Vanesa Sanz

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.

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.

Modification of plasmid DNA topology by 'histone-mimetic' gold nanoparticles

AIMS Our aim is to explore whether gold nanoparticles (AuNPs) functionalized with a carboxylated polyethylene glycol (PEG) and protamine (AuNP@PEG@Prot) can modulate - enhance or restrain - DNA condensation, altering DNA conformation and inducing structural changes. Understanding how these nanoconjugates modulate DNA structure, size and shape of DNA condensates, and enable control over the resulting 3D structures is of major biological and therapeutic importance. MATERIALS & METHODS Citrate-AuNPs were covered with a dense layer of a hetero-functional octa(ethylene glycol) (SH-EG(8)-COOH). Conjugation of protamine to the AuNP@PEG was achieved by taking advantage of the carboxylated surface previously generated on the surface of the NP and the remaining amino groups from the protamine, using carbodiimide and N-hydroxysulfosuccinimide coupling reactions. RESULTS & CONCLUSION AuNP@PEG@Prot modulates the structure and topology of DNA, not only for condensation, but also for decondensation, via formation of higher quantities of dimers and multimers, when compared with AuNP@PEG and free protamine.

Uric acid determination using uricase and the autotransducer molecular absorption properties of peroxidase

Uric acid (UA) is determined using the UV-vis molecular absorption properties of peroxidase (HRP). The method as a whole involves UA oxidation in the presence of uricase (UOx), giving H(2)O(2.) The H(2)O(2) then reacts with HRP forming the compound I species which returns to its initial form by reaction with UA and intramolecular reduction. The molecular absorption changes of HRP at 420nm during the reaction enable the UA to be determined. A mathematical model relating the analytical signal to UA, UOx and HRP has been developed and experimentally validated. The possibility of carrying out both enzymatic reactions sequentially or simultaneously is discussed, the latter option producing better analytical performances. The method permits UA determination in the range 1.5x10(-6)-4.0x10(-5)M, with an R.S.D. of about 3% (n=5, 1.5x10(-6)M UA). It has been applied to analyte determination in synthetic serum samples.

A theoretical approach for designing fluorescent reagentless biosensors : The optical model

In this paper a mathematical model describing the analytical signal obtained in fluorescence sensors is presented and compared with other commonly used models. The model starts from the Kubelka-Munk theory for solid surfaces but incorporates new theoretical improvements, being principally: (a) the increase in the effective optical pathlength due to the Scattering Induced Path Variation (SIPV), the incorporation of this parameter allows us to deduce that the fluorescence intensity from solid surfaces does not linearly change with the fluorophore concentrations; (b) the influence of the inner filter effect and how the error can be rectified and (c) the calculation of the scattering coefficients in sensor films for this kind of sensor. From this model it is possible to predict the effect of the fluorophore concentration, the sensor film scattering coefficient and the sample inner filter effect on the fluorescence signal. The conclusions obtained can be extended to other types of fluorescence measurements from solid surfaces.

Using blood hemoglobin for blood analysis

This paper demonstrates that the spectrophotometric properties of blood hemoglobin (Hb) can be used for the direct determination of biochemical compounds in blood. Glucose is used as a model, but the methodology can be applied to many other compounds (only a previous enzymatic reaction producing H(2)O(2) is needed). In order to develop the method, a model relating the Hb absorbance variation during the reaction with the glucose concentration has been developed to provide theoretical support for the method and to predict its application to other compounds. In addition, clear blood samples need to be prepared without pre-treatment and lateral reactions of H(2)O(2) with other blood constituents need to be blocked; this has been achieved with 100 : 1 v/v blood dilution in bi-distilled water and azide addition. The linear response range of the method can be fitted between 2 and 540 mg dL(-1) glucose relative to the original blood sample (RSD about 4%, 70 mg dL(-1)). The analyte concentration can be obtained by an absolute calibration method or by the standard addition method; both have been applied for direct glucose determination in several blood samples and good correlations with those obtained by an automatic analyzer have been obtained.

Reagentless Optical Biosensors for Organic Compounds Based on Autoindicating Proteins

Optical reagentless biosensors are one of the most promising alternatives for producing selective, sensitive and autonomous sensors for real life applications. These devices are based on the efficient use of the spectroscopic properties of bioreagents, mainly proteins, as transducers; avoiding in this way the use of chemical colorant/fluorophores which usually limit sensors performance. In this paper a brief state of the art of the bioreagents being used in biosensors as well as recent alternatives are discussed. The advantages of flavoenzymes and hemeproteins as the basis for reagentless biosensors are particularly stressed.