Javier Galbán

Direct determination of uric acid in serum by a fluorometric-enzymatic method based on uricase

The present paper describes a method for the fluorometric determination of uric acid in blood serum by its reaction with uricase (UOx). The procedure is based on the changes in fluorescence that take place during the enzymatic reaction of UOx with uric acid when the solution is excited at 287 nm and the emission is measured at 330 nm. A mathematical model which relates the analytical signal to the analyte concentration was developed and the model also served to obtain some of the thermodynamic constants of the system (the Michaelis constant and the turnover number). The optimum reaction conditions and its analytical characteristics were studied, linear response range (3x10(-5)-6x10(-4) M) and reproducibility (4%, n=7). The method was applied to the determination of uric acid in three blood serum samples. The results were compared with those obtained by a commercial clinical analyzer and no systematic errors were observed.

An optical glucose biosensor based on derived glucose oxidase immobilised onto a sol–gel matrix

Abstract Glucose oxidase, firstly covalently bonded to a fluorescein derivative, is immobilised onto a sol–gel matrix. During the enzymatic reaction, glucose reacts with the labelled enzyme and when the oxygen dissolved in the solution is consumed, an increase in the fluorescence intensity of the labelled enzyme is observed. The time at which the variation in the fluorescence intensity is observed is directly related to the glucose concentration, and it was therefore used as the analytical parameter for glucose quantification. The protocol of sol–gel formation is also optimised. The sol–gel presents a dynamic response range from 100 to 1000 mg/l with a lifetime of 15 days.

Sensor film for Vitamin C determination based on absorption properties of polyaniline

An analytical method based on the absorption changes of chemically polymerised polyaniline at 700nm is proposed for the determination of Vitamin C. Vitamin C produces a polyaniline film reduction, originating changes in its absorbance proportional to the Vitamin C concentration. The optimum reaction conditions and the analytical characteristics have been studied. The linear response of the method ranged from 0.10 to 1.0mgl(-1) for a 6min reaction time and from 1.0 to 8.0mgl(-1) for a 2min reaction time. Reproducibility, expressed as the coefficient of variation, was 0.8% (6min reaction time) and 2.3% (2min reaction time) (n = 10). The method has been applied to Vitamin C determination in pharmaceutical preparations and commercial fruit juices. The results were compared with those obtained by the 2,6-dichlorophenolindophenol titration method (the AOAC Official Method) and no systematic errors were observed.

Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration

The chemical derivatization of bilirubin oxidase (BOx) with a fluorescein derivative (FS) yields a chemically modified enzyme (BOx-FS), with excitation and emission maxima at 487 and 520 nm, respectively. During the oxygen oxidation reaction of bilirubins, in the presence of the modified enzyme, the change in the fluorescence of the modified enzyme depends on the concentration and type of bilirubin. This effect can be used for analytical purposes. Firstly, a theoretical-experimental study of the analytical system was carried out. The mechanism responsible for the fluorescence variation was clarified, a mathematical model developed and the variables affecting the fluorescence changes optimized. The concentration ranges in which the model can be applied (up to 12 mg bilirubin l(-1)), and the precision of the measurement (about 4%) were established for the three bilirubins. The application of the methodology to the simultaneous determination of direct and total bilirubins were studied by applying multivariate calibration methods to the whole kinetic profiles. A reduced calibration matrix (derived from a 5(3) base matrix) is proposed for calibration and different numerical methods were tested: Principal Components Regression (PCR), Partial Least Squares Regression (PLS) and Artificial Neural Networks (ANN). The simultaneous determination of direct and total bilirubin (average validation errors of about 9 and 10%, respectively) can be carried out from a single run. Furthermore, a semi-quantitative speciation of the three bilirubins (free, conjugated and albumin-bonded bilirubin) may be simultaneously obtained.

Polycyclic aromatic hydrocarbon emissions from fluidized bed combustion of coal

The most volatile polycyclic aromatic hydrocarbons remaining in the flue gas leaving cyclones placed at the exit of a fluidized bed coal combustor and classified by the US EPA as priority pollutants were trapped on filters and adsorbents, dissolved in dimethylformamide and analysed by synchronous fluorescence spectroscopy. The results are discussed.

Fluorometric sensors based on chemically modified enzymes Glucose determination in drinks.

In this paper an enzymatic fluorometric sensor for glucose determination in drinks is presented. The sensor film was obtained by immobilisation of glucose oxidase chemically modified with a fluorescein derivative (GOx-FS) in a polyacrylamide polymer. During the enzymatic reaction the changes in the fluorescence intensity of the GOx-FS are related to the glucose concentration. Working in FIA mode, the optimum conditions found were: 0.7 ml min(-1) flow rate, 300 mul sample injection and pH 6.5. The sensor responds to glucose concentrations ranging from 400 to 2000 mg l(-1), the reproducibility is around 3% and the life-time is at least 3 months (more than 350 measurements). The sensor was applied to direct glucose determination in drinks with good accuracy; interference caused by the filter effect was avoided by the kinetics of the reaction.

Extraction-atomic-absorption spectrophotometric determination of lead by hydride generation in non-aqueous media

A method for lead hydride generation in a non-aqueous extraction phase is proposed. The lead hydride generation is carried out in an aliquot of lead pyrrolidine-1-carbodithioate extract in chloroform, by the addition of sodium tetrahydroborate(III) solution in dimethylformamide. Lead is determined by atomic-absorption spectrophotometry at 217.0 nm. The proposed method gives improved sensitivity and eliminates interferences, which is necessary because lead hydride generation in aqueous solution occurs in the presence of oxidants such as potassium dichromate or ammonium peroxydisulphate. The method was applied to the determination of lead in BCS standard steels and air particulates. Good accuracy and precision were obtained.

Spectrally matched upconverting luminescent nanoparticles for monitoring enzymatic reactions.

We report on upconverting luminescent nanoparticles (UCLNPs) that are spectrally tuned such that their emission matches the absorption bands of the two most important species associated with enzymatic redox reactions. The core-shell UCLNPs consist of a β-NaYF4 core doped with Yb(3+)/Tm(3+) ions and a shell of pure β-NaYF4. Upon 980 nm excitation, they display emission bands peaking at 360 and 475 nm, which is a perfect match to the absorption bands of the enzyme cosubstrate NADH and the coenzyme FAD, respectively. By exploiting these spectral overlaps, we have designed fluorescent detection schemes for NADH and FAD that are based on the modulation of the emission intensities of UCLNPs by FAD and NADH via an inner filter effect.

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.

Simultaneous determination of inorganic arsenic(III) and arsenic(V) by arsine generation and gas-phase molecular absorption spectrometry

Abstract A method of simultaneous determination of inorganic As(III) and As(V) is described, which involves arsine generation, measurement of the molecular absorption of arsine in the gas phase at 190 nm and quantification of As(III) and As(V) based on different heights and areas of absorbance versus time profiles of arsine generated from both inorganic species. The limits of detection were 0.5 μg of As(III) and 3 μg of As(V) in a sample volume of 1 ml. An interference study for both species was made and the method was applied to the determination of As2O3 and As2O5 in a standard glass sample.