Susana de Marcos

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.

Optical sensing of pH based on polypyrrole films

An optical sensor for pH is presented which is based on the use of a thin polypyrrole (PPy) film obtained by chemical oxidation of pyrrole and deposited as a < 1 μm film on the walls of a polystyrene cuvette. The spectrum of PPy displays strong absorption in the near infrared at around 900 nm, and a characteristic minimum at around 550 nm. The shape and intensity of the spectrum are pH-dependent between pH 6 and 12, the minimum being shifted to shorter wavelengths at higher pH, and the absorbance of the longwave band increasing in the range 600–900 nm range. The apparent pKa is around 8.6, but once exposed to a pH 6, the films need to be reconditioned with 0.1 M HCl in order to give the same response to pH. The films are an interesting alternative to indicator-based pH sensor films in that they do not require a dye to be immobilized, are fully compatible with LED and diode laser light sources, and can easily be prepared.

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.

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.

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.

Characterization of a urea optical sensor based on polypyrrole

A new optical sensor for urea determination is presented. It is based on the enzymatic reaction with urease, which is first photoimmobilized with polyacrylamide onto a chemically polymerized polypyrrole (PPy) film. The main advantage of this sensor is that no indicator dye or pH indicator is needed, because PPy itself acts as the support and the indicator. These PPy films show an absorbance spectrum in the near IR range which is pH dependent. The variation of absorbance is thus directly related to the change of pH caused during the enzymatic reaction, which is also dependent on the urea concentration. The linear range of the sensor is from 0.06 to 1 M of urea, which is the common level of urea concentration found in blood and urine samples.

Enzyme-induced modulation of the emission of upconverting nanoparticles: Towards a new sensing scheme for glucose

A new approach for the design of a fluorometric biosensor for continuous monitoring of glucose levels in biological samples based on near-infrared (NIR) excitation is described. The sensor combines the fluorescence of the enzyme glucose oxidase (GOx) chemically modified with a fluorescein derivative (FS) and the luminescent properties of upconverting luminescent nanoparticles (UCLNPs). Both, the chemically modified enzyme (GOx-FS) and the UCLNPs are immobilized in a poly(acrylamide) film as a physical support. The excitation of the UCLNPs with NIR light is of major advantage, since fluorescence background from the matrix is minimized. The upconverted luminescence is used to excite GOx-FS, which undergoes a change in the fluorescence intensity during the enzymatic reaction with glucose. The sensor comprises sufficient stability and covers the physiological range of glucose levels in blood. Furthermore, in a proof of principle experiment, the sensor system responds linearly to glucose concentrations in the range from 3.3 to 16.6 mM in flow injection analysis mode.

A blood-assisted optical biosensor for automatic glucose determination

A new approach for glucose determination in blood based on the spectroscopic properties of blood hemoglobin (Hb) is presented. The biosensor consists of a glucose oxidase (GOx) entrapped polyacrylamide (PAA) film placed in a flow cell. Blood is simply diluted with bidistilled water (150:1, v:v) and injected into the carrier solution. When reaching the PAA film, the blood glucose reacts with the GOx and the resulting H(2)O(2) reacts with the blood Hb. This produces an absorbance change in this compound. The GOx-PAA film can be used at least 100 times. Lateral reactions of H(2)O(2) with other blood constituents are easily blocked (by azide addition). The linear response range can be fitted between 20 and 1200 mg dL(-1) glucose (R.S.D. 4%, 77 mg dL(-1)). In addition to the use of untreated blood, two important analytical aspects of the method are: (1) the analyte concentration can be obtained by an absolute calibration method; and (2) the signal is not dependent on the oxygen concentration. A mathematical model relating the Hb absorbance variation during the reaction with the glucose concentration has been developed to provide theoretical support and to predict its application to other compounds after changing the GOx by another enzyme. The method has been applied to direct glucose determination in 10 blood samples, and a correlation coefficient higher than 0.98 was obtained after comparing the results with those determined by an automatic analyzer. As well as sharing some of the advantages of disposable amperometric biosensors, the most significant feature of this approach is its reversibility.

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.