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Electrochemical sandwich immunoassay strategies involving the use of carboxyl-functionalized magnetic microbeads (cMBs) and magnetic nanoparticles (cMNPs) have been evaluated and compared. The proteolytically cleaved soluble tyrosine kinase receptor sAXL was used as the target analyte. Antibodies against AXL were covalently immobilized on cMBs or cMNPs. Immunobinding of AXL was detected by means of a secondary biotinylated antibody and a streptavidin-horseradish peroxidase conjugate. The electrochemical transduction was accomplished by capturing the cMBs or cMNPs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by using a small magnet. The amperometric response was measured at −0.20 V (vs the silver pseudo-reference electrode of the SPCE) upon the addition of HO in the presence of hydroquinone as the redox mediator. The calibration plots for AXL extended up to 7.5 ng mL when cMBs were used for the preparation of the immunosensor and up to 40 ng mL in the case of using cMNPs. The respective slope values were 158 (cMBs) and 43 nA mL ng (cMNPs), while the achieved LODs were 74 (cMBs) and 75 pg mL (cMNPs). Although the immunosensors prepared with cMBs provided a shorter range of linearity, they exhibited a 3.7-times larger sensitivity than those constructed with cMNPs. The successful application of the new strategies was demonstrated for the determination of the endogenous content of sAXL in real human serum samples (a cut-off value of 71 ng mL have been established for patients with risk of heart failure). The immunosensors constructed using cMBs or cMNPs can be advanta geously compared, in terms of sensitivity and fabrication time, with the only immunosensor for AXL previously reported. In addition, these new immunosensors took approximately half time than ELISA to perform the assay. [ABSTRACT FROM AUTHOR]

The first electrochemical immunosensor for the determination of peptide YY is reported in this paper. A novel electrochemical platform, prepared by the electrochemical grafting of the diazonium salt of 4-aminobenzoic acid onto a reduced graphene oxide-modified glassy carbon electrode, was used, on which the covalent immobilization of specific anti-PYY antibodies was accomplished. The HOOC-Phe-rGO/GCEs were characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The different variables affecting the preparation of the modified electrodes and the performance of the immunosensor were optimized. Under the optimized conditions, a calibration plot for PYY showing a linear range extending between 10−4 and 102 ng mL−1 was found. This range is adequate for the determination of this protein in real samples, since the expected concentration in human serum is around 100 pg mL−1. The limit of detection was 0.01 pg mL−1 of PYY. The immunosensor exhibited good reproducibility of the PYY measurements, excellent storage stability and selectivity, as well as a shorter assay time than those of ELISA kits. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing human serum samples spiked with PYY at three concentration levels. [ABSTRACT FROM AUTHOR]

A label-free dual electrochemical immunosensor was constructed for the multiplexed determination of human growth (hGH) and prolactin (PRL) hormones. The immunosensor used an electrochemical platform composed of carbon nanotube–screen printed carbon electrodes (CNT/SPCEs) modified with poly(ethylene-dioxythiophene) (PEDOT) and gold nanoparticles, on which the corresponding hGH and PRL antibodies were immobilized. The affinity reactions were monitored by measuring the decrease in the differential pulse voltammetric oxidation response of the redox probe dopamine. The experimental variables involved in the preparation of both AuNP/PEDOT/CNT/SPC modified electrodes and the dual immunosensor were optimized. The immunosensor exhibited an improved analytical performance for hGH and PRL with respect to other electrochemical immunosensor designs, showing wide ranges of linearity and low detection limits of 4.4 and 0.22 pg mL−1, respectively. An excellent selectivity against other hormones and in the presence of ascorbic and uric acids was found. The usefulness of the dual immunosensor for the simultaneous analysis of hGH and PRL was demonstrated by analyzing human serum and saliva samples spiked with the hormones at different concentration levels. [ABSTRACT FROM AUTHOR]

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs-mAbhGH-hGH-pAbhGH-anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL ( r = 0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011-1016, ). Furthermore, good repeatability, with RSD = 3% ( n = 10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96 ± 6% and 99 ± 2%, respectively, were obtained. [ABSTRACT FROM AUTHOR]