Colorimetric enzymatic rapid test for the determination of atropine in baby food using a smartphone.

A method for the enzymatic determination of atropine has been developed, which is based on a sequence of reactions involving (1) the hydrolysis of atropine to give tropine; (2) the enzymatic oxidation of tropine with NAD (catalysed by tropinone reductase); and (3) an indicator reaction, in which the NADH previously formed reduces the dye iodonitrotetrazolium chloride (INT) to a reddish species, the reaction catalysed by diaphorase. The method was first developed in solution (linear response range from 2.4 × 10-6 M to 1.0 × 10-4 M). It was then implemented in cellulose platforms to develop a rapid test where the determination is made by measuring the RGB coordinates of the platforms using a smartphone-based device. The device is based on the integrating sphere concept and contains a light source to avoid external illumination effects. The smartphone is controlled by an app that allows a calibration line to be generated and the atropine concentration to be quantified; moreover, since the app normalizes the CCD response of the smartphone, the results and calibrations obtained with different smartphones are similar and can be shared. Using the G coordinate, the results were shown to have a linear response with the concentration of atropine ranging from 1.2 × 10-5 M to 3.0 × 10-4 M with an RSD of 1.4% (n = 5). The method has been applied to the determination of atropine in baby food and buckwheat samples with good results.

Autoindicating optical properties of laccase as the base of an optical biosensor film for phenol determination.

In the context of sustainable analytical chemistry, phenol has been determined through its enzymatic reaction with laccase. The method has been studied and optimized through the autoindicating optical properties of laccase both by intrinsic molecular absorption and fluorescence. The method shows a linear range from 9.79·10(-6) to 7.50·10(-4) M with a relative standard deviation of 1.07 %. The molecular absorption methodology has been implemented in a polyacrylamide film for the design of an autoindicating optical sensor. In order to increase the lifetime of the sensor, the reversibility study of the enzymatic reaction has proposed, as a novelty, the regeneration of laccase with an oxidase-type enzyme (glucose oxidase). The lifetime of the sensor film has improved from 15 to 30 measurements. The reaction mechanism has also been studied and confirmed by fluorescence and molecular absorption. The method leads to the determination of phenol in environmental samples.

Simultaneous determination of glucose and choline based on the intrinsic fluorescence of the enzymes.

It has been possible to perform the simultaneous determination of choline and glucose using the intrinsic fluorescence of the corresponding enzyme as an analytical signal. This can be done in two ways. First, for low glucose and choline concentrations (about 0.55 mM and 0.75 microM respectively) two differentiated signals, without mutual interference, are obtained for both analytes in the same measurement. Second, when glucose and choline concentrations are higher, a new model has been designed which permits the concentrations to be accurately determined in samples containing from 0.55 mM to 3.75 mM glucose and from 0.75 microM to 11.0 microM choline; the method has been applied to simultaneous glucose and choline determinations in serum samples with good results. This method gives a better performance than multivariate calibration based on Partial Least Squares Regression. The methodology here shown could be also used for the simultaneous determination of other pairs of analytes.

Fluorescence of the Flavin group in choline oxidase. Insights and analytical applications for the determination of choline and betaine aldehyde.

Choline oxidase (ChOx) is a flavoenzyme catalysing the oxidation of choline (Ch) to betaine aldehyde (BA) and glycine betaine (GB). In this paper a fundamental study of the intrinsic fluorescence properties of ChOx due to Flavin Adenine Dinucleotide (FAD) is presented and some analytical applications are studied in detail. Firstly, an unusual alteration in the excitation spectra, in comparison with the absorption spectra, has been observed as a function of the pH. This is ascribed to a change of polarity in the excited state. Secondly, the evolution of the fluorescence spectra during the reaction seems to indicate that the reaction takes place in two consecutive, but partially overlapped, steps and each of them follows a different mechanism. Thirdly, the chemical system can be used to determine the Ch concentration in the range from 5×10(-6)M to 5×10(-5)M (univariate and multivariate calibration) in the presence of BA as interference, and the joint Ch+BA concentration in the range 5×10(-6)-5×10(-4)M (multivariate calibration) with mean errors under 10%; a semiquantitative determination of the BA concentration can be deduced by difference. Finally, Ch has been successfully determined in an infant milk sample.

A modified commercial gas chromatograph for the continuous monitoring of the thermal degradation of sunflower oil and off-line solid phase extraction gas-chromatography-mass spectrometry characterization of released volatiles.

A homemade flow cell attached to a commercial Gas Chromatograph equipped with a Flame Ionization Detector (FID) has been designed for the continuous monitoring of volatile compounds released during heating edible oils. Analytical parameters such as mass of sample, temperature and flow rates have been optimized and the obtained results have been compared with the corresponding thermographs from standard TG systems. Results show that under optimum conditions, the profiles of volatiles released upon heating are comparable to the profiles of TG curves, suggesting that the FID based system could be an alternative to TGA. Additionally, volatiles have been retained in a Lichrolut EN(®) resin, eluted and analyzed by Gas Chromatography-Mass Spectrometry. In this case, forty five compounds have been identified (acids, alcohols, alkanes, aldehydes, ketones and furans) and compared with the FID signals, working both in air or nitrogen atmosphere. It has been concluded that the oxidative thermal degradation is prevented in the presence of a nitrogen atmosphere.

Gas generation and preconcentration coupled to gas phase molecular absorption spectrometry: a powerful tool for the simultaneous determination of analytes forming volatile compounds.

Different methods for a simultaneous determination of several analytes forming volatile compounds at room temperature are described. The main steps of these methods are: continuous generation, collection in a cryogenic trap, revolatilization, measurement of the volatile compounds by Gas Phase Molecular Absorption Spectrometry and resolution by multi-wavelength methods. Several mixtures containing 2, 3 or 4 components have been studied: 1) elements forming covalent hydrides; 2) arsenic organometallic compounds forming volatile gases with a similar structure to arsine; and 3) sulphur species that can evolve volatile compounds. Under the optimum conditions obtained for each mixture, detection limits range from 0.8 ng/mL (dimethylarsinic acid) to 2 microg/mL (SCN(-)).

Simultaneous determination of arsenic, antimony, selenium and tin by gas phase molecular absorption spectrometry after two step hydride generation and preconcentration in a cold trap system.

A cold trap system for the simultaneous determination of arsenic, antimony, selenium and tin by continuous hydride generation and gas phase molecular absorption spectrometry is described. The hydride generation is carried out in two steps; first, tin hydride is generated at low acidity and second, arsenic, antimony and selenium hydrides are formed at higher acidity. All the hydrides are collected in a liquid nitrogen cryogenic trap and transported to the flow cell of a diode array spectrophotometer, where molecular absorption spectra are obtained in the 190-250 nm range. Five calibration solutions containing arsenic, antimony, selenium and tin are solved using multiple linear regression analysis. Tests are performed in order to extend the same manifold to other hydrides but no signals are obtained for bismuth, cadmium, lead, tellurium and germanium. Under the optimum conditions found and using the wavelengths of maximum sensitivity (190, 198, 220 and 194 nm), the analytical characteristics of each element are calculated. The detection limits are 0.050, 0.020, 0.12 and 1.1 mug ml(-1) and the RSD values are 3.7, 3.1, 3.5 and 3.0% for As, Sb, Se and Sn, respectively. The method is applied to As, Sb, Se and Sn determination in natural spiked water samples.

Fluorometric-enzymatic lactate determination based on enzyme cytochrome b2 fluorescence.

This paper presents a procedure for fluorometric-enzymatic lactate determination based on the modification of the fluorometric properties of the enzyme L-lactic dehydrogenase (cytochrome b2). during the enzymatic oxidation of the analyte with ferricyanide. During the reaction one can observe an irreversible fall in the intensity of the enzyme's fluorescence, the rate of which is proportional to the concentration of the lactate. The source of this signal has been investigated and it has been shown that, besides the formation of a complex between the enzyme and the ferricyanide (the constant of which can be determined), this signal loss can be explained by simultaneous inner filter effects caused by the ferricyanide and the ferrocyanide (generated in the enzymatic reaction). A mathematical model has been developed which makes it possible to establish a linear response between the enzyme's analytical signal of fluorescence and the concentrations of the lactate, the cytochrome, and the ferricyanide. The procedure makes it possible to determine the lactate in concentrations ranging from 0.2 to 45 mg/L. Determination of the analyte has been carried out in milk samples with great precision and accuracy.

Determination of direct-bilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase.

A method for the determination of direct bilirubin by reaction with bilirubin oxidase (BOx) is reported. The procedure is based on the changes in fluorescence which take place during the enzymatic reaction of BOx with any of the three forms of bilirubin (free, conjugated and with albumin) when the solution is excited at 240 nm and the emission is measured at 440 nm. The change in fluorescence was studied thoroughly. It seems mainly due to the fluorescence of one of the reaction products. A theoretical study was carried out to relate the changes in fluorescence observed to the species taking part in the reaction and to establish some of the enzymatic reaction constants. The optimum reaction conditions were studied for each of the three types of bilirubin together with their analytical characteristics (linear range and precision). Selective determination of direct bilirubin was carried out for various synthetic samples with good results. A linear response up to 7 mg L(-1) of direct bilirubin was obtained. Using optimum conditions, the precision for free and conjugated bilirubin was 3.4% (n = 5) and 3.0% (n = 5), respectively.

Intrinsic molecular fluorescence of lactate dehydrogenase: an analytical alternative for enzymic determination of pyruvate.

A method for the enzymic determination of pyruvate based on changes in the fluorescence intensity of lactate dehydrogenase (LDH) is described. These changes are due to the differential quenching effect produced by NAD and NADH on the LDH fluorescence. The NADH quenching is due to both an inner filter effect and LDH-NADH complex formation; the LDH-NADH complex is also fluorescent. However, the NAD quenching is based only on the inner filter effect. From these suppositions, the equilibrium constant of the reaction and the formation constant of the LDH-NADH complex were obtained. Given this, an appropriate analytical signal for the quantification of pyruvate and a mathematical model explaining the effect of each parameter are proposed. The linear response range of the method depends on the NADH concentration used during the determination; it is possible to determine pyruvate concentrations down to 8.8 x 10(-7) mol dm-3. The method was applied to the determination of pyruvate in synthetic blood samples with good accuracy.

Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review.

In recent years our research group has developed new alternatives for fluorescence enzymatic determinations. First, we observed that the intrinsic fluorescence of enzymes changes during enzymatic reactions, proportionally to the substrate concentration, avoiding the combination of the enzymatic reaction with a fluorophore-involving reaction. The main disadvantage of this method is that the excitation and emission wavelengths of the enzymes are in the UV region of the spectrum. An alternative to overcome this problem consisted of covalently bonding the enzyme to a fluorophore. In this paper, an overview is given of all of the applications and future developments on both types of alternatives that we have developed. Apart from the analytical characteristics of the methods, we have also reviewed all of the information about mathematical models we have elaborated to date.

Removal of naphthalene, phenanthrene, and pyrene by sorbents from hot gas.

It is the first time that the removal of polycyclic aromatic hydrocarbons (PAH) containing different aromatic rings number [naphthalene (Np), phenanthrene (Phe), and pyrene (Py)] from combustion hot gas has been carried out. The aim was to relate the sorbents textural characteristics with the adsorption capacity of these 2-4-ring PAH at the conditions emitted at energy generation. The sorbents textural parameters [total micropore volume (VN2), narrow micropore volume (VCO2), mesopore volume (VBJH), and the free active sites] were analyzed trying to correlate them with their Np, Phe, and Py adsorption capacities. To get this aim, single and multiple linear regressions (MLR) were applied to the three PAH. A principal component analysis was performed to generate new and uncorrelated variables. It enabled us to show that the relations between the textural parameters were analyzed using a principal components regression (PCR). The PCR analysis had a good statistical quality, but neither did it allow differentiating free active site types nor did VN2 and VCO2. The correlations were thus set up applying a MLR to the original variables. The regression statistical quality was similar to the PCR analysis, and it could give an easier explanation of the parameters that affected the adsorption. In Np adsorption, the 87% data variance was explained, and the adsorption was positively correlated to VCO2 and the micropore mean diameter (I.). In the Phe regression there was 98% variance explained, and its adsorption was positively correlated to the VN2 and the micropore distribution, n. Finally, in the Py adsorption, the 96% data variance was explained, and this adsorption was positively correlated to VN2 and VBJH. These dependencies were according to the molecular parameters of these compounds (molecular diameter and volatility) because the higher the number of aromatic rings of the PAH, the more favored the adsorbate-adsorbate interactions. Besides, the higher the mean diameter micropores, the lower the diffusional problems showed by Np, Phe, and Py.

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.