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.

Design of electrochemical immunosensors using electro-click chemistry. Application to the detection of IL-1ß cytokine in saliva.

Electro-click methodology was employed to prepare an electrochemical immunosensor for the cytokine interleukin 1ß (IL-1ß). The strategy involved binding of ethynylated IgG to azide-MWCNTs modified electrodes by Cu(I) catalyzed-cycloaddition reaction where the catalyst was electrochemically synthesized. This electro-click protocol is significantly faster and greener than the methods for catalyst generation through chemical reduction. The oriented immobilization of the capture antibody onto IgG-MWCNTs conjugates allowed the preparation of a sandwich-type immunosensor using biotinylated anti-IL-1ß as detector antibody labeled with alkaline phosphatase-streptavidin (AP-strept). Differential pulse voltammetric transduction through the 1-naphthylphosphate/1-naphthol system was carried out. The analytical characteristics achieved with the electrochemical immunosensor showed a calibration curve exhibiting two linear ranges between 10 and 200 pg mL-1 (r2 = 0.998), and from 200 to 1200 pg mL-1 (r2 = 0.998), and a LOD value of 5.2 pg mL-1, an improvement compared with those claimed for commercial ELISA kits. In addition, the assay time was at least one hour shorter. Excellent performance was observed in the determination of IL-1ß in saliva with no need for sample treatment, and by simple interpolation using a calibration plot constructed with standard solutions of the target cytokine.

Oxidative grafting vs. monolayers self-assembling on gold surface for the preparation of electrochemical immunosensors. Application to the determination of peptide YY.

A comparison of the performance of two electrochemical immunosensors for the determination of the anorexigen biomarker peptide YY (PYY) is reported by using as scaffolds screen printed gold electrodes modified either by oxidative grafting of p-aminobenzoic acid (p-ABA) or by assembling of a 4-mercaptobenzoic acid (4-MBA) SAM. Covalent immobilization of capture antibodies on the surface-confined carboxyl groups was carried out by EDC/NHSS chemistry, and competitive immunoassays between target PYY and Biotin-PYY were implemented. Upon labeling with alkaline phosphatase (AP)-streptavidin conjugate and 1-naphtyl phosphate addition, differential pulse voltammograms recorded between -0.2 and +0.7 V were used as analytical readout. All the steps involved in the functionalization of the electrodes and the preparation of the immunosensors were monitored by electrochemical impedance spectroscopy. The calibration plot for PYY using the AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor provided a linear current vs. log [PYY] plot extending between 10-6 and 103 ng/mL PYY with a detection limit of 3 × 10-7 ng/mL. These analytical characteristics are remarkably better than those obtained with the immunosensor prepared with 4-MBA SAM-SPAuEs. The AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor was used to analyze human serum and saliva samples spiked with PYY at concentrations fitting with normal levels in these biological fluids.

Electrochemical immunosensor for sensitive determination of the anorexigen peptide YY at grafted reduced graphene oxide electrode platforms.

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 10(2) 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.

Multiplexed determination of human growth hormone and prolactin at a label free electrochemical immunosensor using dual carbon nanotube-screen printed electrodes modified with gold and PEDOT nanoparticles.

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.

Biosensors in forensic analysis. A review.

Forensic analysis is an important branch of modern Analytical Chemistry with many legal and socially relevant implications. Biosensors can play an important role as efficient tools in this field considering their well known advantages of sensitivity, selectivity, easy functioning, affordability and capability of miniaturization and automation. This article reviews the latest advances in the use of biosensors for forensic analysis. The different methodologies for the transduction of the produced biological events are considered and the applications to forensic toxicological analysis, classified by the nature of the target analytes, as well as those related with chemical and biological weapons critically commented. The article provides several Tables where the more relevant analytical characteristics of the selected reported methods are gathered.

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.

Ultrasensitive determination of human growth hormone (hGH) with a disposable electrochemical magneto-immunosensor.

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. TsMBs­mAbhGH­hGH­pAbhGH­anti-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, 2009). 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.

Voltammetry and amperometric detection of tetracyclines at multi-wall carbon nanotube modified electrodes.

The voltammetric behaviour and amperometric detection of tetracycline (TC) antibiotics at multi-wall carbon nanotube modified glassy carbon electrodes (MWCNT-GCE) are reported. Cyclic voltammograms of TCs showed enhanced oxidation responses at the MWCNT-GCE with respect to the bare GCE, attributable to the increased active electrode surface area. Hydrodynamic voltammograms obtained by flow-injection with amperometric detection at the MWCNT-GCE led us to select a potential value E(det) = +1.20 V. The repeatability of the amperometric responses was much better than that achieved with bare GCE (RSD ranged from 7 to 12%), with RSD values for i (p) of around 3%, thus demonstrating the antifouling capability of MWCNT modified electrodes. An HPLC method with amperometric electrochemical detection (ED) at the MWCNT-GCE was developed for tetracycline, oxytetracycline (OTC), chlortetracycline and doxycycline (DC). A mobile phase consisting of 18:82 acetonitrile/0.05 mol L(-1) phosphate buffer of pH 2.5 was selected. The limits of detection ranged from 0.09 micromol L(-1) for OTC to 0.44 micromol L(-1) for DC. The possibility to carry out multiresidue analysis is demonstrated. The HPLC-ED/MWCNT-GCE method was applied to the analysis of fish farm pool water and underground well water samples spiked with the four TCs at 2.0 x 10(-7) mol L(-1). Solid-phase extraction was accomplished for the preconcentration of the analytes and clean-up of the samples. Recoveries ranged from 87 +/- 6 to 99 +/- 3%. Under preconcentration conditions, limits of detection in the water samples were between 0.50 and 3.10 ng mL(-1).

Electrochemical detection of phenolic estrogenic compounds at carbon nanotube-modified electrodes.

The use of a carbon nanotube-modified glassy carbon electrode (CNT-GCE) for the LC-EC detection of phenolic compounds with estrogenic activity is reported. Cyclic voltammograms for phenolic endocrine disruptors and estrogenic hormones showed, in general, an enhancement of their electrochemical oxidation responses at CNT-GCE attributable to the electrocatalytic effect caused by CNTs. Hydrodynamic voltammograms obtained under flow injection conditions lead to the selection of +700mV as the potential value to be applied for the amperometric detection of the phenolic estrogenic compounds, this value being remarkably less positive than those reported in the literature using other electrode materials. Successive injections of these compounds demonstrated that no electrode surface fouling occurred. A mobile phase consisting of a 50:50 (v/v) acetonitrile:0.05moll(-1) phosphate buffer of pH 7.0 was selected for the chromatographic separation of mixtures of these compounds, with detection limits ranging between 98 and 340nmoll(-1). Good recoveries were obtained in the analysis of underground well water and tap water samples spiked with some phenolic estrogenic compounds at a 14nmoll(-1) concentration level.

Electrochemical determination of homocysteine at a gold nanoparticle-modified electrode.

The construction of a colloidal gold-cysteamine-carbon paste electrode, Au(coll)-Cyst-CPE, for the electrochemical determination of homocysteine is reported. The improved voltammetric behaviour of homocysteine at Au(coll)-Cyst-CPE with respect to that observed at a gold disk electrode is attributed to an enhanced electron transfer kinetics as a consequence of the array distribution of gold nanoparticles immobilized onto the Cyst SAM. Cyclic voltammetry of homocysteine showed an adsorption-controlled current for scan rates between 500 and 5000 mV s(-1). The hydrodynamic voltammogram constructed for homocysteine allowed the selection of a potential value of +600 mV, where the background current is negligible, for the amperometric detection of the analyte at the Au(coll)-Cyst-CPE. Using a flow rate of 0.8 ml min(-1), the R.S.D. value for i(p) after 25 repetitive injections of homocysteine was of 4.3%, and one single electrode could be used for more than 15 days without any treatment or regeneration procedure of the modified electrode surface. An HPLC method for the separation and quantification of homocysteine and related thiols, using amperometric detection at the modified electrode has been developed. A mobile phase consisting of 2:98% (v/v) acetonitrile:0.05 mol l(-1) buffer solution of pH 2.0, and a detection potential of +0.80 V were selected. Separation with baseline resolution and retention times of 3.00, 3.60, 4.52, 5.71 and 7.79 min were obtained for cysteine, homocysteine, glutathione, penicillamine and N-acetyl-cysteine, respectively. Calibration graphs were constructed for all the separated compounds. Detection limits ranged between 20 nM for cysteine and 120 nM for penicillamine, with a value for homocysteine of 30 nM. These values compare advantageously with those achieved with previously reported HPLC methods using electrochemical, UV, fluorescence and MS detection modes. The developed method was applied to the determination of cysteine and homocysteine serum samples with good results.

Colloidal-gold cysteamine-modified carbon paste electrodes as suitable electrode materials for the electrochemical determination of sulphur-containing compounds Application to the determination of methionine.

The suitability of colloidal-gold cysteamine-modified carbon paste electrodes (nAu-Cyst-CPE) for the electrochemical determination of sulphur-containing compounds is illustrated in this work by determining the amino acid methionine in real samples, as well as a methionine-based peptide. Voltammograms from methionine solutions at nAu-Cyst-CPE exhibited improved electroanalytical characteristics when compared with colloidal-gold cysteamine-modified Au disk electrodes (nAu-Cyst-AuE). Differential pulse voltammetry in phosphate buffer of pH 7.0 was used for the determination of methionine, with a range of linearity of (1.0-100) x 10(-6)moll(-1) and a detection limit of 5.9 x 10(-7)moll(-1). This detection limit is remarkably lower than those reported previously using other modified electrodes or amperometric detection. Methionine peptides also exhibited anodic peaks suitable to detect this kind of molecules. Methionine was determined with good results in a pharmaceutical product containing several vitamins, amino acids and other compounds, and in spiked meat peptone, a complex sample containing enzimatically digested protein.

Molecularly imprinted polymers for on-line clean up and preconcentration of chloramphenicol prior to its voltammetric determination.

The performance of a molecularly imprinted polymer (MIP) as a selective solid-phase extraction sorbent for the clean-up and preconcentration of the antibiotic chloramphenicol is described. The MIP was prepared using chloramphenicol as the template, (diethylamino)ethyl methacrylate as the functional monomer, and ethylene glycol dimethacrylate as the cross-linking monomer, and using tetrahydrofuran as the solvent. Detection of chloramphenicol was carried out by square-wave voltammetry at electrochemically activated carbon fiber microelectrodes. Chloramphenicol was eluted from the MIP microcolumn with methanol. Different experimental variables (sample pH, eluent volume, analyte and eluent flow rates and sample volume) associated with the rebinding/elution process were optimized. For a 250 mL sample, a nominal enrichment factor of 500 was attained, and for a chloramphenicol concentration of 3.0x10(-8) mol L(-1) (9.7 microg L(-1)) a recovery of 96+/-4% was obtained. A range of linearity for chloramphenicol between 3.0x10(-8) and 1.0x10(-5) mol L(-1) was obtained by loading 17 mL of analyte solutions of different concentration, eluting with 0.5 mL methanol, evaporating under a stream of nitrogen and dissolving the residue in phosphate buffer of pH 7.8. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of chloramphenicol. The applicability of the MIP for both clean up and preconcentration was demonstrated by determining chloramphenicol in ophthalmic solutions and spiked milk at different concentration levels.