Esteve Fàbregas

Toward a fast, easy, and versatile immobilization of biomolecules into carbon nanotube/polysulfone-based biosensors for the detection of hCG hormone.

The aim of this study was the fabrication and characterization of biomembranes by the phase inversion (PI) method followed by their subsequent casting onto screen-printed electrodes (SPE) for biomedical applications. The combination of multiwalled carbon nanotubes (MWCNT) as a transducer with polysulfone (PSf) polymer enables easy incorporation of biological moieties (hormones or antibodies), providing a 3D composite with high electrochemical response to corresponding analytes. Antibody/MWCNT/PSf biosensors were characterized by confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM), and electrochemical methods. For biomedical purposes, human chorionic gonadotropin (hCG) hormone was tested by competitive immunoassay. The detection limit was determined to be 14.6 mIU/mL with a linear range up to 600 mIU/mL. We concluded that the easy and fast incorporation of biomolecules by the PI method, as well as their stability and distribution throughout the 3D polysulfone composite, are testament to the utility for the versatile fabrication of biosensors for clinical diagnosis.

Carbon nanotube/polysulfone composite screen-printed electrochemical enzyme biosensors

The fabrication, evaluation and attractive performance of multiwall carbon nanotube(MWCNT)/polysulfone biocomposite membrane modified thick-film screen-printed electrochemical biosensors are reported. The fabricated carbon nanotube/polysulfone (CNT/PS) strips combine the attractive advantages of carbon nanotube materials, polysulfone matrix and disposable screen-printed electrodes. Such thick-film carbon nanotubes/polysulfone sensors have a well defined performance, are mechanically stable, and exhibit high electrochemical activity. Furthermore, biocompatibility of CNT/PS composite allows easy incorporation of biological functional moiety of horseradish peroxidase by phase inversion technique. The comparison of graphite with MWCNT as conductor material is described in this paper. The proposed H(2)O(2) biosensor exhibited a linear range (applied potential, -0.2 V) from 0.02 to 0.5 mM and a K(M)(app) of 0.71 mM.

Development of electrochemical immunosensing systems with renewable surfaces

The repeated use of immunochemically modified solid phases in electrochemical immunosensor analysis is the driving interest of this work. Two new strategies have been developed. One of these strategies is aimed at the development of a manual methodology. It comprises the construction of amperometric immunosensors based on rigid biocomposites. These biocomposites are formed by a conducting polymer composite matrix that acts as a reservoir of an immobilized immunologic material. The surface of the biocomposite can be renewed by a simple polishing procedure. The second strategy involves the design of an automatic methodology. It features an immunochemical analytical system using flow injection techniques. The potentiometric detection uses a solid phase formed by immunologic reagents immobilized in magnetic particles. These particles are fixed to the sensor with the use of a magnetic field. The renewal of the reactive surface is achieved by the release and activation of the restraining magnetic field and the manipulation of the flow. The analytical properties of these immunosensors were evaluated measuring RIgG using a competitive technique and measuring GaRIgG with a sandwich methodology. The labelling enzymes of the immunoconjugates were peroxidase in amperometric measurements and urease in potentiometric measurements.

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

Evaluation of different mediator-modified screen-printed electrodes used in a flow system as amperometric sensors for NADH

This work presents a comparative study between two different methods for the preparation of mediator-modified screen-printed electrodes, to be used as detectors in a reliable flow injection system for the determination of the nicotinamide adenine dinucleotide (NADH) coenzyme. The best strategy was selected for the final development of compact biosensors based on dehydrogenase enzymes. For the first immobilisation strategy, different redox mediators were electropolymerised onto the SPE surface. The second immobilisation strategy was carried out using polysulfone-graphite composites, which were deposited by screen-printing technology onto the screen-printed electrode (SPE) surface. Both methods achieved an effective and reliable incorporation of redox mediators to the SPE configuration. Finally, a flow system for ammonium determination was developed using a glutamate dehydrogenase (GlDH)-Meldolas Blue (MB)-polysulfone-composite film-based biosensor. The stability of the redox mediators inside the composite films as well as the negligible fouling effect observed on the electrode surface improve the repeatability and reproducibility of the sensors, important features for continuous analysis in flow systems. Furthermore, the optimised bio/sensors, incorporated in a flow injection system, showed good sensitivities and short response times. Such a good analytical performance together with the simple and fast sensor construction are interesting characteristics to consider the polysulfone-composite films as attractive electrochemical transducer materials for the development of new dehydrogenase-based SPEs.

Determination of tryptophan in feed samples by cyclic voltammetry and multivariate calibration methods

A voltammetric method for the determination of tryptophan (Trp) in feed samples using multivariate calibration techniques is proposed. The multivariate data consisted of the oxidation wave of cyclic voltammograms registered from 400 to 925 mV using a graphite–methacrylate composite electrode, at which Trp was oxidized around a potential of 900 mV. The interference from other oxidizable amino acids as well as the feed sample matrix was circumvented by using multivariate calibration methods. Principal component regression (PCR) and partial least squares regression (PLS) and non-linear PLS (NL-PLS) were checked for quantifying Trp in these samples. The results obtained using NL-PLS were in agreement with those obtained with the standard method for the determination of amino acids, with an overall prediction error of 7.9%.

Amperometric biosensor for the determination of histamine in fish samples

A bienzymatic biosensor employing diamine oxidase (DOx) and horseradish peroxidase (HRP) for the detection of histamine in fish samples has been developed and optimized in this work. These enzymes have been co-immobilized into a polysulfone/carbon nanotubes/ferrocene membrane by means of phase inversion technique onto screen-printed electrodes. The electrochemical measurements have been carried out in phosphate buffer solution at pH 8.0 in batch mode and low applied potential (-50 mV vs. Ag/AgCl, KCl 0.1 M) to minimize the interferences. Developed biosensor exhibits high sensitivity (1.9×10(7) nA(-1)), low limit of detection (1.7×10(-7) M), high storage stability and excellent reproducibility, obtaining a linear interval range from 3×10(-7) to 2×10(-5) M. Finally, applicability of the biosensor to the estimation of histamine content in different fish samples has been assessed; obtaining a good correlation between results obtained with the biosensor and those obtained with the reference method (ELISA) in case of sardines, mackerel and greater weever.

Development of a biparametric bioanalyser for creatinine and urea. Validation of the determination of biochemical parameters associated with hemodialysis

The construction and evaluation of an automated urea and creatinine biparametric biosystem using flow injection analysis (FIA) are described. The biosystem uses enzyme reactions that hydrolyse urea and creatinine producing ammonium ions. The enzymes used were creatinine deiminase and urease, which are immobilized covalently in flow reactors. The reactor with creatinine deiminase has the enzyme immobilized on controlled-pore glass beads, whereas urease is immobilized on a nylon open tubular reactor. Detection is realised with a flow-through ammonium ion-selective electrode with an inner solid-state contact (graphite-epoxy composite). Ammonium ions are separated from alkali ion interferents through a gas-diffusion cell. The bioanalyser is fully automated using software and electronics developed ex profeso in our laboratories. The analyser was validated off-line by measuring urea and creatinine from discrete effluent samples from hemodialysis equipment. Results agreed with concurrent analyses realised using hospital laboratory methods. There were no significant differences between the two sets of results at the 95% confidence level. Finally, the biparametric bioanalyser was validated on-line by measuring creatinine and urea levels in artificial kidney effluents. These measurements were useful in the determination of key biochemical parameters of clinical interest such as the mass of urea and creatinine extracted from the patient as well as the initial concentration of creatinine and urea in blood plasma. When the results of the bioanalyser were compared with those yielded by the usual methods, they showed no significant differences at the 95% confidence level when determining the mass of the analytes extracted by the hemodialyser or when determining the urea concentration in blood plasma. However, when measuring the creatinine concentration in blood plasma using the developed bioanalyser, significant differences appeared.

Novel potentiometric sensors based on polysulfone immobilized metallothioneins as metal-ionophores.

We show here the use of immobilized metal-binding biomolecules for metal analysis by using novel potentiometric sensors. To this end and as a model, Ag(+)-ISEs were developed using polysulfone matrix embedding metallothioneins as ionophores (mouse MT1 (P1) or sea urchin SpMTA (P2)). Polysulfone, a porous polymer that was not used until the present in potentiometric biosensors, has the advantage of being compatible with biological materials. Also, the phase inversion procedure allows protein incorporation into the membrane with minima alterations, since it always remains in the aqueous phase. Construction of these biosensors required small amounts of protein; they can be dry-stored and have long lifetimes. They exhibited linear responses with slopes of ca. 61mV per decade within the 10(-5) to 10(-2)M Ag(+) concentration range, detection limits of about 10(-5)M, and worked in the 2-to-8 pH range. Except for Hg(2+), the Pb(2+), Zn(2+), Cd(2+), Cu(2+) cations do not interfere with Ag(+) determination. Significantly, different affinities of Pb(2+) and Zn(2+) towards P1- and P2-ISE were found, in good correlation with the higher affinity of these cations towards SpMTA than to MT1. Consequently, the distinct metal-binding features of each MT are conserved and determine the differential properties of their biosensors. These results open a broad range of possibilities for the use of proteins as ionophores in what could be considered a new type of potentiometric biosensor if their response mechanism is taken into account.

Amperometric bienzymatic biosensor for L-lactate analysis in wine and beer samples

A novel amperometric bienzymatic biosensor has been developed based on the incorporation of Lactate Oxidase (LOx) and Horseradish Peroxidase (HRP) into a carbon nanotube/polysulfone membrane by the phase inversion technique onto screen-printed electrodes (SPEs). In order to improve the sensitivity and reduce the working potential, experimental conditions have been optimized and ferrocene has also been incorporated into the membrane as a redox mediator of the enzymatic reactions, which allows the reduction of H(2)O(2) at -100 mV. Measurements were carried out in phosphate buffer solution at pH 7.5 and under batch conditions. The biosensor response time to L-lactate was only 20 s and showed a good reproducibility (RSD 2.7%). Moreover, the detection limit was 0.05 mg L(-1) of l-lactate with a linear interval range from 0.1 mg L(-1) to 5 mg L(-1). Finally, the biosensor has been applied to the determination of l-lactic acid in different wine and beer samples. Then, the results obtained with the biosensor were compared with the ones obtained using, as a reference method, a commercial kit based on spectrophotometric measurements, obtaining an excellent agreement between the results, validating our approach.