Olga Domínguez-Renedo

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 microM and from 0.4 to 2.4 microM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18+/-0.01 microM in the case of DAO/HRP and 0.40+/-0.04 microM (alpha=0.05 and beta=0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.

Electrochemical determination of chromium(VI) using metallic nanoparticle-modified carbon screen-printed electrodes

Carbon screen-printed electrodes (CSPEs) modified with metal nanoparticles present an interesting alternative in the determination of chromium(VI) by differential pulse voltammetry (DPV). Metallic silver and gold nanoparticle deposits have been obtained by electrochemical deposition. Scanning electron microscopy measurements show that the electrochemically synthesized silver and gold nanoparticles are deposited in aggregated form. The detection limit for the analytical procedures developed in this work were 8.5 x 10(-7) and 4.0 x 10(-7)M for silver and gold nanoparticle-modified CSPE, respectively. In terms of reproducibility, the precision of the above-mentioned method was calculated at 6.7% in %R.S.D. values for silver and 3.21% for gold nanoparticle CSPE.

Sensitive enzyme-biosensor based on screen-printed electrodes for Ochratoxin A

Horseradish peroxidase has been successfully immobilized in a polypyrrole matrix onto disposable screen-printed carbon electrodes for the selective detection of Ochratoxin A. The chronoamperometric determination of this mycotoxin has been optimized by experimental design methodology, which implied the join evaluation of pH of the buffer solution, applied potential and concentration of H(2)O(2). The slopes of the calibration curves built under the optimum conditions of the experimental variables have been used to estimate the reproducibility and the repeatability of the developed biosensor for Ochratoxin A. Relative standard deviation values of 1.9% (n=5 and alpha=0.05) and 7.1% (n=4 and alpha=0.05) were obtained for reproducibility and repeatability, respectively. The capability of detection for this method was 0.1 ng mL(-1) (alpha=0.05 and beta<0.05). The viability of the developed biosensor in the determination of Ochratoxin A in spiked beer and in roasted coffee has been shown, yielding average recoveries of 103% and 99%, in that order, with relative standard deviation values less than 5% (n=3 and alpha=0.05) in both cases.

CYP450 2B4 covalently attached to carbon and gold screen printed electrodes by diazonium salt and thiols monolayers.

An easy covalent immobilization method used to develop enzyme biosensors based on carbon and gold screen printed electrodes (SPCEs and gold SPEs) is described. The linkage of biomolecules through 4-nitrobenzenediazonium tetrafluoroborate, mercaptopropionic acid and thioctic acid monolayers has been attempted using bare SPCEs and gold SPEs, as well as gold nanoparticles (AuNPs) modified SPCEs and gold SPEs. Direct covalent attachment of Cytochrome P450 2B4 (CYP450 2B4) to the transducer has been carried out by carbodiimide and hydroxysuccinimide. Experimental variables in the immobilization process and in the chronoamperometric determination of Phenobarbital (PB) have been optimized by the experimental design methodology. Reproducibility of the different biosensors has been checked under the optimum conditions, yielding values lower than 6%. Their performances have been shown by the determination of PB in pharmaceutical drugs.

Immobilization of Acetylcholinesterase on Screen-Printed Electrodes. Application to the Determination of Arsenic(III)

Enzymatic amperometric procedures for measuring arsenic, based on the inhibitive action of this metal on acetylcholinesterase enzyme activity, have been developed. Screen-printed carbon electrodes (SPCEs) were used with acetylcholinesterase covalently bonded directly to its surface. The amperometric response of acetylcholinesterase was affected by the presence of arsenic ions, which caused a decrease in the current intensity. The experimental optimum working conditions of pH, substrate concentration and potential applied, were established. Under these conditions, repeatability and reproducibility of biosensors were determined, reaching values below 4% in terms of relative standard deviation. The detection limit obtained for arsenic was 1.1 × 10−8 M for Ach/SPCE biosensor. Analysis of the possible effect of the presence of foreign ions in the solution was performed. The method was applied to determine levels of arsenic in spiked tap water samples.

Development of urease based amperometric biosensors for the inhibitive determination of Hg (II).

Enzymatic amperometric procedures for measurement of Hg (II), based on the inhibitive action of this metal on urease enzyme activity, were developed. Screen-printed carbon electrodes (SPCEs) and gold nanoparticles modified screen-printed carbon electrodes (AuNPs/SPCEs) were used as supports for the cross-linking inmobilization of the enzyme urease. The amperometric response of urea was affected by the presence of Hg (II) ions which caused a decreasing in the current intensity. The optimum working conditions were found using experimental design methodology. Under these conditions, repeatability and reproducibility for both types of biosensors were determined, reaching values below 6% in terms of residual standard deviation. The detection limit obtained for Hg (II) was 4.2x10(-6)M for urease/SPCE biosensor and 5.6x10(-8)M for urease/AuNPs/SPCE biosensor. Analysis of the possible effect of the presence of foreign ions in the solution was performed. The method was applied to determine levels of Hg (II) in spiked human plasma samples.

Horseradish peroxidase-screen printed biosensors for determination of Ochratoxin A.

This work summarizes the manufacturing procedure of Horseradish peroxidase (HRP) based biosensors for the determination of the mycotoxin Ochratoxin A (OTA). The biosensors have been fabricated using the single technology of screen-printing. That is to say, an HRP containing ink has been directly screen-printed onto carbon electrodes, which offers a higher rapidity and simplicity in the manufacturing process of biosensors for OTA determination. The formal redox potential of the Fe(III/II) moiety of HRP has been used to demonstrate the effective loading of enzyme into the ink. The chronoamperometric oxidation current registered has been successfully related to the concentration of OTA in solution from different samples, including beer ones. Under the optimum conditions of the experimental variables, precision in terms of reproducibility and repeatability has been calculated in the concentration range from 23.85 to 203.28 nM. A relative standard deviation for the slopes of 10% (n = 4) was obtained for reproducibility. In the case of repeatability, the biosensor retained a 30% of the initial sensitivity after the third calibration. The average capability of detection for 0.05% probabilities of false positive and negative was 26.77 ± 3.61 nM (α = 0.05 and β=0.05, n = 3).

CYP450 biosensors based on screen-printed carbon electrodes for the determination of cocaine.

A new electrochemical method has been described and characterized for the determination of cocaine using screen-printed biosensors. The enzyme cytochrome P450 was covalently attached to screen-printed carbon electrodes. Experimental design methodology has been performed to optimize the pH and the applied potential, both variables that have an influence on the chronoamperometric determination of the drug. This method showed a reproducibility of 3.56% (n=4) related to the slopes of the calibration curves performed in the range from 19 up to 166nM. It has been probed the used of this kind of biosensors in the determination of cocaine in street samples, with an average capability of detection of 23.05±3.53nM (n=3, α=β=0.05).

Electrochemical determination of levetiracetam by screen-printed based biosensors.

This work shows an easy and fast electrochemical method for Levetiracetam (LEV) determination, which is a novel antiepileptic. Most of the methods used up to now for its determination required a pre-treatment of the sample. It is shown here that the developed Peroxidase based biosensors avoid this kind of drawbacks. Screen-printed carbon electrodes have been used as transducers for the Peroxidase immobilization by pyrrole electropolymerization. Experimental variables that can affect LEV chronoamperometric response, such as hydrogen peroxide concentration, pH and applied potential, have been optimized in order to perform a selective LEV determination. Under these conditions, the performance of the biosensors has been tested. The residual standard deviation (RSD) of the slopes of different calibration curves was 9.77% (n=4 and alpha=0.05) for the reproducibility and 7.73% (n=4 and alpha=0.05) in the case of the repeatability. An average limit of detection of 9.81x10(-6) M (alpha=beta=0.05) was obtained. The biosensors have been finally applied to the determination of LEV in complex matrices, such as pharmaceutical drugs and spiked human plasma samples, yielding successful results.

Determination of Antimony (III) in Real Samples by Anodic Stripping Voltammetry Using a Mercury Film Screen-Printed Electrode

This paper describes a procedure for the determination of antimony (III) by differential pulse anodic stripping voltammetry using a mercury film screen-printed electrode as the working electrode. The procedure has been optimized using experimental design methodology. Under these conditions, in terms of Residual Standard Deviation (RSD), the repeatability (3.81 %) and the reproducibility (5.07 %) of the constructed electrodes were both analyzed. The detection limit for Sb (III) was calculated at a value of 1.27×10−8 M. The linear range obtained was between 0.99 × 10−8 − 8.26 × 10−8 M. An analysis of possible effects due to the presence of foreign ions in the solution was performed and the procedure was successfully applied to the determination of antimony levels in pharmaceutical preparations and sea water samples.