Simultaneous electrochemical immunosensing of relevant cytokines to diagnose and track cancer and autoimmune diseases.

This work reports the first dual magnetic beads (MBs) assisted immunoplatform for the simultaneous determination of BAFF (B cell activation factor) and APRIL (a proliferation-induced ligand), two cytokines related to immunity and tumour invasion, growth and metastasis. The electrochemical immunoplatform involves sandwich-type immunoassays implemented on magnetic microparticles functionalized with neutravidin (NA-MBs) or carboxylic groups (HOOC-MBs), and amperometric detection (Eapp =  - 0.20 V vs. Ag pseudo-reference electrode) at screen-printed dual carbon electrodes (SPdCE) using the H2O2/hydroquinone (HQ) system. The developed immunosensors provide improved sensitivity, with LOD values of 0.33 and 16.4 pg mL-1 for BAFF and APRIL, respectively, and much shorter assay time that those claimed for ELISA kits and allow their simultaneous determination. The dual immunosensor permits discrimination between healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) or colorectal cancer (CRC) through the determination of both cytokines in 100-times diluted human sera with results in agreement with those provided by the individual ELISA methodologies.

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.

First electrochemical immunosensor for the rapid detection of mustard seeds in plant food extracts.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL-1 (20.5 pg of protein in 25 µL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

Enlightening the advancements in electrochemical bioanalysis for the diagnosis of Alzheimer's disease and other neurodegenerative disorders.

Neurodegenerative disorders (NDD), and particularly Alzheimer's disease (AD), are one of the greatest challenges facing our current medicine and society because of its increasing incidence and the high burden imposed both on patients' families and health systems. Despite this, their accurate diagnosis, mostly conducted by cerebrospinal fluid (CSF) analysis or neuroimaging techniques, costly, time-consuming, and unaffordable for most of the population, remains a complex task. In this situation, electrochemical biosensors are flourishing as promising alternative tools for the simple, fast, and low-cost diagnosis of NDD/AD. This review article provides the relevant clinical details of NDD/AD along with the closely related genetic (genetic mutations, polymorphisms of ApoE and specific miRNAs) and proteomic (amyloid-ß peptides, total and phosphorylated tau protein) biomarkers circulating mostly in CSF. In addition, the article systematically enlightens a general view of the electrochemical affinity biosensors (mostly aptasensors and immunosensors) reported in the past two years for the determination of such biomarkers. The different developed strategies, analytical performances and applications are comprehensively discussed. Recent advancements in signal amplification methodologies involving smart designs and the use of nanomaterials and rational surface chemistries, as well as the challenges that must be struggled and the prospects in electrochemical affinity biosensing to bring more accessibility to NDD/AD diagnosis, prognosis, and follow-up, are also pointed out.

Electrochemical biosensing to move forward in cancer epigenetics and metastasis: A review.

Early detection and effective treatment are crucial to reduce the physical, emotional, and financial pressure exerted by growing cancer burden on individuals, families, communities, and health systems. Currently, it is clear that the accurate analysis of emerging cancer epigenetic and metastatic-related biomarkers at different molecular levels is envisaged as an exceptional solution for early and reliable diagnosis and the improvement of therapy efficiency through personalized treatments. Within this field, electrochemical biosensing has demonstrated to be competitive over other emerging and currently used methodologies for the determination of these biomarkers accomplishing the premises of user-friendly, multiplexing ability, simplicity, reduced costs and decentralized analysis, demanded by clinical oncology, thus priming electrochemical biosensors to spark a diagnostic revolution for cancer prediction and eradication. This review article critically discusses the main characteristics, opportunities and versatility exhibited by electrochemical biosensing, through highlighting representative examples published during the last two years, for the reliable determination of these emerging biomarkers, with great diagnostic, predictive and prognostic potential. Special attention is paid on electrochemical affinity biosensors developed for the single or multiplexed determination of methylation events, non-coding RNAs, ctDNA features and metastasis-related protein biomarkers both in liquid and solid biopsies of cancer patients. The main challenges to which further work must be addressed and the impact of these advances should have in the clinical acceptance of these emerging biomarkers are also discussed which decisively will contribute to understand the molecular basis involved in the epigenetics and metastasis of cancer and to apply more efficient personalized therapies.

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.

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.

Copper(I)-Catalyzed Click Chemistry as a Tool for the Functionalization of Nanomaterials and the Preparation of Electrochemical (Bio)Sensors.

Proper functionalization of electrode surfaces and/or nanomaterials plays a crucial role in the preparation of electrochemical (bio)sensors and their resulting performance. In this context, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been demonstrated to be a powerful strategy due to the high yields achieved, absence of by-products and moderate conditions required both in aqueous medium and under physiological conditions. This particular chemistry offers great potential to functionalize a wide variety of electrode surfaces, nanomaterials, metallophthalocyanines (MPcs) and polymers, thus providing electrochemical platforms with improved electrocatalytic ability and allowing the stable, reproducible and functional integration of a wide range of nanomaterials and/or different biomolecules (enzymes, antibodies, nucleic acids and peptides). Considering the rapid progress in the field, and the potential of this technology, this review paper outlines the unique features imparted by this particular reaction in the development of electrochemical sensors through the discussion of representative examples of the methods mainly reported over the last five years. Special attention has been paid to electrochemical (bio)sensors prepared using nanomaterials and applied to the determination of relevant analytes at different molecular levels. Current challenges and future directions in this field are also briefly pointed out.

Pushing the limits of electrochemistry toward challenging applications in clinical diagnosis, prognosis, and therapeutic action.

Constant progress in the identification of biomarkers at different molecular levels in samples of different natures, and the need to conduct routine analyses, even in limited-resource settings involving simple and short protocols, are examples of the growing current clinical demands not satisfied by conventional available techniques. In this context, the unique features offered by electrochemical biosensors, including affordability, real-time and reagentless monitoring, simple handling and portability, and versatility, make them especially interesting for adaptation to the increasingly challenging requirements of current clinical and point-of-care (POC) diagnostics. This has allowed the continuous development of strategies with improved performance in the clinical field that were unthinkable just a few years ago. After a brief introduction to the types and characteristics of clinically relevant biomarkers/samples, requirements for their analysis, and currently available methodologies, this review article provides a critical discussion of the most important developments and relevant applications involving electrochemical biosensors reported in the last five years in response to the demands of current diagnostic, prognostic, and therapeutic actions related to high prevalence and high mortality diseases and disorders. Special attention is paid to the rational design of surface chemistry and the use/modification of state-of-the-art nanomaterials to construct electrochemical bioscaffolds with antifouling properties that can be applied to the single or multiplex determination of biomarkers of accepted or emerging clinical relevance in particularly complex clinical samples, such as undiluted liquid biopsies, whole cells, and paraffin-embedded tissues, which have scarcely been explored using conventional techniques or electrochemical biosensing. Key points guiding future development, challenges to be addressed to further push the limits of electrochemical biosensors towards new challenging applications, and their introduction to the market are also discussed.

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.

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.

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.

Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level.

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion (catalytic, magnetic, acoustic or biological) and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes (drugs, functional proteins, oligonucleotides and cells) for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed.

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.

Hybrid 2D-nanomaterials-based electrochemical immunosensing strategies for clinical biomarkers determination.

Owing to the outstanding conductivity and biocompatibility as well as numerous other fascinating properties of two-dimensional (2D)-nanomaterials, 2D-based nanohybrids have shown unparalleled superiorities in the field of electrochemical biosensors. This review highlights latest advances in electrochemical immunosensors for clinical biomarkers based on different hybrid 2D-nanomaterials. Particular attention will be given to hybrid nanostructures involving graphene and other graphene-like 2D-layered nanomaterials (GLNs). Several recent strategies for using such 2D-nanomaterial heterostructures in the development of modern immunosensors, both for tagging or modifying electrode transducers, are summarized and discussed. These hybrid nanocomposites, quite superior than their rival materials, will undoubtedly have an important impact within the near future and not only in clinical areas. Current challenges and future perspectives in this rapidly growing field are also outlined.

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.

Competitive RNA-RNA hybridization-based integrated nanostructured-disposable electrode for highly sensitive determination of miRNAs in cancer cells.

A new method for the detection of miRNAs making use of a competitive RNA/RNA hybridization configuration is described in this work. A biotinylated miRNA (biotin-miRNA) of identical sequence to that of the target miRNA is mixed with the samples to be analyzed allowing competition to be accomplished with the target miRNA for a thiolated RNA probe assembled onto a gold nanoparticles (AuNPs) modified screen-printed electrode. After labeling the hybridized biotin-miRNA with streptavidin-HRP conjugates, amperometric detection at -0.20V was carried out using the H2O2/hydroquinone (HQ) system. The decrease in the amperometric response was proportional to the concentration of model target miRNA-21 in the 100 fM to 25.0 pM range. The integrated sensor provided a very low detection limit (25 fM, 0.25 attomol in 10µL sample) for miRNA-21 without any amplification step, a complete discrimination against single nucleotide mismatched sequences under practical conditions and high storage stability. The usefulness of the developed method was demonstrated by determining the endogenous levels of the mature target miRNA in total RNA (RNAt) extracted from cancerous and non-cancerous cells.

Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification.

A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0nM with a 60pM (1.5fmol in 25µL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1h 45min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.

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.