometer with a pumpprobe coaxial configuration, pumped by a pulsed Nd-YAG laser operating at the fundamental wavelength of 1064 nm. As a preliminary step, several compounds were studied in batch regime using carbon tetrachloride as solvent, in order to observe the influence of overtones and combinations involving distinct chemical bonds on thermal lens spectrometry (TLS). Several factors related with supercritical fluid extraction (SFE) under hydrodynamic conditions were studied in order to establish their influence both in the extraction yield and thermal lens signal magnitude obtained. The advantages and limitations of the hyphenated SFE-TLS technique proposed are discussed, and the possibility of on-line detection in SFE with a pulse thermal lens spectrometer was demonstrated.
Talanta 08/1999; 49(4):813-23. · 3.79 Impact Factor
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ABSTRACT: A new method for the determination of the hydroxymetabolites of vitamin D(3) (24,25-(OH)(2)-D(3), 1,25-(OH)(2)-D(3) and 25-OH-D(3)) in plasma is reported. The method is based on the integration of three subsystems: continuous cleanup/preconcentration, HPL separation and post-column fluorimetric derivatisation. The derivatising subsystem is based on the dehydration reaction undergone by the secosteriod molecules in a strong-acid medium. The calibration graphs were run between 0.1 pg ml(-1) and 100 ng ml(-1) for each analyte with excellent regression coefficients (>/=0.9933) in all cases. The precision at two concentration levels was established with acceptable RSDs (%) in all instances (values between 2.1 and 5.2%). The method was also checked by applying it to human plasma samples spiked with the target analytes and the recoveries ranged between 86 and 106%.
Talanta 08/1999; 50(1):57-66. · 3.79 Impact Factor
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ABSTRACT: The steps involved in the methods for the determination of vitamin D3 metabolites (namely, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3) mainly in clinical samples are critically reviewed. Sample pretreatment (e.g. deproteinization, saponification, liquid liquid and liquid solid extraction, etc.) as a function of both type of sample and detection system, quantitation based on protein saturation and liquid as well as gas chromatography are discussed. The chemical principles on which the methods are based and the derivatization procedures, which facilitate separation and/or detection, are also commented upon. Finally, the future prospects of the research on methods for the determination of these metabolites are outlined.
Journal of Pharmaceutical and Biomedical Analysis 07/1999; 20(1-2):1-17. · 2.97 Impact Factor
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ABSTRACT: A method for the determination of mercury in solid samples using laser ablation coupled with atomic fluorescence spectroscopy
has been developed. An Nd-YAG laser was used for ablation and the vaporised and atomised material was rapidly led to an atomic
fluorescence detector, where excitation and emission took place. The experimental approach was applied to the assessment of
different procedures as sensitive as possible for implementing standard addition methods. Calibration curves were recorded
using the prepared standards, which exhibited linear ranges between 0.5–100 μg/g, with excellent regression coefficients in
all instances (0.9907). The precision, expressed as RSD %, was 3 and 4% for contents of 1 and 30 μg/g, respectively, in the
same pellet; and 7 and 12% for the same contents and different type of pellets. The method has been applied to the determination
of mercury in CRM of sewage sediment and a sludge sample with a known amount of mercury determined by an interlaboratory study.
The results obtained show good agreement with those expected.
Fresenius Journal of Analytical Chemistry 04/1999; 365(4):320-324.
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ABSTRACT: A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.
The Analyst 03/1999; 124(3):401-6. · 4.23 Impact Factor
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ABSTRACT: A sensitive method for the determination of hydroxymetabolites of vitamin D3, parficularly calcitriol (1,25-(OH)2-D3), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to
conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system.
The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature
in a strong-acid medium. A LOD of 0.01 pg mL−1 (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients
(≥0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n=5) were acceptable
(between 2.6 and 4.7%). The method was also checked by applying it to human plasma spiked with the target analytes and excellent
recoveries were obtained. This is the first time that these species have been determined at the sub-pg mL−1 level.
Chromatographia 01/1999; 50(7):399-406. · 1.20 Impact Factor
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ABSTRACT: An affinity flow-through sensor system based on a heterogeneous competitive affinity assay for the determination of low molecular weight compounds is described using the examples of biotin and atrazine determination. The binding proteins, either streptavidin or a biotinylated monoclonal antibody, were immobilized on a biotinylated screen-printed electrode, where the competition between the analyte and an analyte-enzyme-conjugate took place. Determination of the bound enzyme was done through the supply of suitable enzyme substrates and electrochemical determination of an enzyme reaction product. In the assays described here, peroxidase was used as enzyme label. As hydrogen peroxide and hydroquinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrically at a reducing potential of -600 mV vs a screen-printed platinum electrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserted. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of required reagents. The system was optimized mainly through the determination of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. The method showed a linear range from 0.045 to 2 micrograms/l (r2 = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by using a biotinylated monoclonal antibody against atrazine for analyte recognition and performing a competitive assay between atrazine and a triazine-peroxidase-conjugate. The linear range was from 0.01 to 10 micrograms/l, with IC50 = 0.4 microgram/l and RSD lower than 4.6%. The method was also applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays.
Biosensors and Bioelectronics 12/1998; 13(10):1107-15. · 5.60 Impact Factor
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ABSTRACT: Approaches based on continuous separation units coupled to either liquid or gas chromatography for improving the features of analytical methods are proposed. Examples of solid-phase separation-liquid chromatography for the determination of fat-soluble vitamins and their metabolites in clinical samples, and pervaporation-gas chromatography for the determination of volatile compounds in solid environmental samples are described. The clean-up and preconcentration effect achieved by the former coupling and the easy and effective solid-sample pretreatment in the latter clearly show their utility. The use of pervaporation as an advantageous alternative to headspace is demonstrated.
Journal of Chromatography 10/1998; 819(1-2):25-33. · 4.53 Impact Factor
