Monitoring autoimmune diseases by bioelectrochemical detection of autoantibodies. Application to the determination of anti-myelin basic protein autoantibodies in serum of multiple sclerosis patients.

This work reports an amperometric bioplatform for the determination of anti-myelin basic protein autoantibodies (anti-MBP), a relevant biomarker for multiple sclerosis (MS) autoimmune disease. The developed configuration involves the use of carboxylated magnetic microparticles (cMBs) where the protein for specific capture of the target autoantibodies was covalently attached. The immobilized anti-MBP were further conjugated with a secondary antibody labelled with horseradish peroxidase (HRP-anti-hIgG) and amperometric transduction was performed by adding hydrogen peroxide and using hydroquinone (HQ) as redox mediator. The cathodic current resulting from the reduction of the corresponding quinone was directly proportional to the logarithmic concentration of the target autoantibodies. The analytical performance of the developed method for the determination of anti-MBP is competitive in terms of sensitivity and range of linearity with that claimed for the only biosensor reported so far in the literature, as well as with commercially available ELISA kits showing a remarkably shorter assay time. The bioplatform was applied to the analysis of serum samples of healthy individuals and patients diagnosed with MS providing results in agreement with the ELISA methodology.

Simultaneous determination of CXCL7 chemokine and MMP3 metalloproteinase as biomarkers for rheumatoid arthritis.

This paper reports the preparation of the first dual electrochemical immunosensor for the simultaneous determination of the CXCL7 chemokine and the MMP3 metalloproteinase as relevant biomarkers for the better diagnosis and monitoring of rheumatoid arthritis derived from the multiple biomarkers measurement. The developed immunosensor involves the use of carboxylated magnetic beads (MBs) and dual screen-printed carbon electrodes (SPdCEs). Sandwich-type configurations implied the covalent immobilization of specific anti-CXCL7 (cAb1) or anti-MMP3 (cAb2) capture antibodies onto MBs and the use of biotinylated detection antibodies with further labelling with HRP-Strept conjugates. The resulting MBS bioconjugates were magnetically captured on the respective working electrode of the SPdCE and the determination of the antigens was accomplished by measuring the amperometric responses of H2O2 mediated by hydroquinone (HQ) at a potential value of -0.20 V. The dual immunosensor provided calibration plots with linear ranges between 1 and 75 ng mL-1 (CXCL7) (R2 = 0.997) and from 2.0 to 2000 pg mL-1 (MMP3) (R2 = 0.998) with detection limits of 0.8 ng mL-1 and 1.2 pg mL-1, respectively. The assay took 2 h 20 min for the simultaneous determination of both biomarkers. The dual immunosensor was successfully applied to the analysis of human serum from positive and negative RA patients.

Electrochemical immunosensor for the determination of the cytokine interferon gamma (IFN-γ) in saliva.

A simple, fast and sensitive amperometric immunosensing method for the determination of the clinically relevant cytokine interferon gamma (IFN-γ) in saliva complying the requirements demanded for this kind of sample is reported. The target analyte was sandwiched between a specific capture antibody covalently immobilized on a screen-printed electrode functionalized by the diazonium salt grafting of p-aminobenzoic acid, and a biotinylated detector antibody labeled with a streptavidin-horseradish peroxidase conjugate. The amperometric responses measured at - 0.20 V vs Ag pseudo-reference electrode upon addition of hydrogen peroxide in the presence of hydroquinone as the redox mediator allowed a calibration plot with a linear range between 2.5 and 2000 pg mL-1 and a low limit of detection (1.6 pg mL-1) to be obtained. In addition, a good selectivity against other non-target proteins was achieved. The developed method was validated by analyzing a WHO 1st International Standard for IFN-γ. In addition, the immunosensor was used for the determination of the endogenous IFN-γ in saliva with results in excellent agreement with those obtained by a commercial ELISA kit.

Electrochemical immunosensor for simultaneous determination of interleukin-1 beta and tumor necrosis factor alpha in serum and saliva using dual screen printed electrodes modified with functionalized double-walled carbon nanotubes.

Dual screen-printed carbon electrodes modified with 4-carboxyphenyl-functionalized double-walled carbon nanotubes (HOOC-Phe-DWCNTs/SPCEs) have been used as scaffolds for the preparation of electrochemical immunosensors for the simultaneous determination of the cytokines Interleukin-1ß (IL-1ß) and factor necrosis tumor α (TNF-α). IL-1ß. Capture antibodies were immobilized onto HOOC-Phe-DWCNTs/SPCEs in an oriented form making using the commercial polymeric coating Mix&Go™. Sandwich type immunoassays with amperometric signal amplification through the use of poly-HRP-streptavidin conjugates and H2O2 as HRP substrate and hydroquinone as redox mediator were implemented. Upon optimization of the experimental variables affecting the immunosensor performance, the dual immunosensor allows ranges of linearity extending between 0.5 and 100 pg/mL and from 1 to 200 pg/mL for IL-1ß and TNF-α, respectively, these ranges being adequate for the determination of the cytokines in clinical samples. The achieved limits of detection were 0.38 pg/mL (IL-1ß) and 0.85 pg/mL (TNF-α). In addition, the dual immunosensor exhibits excellent reproducibility of the measurements, storage stability of the anti-IL-Phe-DWCNTs/SPCE and anti-TNF-Phe-DWCNTs/SPCE conjugates, and selectivity as well as negligible cross-talking. The dual immunosensor was applied to the simultaneous determination of IL-1ß and TNF-α in human serum spiked at clinically relevant concentration levels and in real saliva samples.

Electrochemical immunosensor for sensitive determination of transforming growth factor (TGF) - ß1 in urine.

The first amperometric immunosensor for the quantification of TGF-ß1, a cytokine proposed as a biomarker for patients having or at risk for renal disease, is described in this work. The immunosensor design involves disposable devices using carboxylic acid-functionalized magnetic microparticles supported onto screen-printed carbon electrodes and covalent immobilization of the specific antibody for TGF-ß1 using Mix&Go polymer. A sandwich-type immunoassay was performed using biotin-anti-TGF and conjugation with peroxidase-labeled streptavidin (poly-HRP-Strept) polymer. Amperometric measurements were carried out at -0.20V by adding hydrogen peroxide solution onto the electrode surface in the presence of hydroquinone as the redox mediator. The calibration plot allowed a range of linearity extending between 15 and 3000pg/mL TGF-ß1 which is adequate for the determination of the cytokine in plasma and urine. The limit of detection, 10pg/mL, is notably improved with respect to those obtained with ELISA kits. The usefulness of the immunosensor for the determination of low TGF-ß1 concentrations in real samples was evaluated by analyzing spiked urine at different pg/mL concentration levels.

Carbon nanotubes functionalized by click chemistry as scaffolds for the preparation of electrochemical immunosensors. Application to the determination of TGF-beta 1 cytokine.

An electrochemical immunosensor for the determination of the multifunctional Transforming Growth Factor ß1 (TGF-ß1) cytokine has been prepared using multi-walled carbon nanotube (MWCNT)-modified screen-printed carbon electrodes. MWCNTs were functionalized by means of copper(i) catalyzed azide-alkyne cycloaddition ("click" chemistry) as an efficient strategy for the covalent immobilization of immunoreagents without altering their configurations and preserving their biological activity. Alkyne-functionalized IgGs were also prepared and used to assemble IgG-alkyne-azide-MWCNT conjugates used as scaffolds for the immunosensor preparation. After a blocking step with casein, anti-TGF was immobilized and the target cytokine was sandwiched with biotinylated anti-TGF labeled with poly-HRP-labeled streptavidin. The affinity reaction was monitored amperometrically at -0.20 V using the hydroquinone (HQ)/H2O2 system. The calibration plot for TGF-ß1 exhibited a range of linearity (r2 = 0.995) extending between 5 and 200 pg mL-1 which is suitable for the determination of the target cytokine in human serum. A limit of detection of 1.3 pg mL-1 was achieved. The analytical performance of the immunosensor can be advantageously compared with that claimed for ELISA kits. The immunosensor was applied to the analysis of spiked human serum samples at different concentration levels with excellent recoveries.