V. Serafín

Electrochemical immunosensor for the determination of insulin-like growth factor-1 using electrodes modified with carbon nanotubes-poly(pyrrole propionic acid) hybrids.

An amperometric immunosensor for the determination of the hormone insulin-like growth factor 1 (IGF1) is reported for the first time in this work. As electrochemical transducer, a multiwalled carbon nanotubes-modified glassy carbon electrode on which poly(pyrrole propionic acid) was electropolymerized was prepared. This approach provided a high content of surface confined carboxyl groups suitable for direct covalent binding of anti-IGF1 monoclonal antibody. A sandwich-type immunoassay using a polyclonal antibody labeled with peroxidase, hydrogen peroxide as the enzyme substrate and catechol as redox mediator was employed to monitor the affinity reaction. All the variables involved in the preparation of the modified electrode were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Moreover, the different experimental variables affecting the amperometric response of the immunosensor were also optimized. The calibration graph for IGF1 showed a range of linearity extending from 0.5 to 1000 pg/mL, with a detection limit, 0.25 pg/mL, more than 100 times lower than the lowest values reported for the ELISA immunoassays available for IGF1 (30 pg/mL, approximately). Excellent reproducibility for the measurements carried out with different immunosensors and selectivity against other hormones were also evidenced. A commercial human serum spiked with IGF1 at different levels between 0.01 and 10.0 ng/mL was analyzed with good results.

An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL-1, and a LOD of 4pgmL-1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment.

Ultrasensitive determination of receptor tyrosine kinase with a label-free electrochemical immunosensor using graphene quantum dots-modified screen-printed electrodes

A new label-free electrochemical immunosensor is constructed for the selective and sensitive determination of the clinically relevant biomarker receptor tyrosine kinase (AXL) in human serum. The disposable immunosensing platform is prepared by immobilization of the specific anti-AXL antibody onto amine functionalized graphene quantum dots (fGQDs)-modified screen-printed carbon electrodes (SPCEs). The affinity reactions were monitored by measuring the decrease in the differential pulse voltammetric (DPV) response of the redox probe Fe(CN)63-/4-. All the experimental variables involved in the preparation of the modified electrodes and in the immunosensor performance were optimized. The as prepared immunosensor exhibits an improved analytical performance with respect to other electrochemical immunosensors reported so far, with a wider range of linearity and a lower detection limit, 0.5 pg mL-1, which is more than one hundred thousand times lower than the established cut-off value for heart failure (HF) diagnosis in serum (71 ng mL-1). The developed immunosensor was successfully applied to the determination of the endogenous content of AXL in serum of HF patients without any matrix effect observed after just a sample dilution.

Amperometric detection at carbon felt electrodes. Application to the determination of nitro musk derivatives and phenolic endocrine disruptors

The use of a carbon-felt electrode (CFE) of small dimensions for the amperometric detection of various nitro musk fragrances and phenolic compounds with endocrine disrupting properties is reported. The electrode material is composed of multiple disordered carbon fibres of micrometre diameter. The use of CFE for the detection of nitro musk derivatives implied the electrochemical activation by applying successive SW voltammetric scans between 0.0 and +2.6 V vs. Ag/AgCl in 0.1 M PBS of pH 7.0. At the activated CFE (aCFE), cyclic voltammograms for nitro musks derivatives in 0.1 M PBS of pH 7.0, showed a non-reversible behaviour with two or three cathodic peaks corresponding to the number of nitro groups present in the molecule. Hydrodynamic voltammograms from nitro musk solutions obtained under flow injection conditions at the aCFE led to the selection of a cathodic potential of −800 mV vs. Ag/AgCl as the value to be applied for the amperometric detection of these compounds. A calibration graph for musk ketone was obtained between 0.1 and 10 μg mL−1 with a detection limit of 50 ng mL−1. A RSD value of 3.7% was calculated for 10 successive measurements of 500 ng mL−1 musk ketone with no need for cleaning the electrode surface. Similar results to those obtained by GC-MS were found for the analysis of incense samples by FI with amperometric detection at the activated CFE. FI hydrodynamic voltammograms for estrogenic phenols at non-activated CFE exhibited current anodic plateaux from which a detection potential of +700 mV was chosen. Liquid chromatography with amperometric detection at the CFE was accomplished to analyze mixtures of estrogenic compounds. A mobile phase consisting of 40 : 60 (v/v) acetonitrile : 0.05 M PBS of pH 7.0 was employed. Detection limits ranging between 102 nM for bisphenol A (BPA) and 182 nM for 17α-ethynylestradiol (EE2) were achieved. Good recoveries were obtained in the analysis of well water and tap water samples spiked with five phenolic estrogenic compounds at a 14 nM each concentration level.

Determination of progesterone in saliva using an electrochemical immunosensor and a COTS-based portable potentiostat

This paper describes the reliable determination of progesterone (P4) in undiluted saliva making use of a disposable amperometric immunosensors implemented on low-cost and portable device/potentiostat constructed with commercial-off-the-shelf (COTS) components. The immunosensor allows the fast (45 min), selective and sensitive determination (5 pg mL-1 LOD) of P4 using amperometry in stirred solutions. The immunosensor was coupled to the COTS-based potentiostat and amperometry was made into drops of quiescent solutions. No significant differences were apparent between the analytical performance achieved with the immunosensor for P4 using both a conventional and the COST-based potentiostats. The practical applicability of the immunosensor coupled with the COTS-based potentiostat was demonstrated by determining the endogenous P4 content in different undiluted saliva samples with highly variable endogenous contents of the target hormone. The obtained results were in good agreement with those provided by the conventional ELISA methodology and with the contents reported in the literature for samples with similar characteristics. This validated the combined device for the reliable and minimally invasive determination of the target hormone involving a very simple protocol and taking only 45 min.