María Moreno-Guzmán

An electrochemical immunosensor for testosterone using functionalized magnetic beads and screen-printed carbon electrodes

A disposable electrochemical immunosensor using screen-printed carbon electrodes (SPCEs) and protein A-functionalized magnetic beads (MBs) was developed for the determination of testosterone. Anti-testosterone was immobilized onto MBs and a direct competitive immunoassay involving testosterone labeled with peroxidase (HRP) was performed. The resulting conjugate was trapped on the SPCE with a small magnet. Testosterone determination was carried out by amperometry at -0.2V upon H2O2 additions using hydroquinone (HQ) as the redox mediator. The experimental variables involved in the immunosensor response to testosterone were evaluated. Under the optimized conditions, a calibration plot for testosterone was obtained with a linear range between 5.0×10(-3) and 50 ng/mL (r=0.995). The detection limit was 1.7 pg/mL and the EC50 was 0.25±0.04 ng/mL. These characteristics are notably better than those achieved with other reported immunosensors. Furthermore, anti-testosterone/MBs conjugates were shown to be stable for at least 25 days. A good selectivity was also found against other steroid hormones. The usefulness of the immunosensor was demonstrated by analyzing human serum spiked with 1 and 10 ng/mL testosterone.

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors.

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.

Electrochemical immunosensor for rapid and sensitive determination of estradiol

This work describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin (Strept) and immobilization of biotinylated anti-estradiol (anti-estradiol-Biotin). The hormone determination was performed by applying a competitive immunoassay with peroxidase-labelled estradiol (HRP-estradiol) and measurement of the amperometric response at -200 mV using hydroquinone (HQ) as redox mediator. The calibration curve for estradiol exhibited a linear range between 1 and 250 pg mL(-1) (r=0.990) and a detection limit of 0.77 pg mL(-1) was achieved. Cross-reactivity studies with other hormones related with estradiol at physiological concentration levels revealed the practical specificity of the developed method for estradiol. A good reproducibility, with RSD=5.9% (n=8) was also observed. The operating stability of a single bioelectrode modified with anti-estradiol-Biotin-Strept was nine days when it was stored at 8°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of certified serum and spiked urine samples with good results.

A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.

Carbon Nanohorns as a Scaffold for the Construction of Disposable Electrochemical Immunosensing Platforms. Application to the Determination of Fibrinogen in Human Plasma and Urine

We describe in this work a novel electrochemical immunosensor design making use of carbon nanohorns (CNHs) as a scaffold for the preparation of disposable immunosensing platforms for the determination of fibrinogen (Fib). The approach involved the immobilization of Fib onto activated CNHs deposited on screen-printed carbon electrodes (SPCEs) and the implementation of an indirect competitive assay using anti-Fib labeled with horseradish peroxidase (HRP) and hydroquinone (HQ) as the redox mediator. Both CNHs and the Fib-CNHs covalent assembly were characterized by microscopic and electrochemical techniques. The different variables affecting the analytical performance of the amperometric immunosensing strategy were optimized. The calibration plot for Fib allowed a range of linearity between 0.1 and 100 μg/mL (r(2) = 0.994) and a detection limit of 58 ng/mL to be achieved. The Fib-CNHs/SPCEs exhibited an excellent storage stability of at least 42 days. The developed immunosensor provides, in general, an analytical performance better than that reported for other Fib immunosensors and commercial ELISA kits. This simple and relatively low cost immunosensor configuration permitted the sensitive and selective determination of Fib in human plasma and urine.

Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma.

Two different fibrinogen (Fib) amperometric immunosensing designs based on the use of magnetic beads (MBs) and a novel specific nanobody (Nb) expressed in Escherichia coli are described for the first time. The immunological reaction for Fib detection was performed on COOH-MBs or His-Tag-Isolation-MBs as solid support for the immobilization of the antigen or the captured Nb. Direct and indirect competitive magnetoimmunosensing configurations have been tested and compared. In the former one, Fib and biotinylated Fib competed for the immobilized Nb binding sites while the latter configuration involved competition of free Fib in solution and immobilized Fib for binding to a fixed amount of the specific biotinylated Nb. Labeling of the captured biotinylated Nb or antigen was made with streptavidin-HRP. The modified magnetic beads were captured by a neodymium magnet on the surface of screen-printed carbon electrodes (SPCEs). Amperometric detection was accomplished at -0.20 V (vs. Ag pseudo-reference electrode) by measuring the catalytic current arising upon addition of H2O2 and using hydroquinone (HQ) as redox mediator in solution. A better analytical performance was achieved with the indirect competitive immunoassay with a detection limit of 0.044 μg mL(-1) Fib. The usefulness of both approaches was successfully demonstrated by analyzing an international standard for Fib plasma. The assays could be carried out in diluted plasma samples in a total analysis time of 90 min.

Enzyme biosensor for androsterone based on 3α-hydroxysteroid dehydrogenase immobilized onto a carbon nanotubes/ionic liquid/NAD+ composite electrode

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples.

Self-Propelled Enzyme-Based Motors for Smart Mobile Electrochemical and Optical Biosensing.

A millimeter-sized tubular motor for mobile biosensing of H2O2 in environmental and relevant clinical samples is reported. The concept relies on the self-propelled motion by the Marangoni effect, where the asymmetric release of SDS surfactant induces fluid convection and rapid dispersion of horseradish peroxidase (HRP) enzyme into the sample solution. This efficient movement together with the continuous release of fresh enzyme leads to greatly accelerated enzymatic reaction processes without the need of external stirring or chemical and physical attachment of the enzyme as in common classical biosensing approaches. In this strategy, the use of a single millimeter-sized tubular motor during 120 s allows the reliable and accurate quantification of hydrogen peroxide in a set of different matrices such as tap and mineral waters, urine, plasma, and tumor cell cultures treated with antineoplasic Cisplatin without any previous sample preparation. Furthermore, detection can be performed electrochemically, optically, and via visual detection, which makes this approach a clear candidate as a point-of-care analytical tool.

Amperometric immunosensor for the determination of ceruloplasmin in human serum and urine based on covalent binding to carbon nanotubes-modified screen-printed electrodes

A novel electrochemical immunosensor for the determination of ceruloplasmin (Cp) in human serum and urine is reported. The immunosensor configuration involves an indirect competitive immunoassay implying covalent immobilization of Cp on activated carboxylic groups at carbon nanotubes-modified screen-printed electrodes (CNTs/SPE). After Cp immobilization and reaction between the target analyte and anti-ceruloplasmin antibodies in solution, the remaining non-conjugated antibody is attached on the Cp-CNTs modified electrode. Monitoring of Cp is performed by means of a secondary antibody labeled with peroxidase (HRP-anti-IgG) and measurement of the amperometric current resulting from the addition of hydrogen peroxide in the presence of hydroquinone as the redox mediator. The experimental variables affecting the analytical performance of the immunosensor were optimized. Calibration curves for Cp provided a linear range between 0.07 and 250 μg/mL (r=0.997). The limit of detection achieved was 21 ng/mL. These analytical characteristics allow the immunosensor to be successfully used for the determination of Cp in spiked human serum and urine at various concentration levels.

A disposable electrochemical immunosensor for the determination of leptin in serum and breast milk

The preparation of a disposable electrochemical immunosensor for the quantification of the hormone leptin is described in this work. The preparation approach involved immobilization of a specific biotinylated anti-leptin antibody on the surface of streptavidin-functionalized magnetic beads (Strept-MBs) and a sandwich-type immunoassay involving the target analyte, monoclonal anti-leptin, and IgG labeled with alkaline phosphatase (AP-IgG). The electrochemical transduction step was accomplished by trapping the MBs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by means of an Nd magnet and measuring the electrochemical oxidation of the 1-naphthol generated in the AP enzyme reaction upon 1-naphthyl phosphate (1-NPP) additions by differential pulse voltammetry (DPV). A calibration plot with a linear range between 5 and 100 pg mL(-1) as well as a detection limit of 0.5 pg mL(-1) (3sb/m) were achieved. This value is more than 27 times lower than that reported in the only voltammetric immunosensor for leptin described in the literature until now. The usefulness of the immunosensor was demonstrated by analyzing different types of real samples: human serum, infant powdered milk, and breast milk from a nursing mother with two months of breastfeeding.