Label-free electrochemical immunosensing of glial fibrillary acidic protein (GFAP) at synthesized rGO/MoS2/AgNPs nanocomposite. Application to the determination in human cerebrospinal fluid.

An electrochemical bioplatform involving screen-printed carbon electrodes modified with rGO/MoS2/AgNPs nanocomposites, the covalent immobilization of the specific capture antibody, and label-free detection has been developed for the determination of Glial Fibrillary Acidic Protein (GFAP). The resulting immunosensor profits the benefits of the rGO high conductivity, the pseudo-peroxidase activity of MoS2 and the electrocatalytic effect provided by AgNPs for improving the reduction current responses of hydrogen peroxide at the electrode surface. GFAP is a biomarker of central nervous system injuries has been proposed for the detection and monitoring of neurological diseases as epilepsy, encephalitis, or multiple sclerosis. For the first time, amperometric detection of the immunosensing event was performed by measuring the electrocatalytic response of hydrogen peroxide reduction at the modified electrode. Several techniques including scanning (SEM) and transmission (TEM) electron microscopies were used for the characterization of the synthesized composite whilst electrochemical impedance spectroscopy (EIS) using the redox probe Fe(CN)63-/4- was employed to evaluate the success of the steps implied in the fabrication of the immunosensor. After optimization of the involved experimental variables, a linear calibration plot for GFAP was constructed over the 0.6-100 ng mL-1 range, and a detection limit of 0.16 ng mL-1 was achieved. The developed immunosensor was successfully applied to the determination of GFAP in human cerebrospinal fluid (CSF) of patients diagnosed with encephalitis.

Serum Autoantibody Biomarkers for Management of Rheumatoid Arthritis Disease.

Rheumatoid arthritis (RA) is a systemic chronic autoimmune inflammatory disease that is characterized by the destruction of bone and production of autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPAs). The high prevalence of this disease and the need of affordable tools for its early detection led us to prepare the first electrochemical immunoplatform for the simultaneous determination of four RA biomarkers, the autoantibodies: RF, anti-peptidyl-arginine deiminase enzyme (anti-PAD4), anti-cyclic citrullinated peptide (anti-CCP), and anti-citrullinated vimentin (anti-MCV). Functionalized magnetic beads (MBs) were used to immobilize the specific antigens, and sandwich-type immunoassays were implemented for the amperometric detection of the four autoantibodies, using the horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) system. The immunoplatform was applied to the determination of the biomarkers in human serum of twenty-two patients diagnosed with RA and four healthy individuals, and the results were validated against ELISA tests and the certified values.

Electrochemical (Bio)Sensing Devices for Human-Microbiome-Related Biomarkers.

The study of the human microbiome is a multidisciplinary area ranging from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers to improve the diagnosis and monitoring of diseases (e.g., cancer), health conditions (e.g., obesity) or relevant processes (e.g., aging) has raised great expectations, also in the field of bioelectroanalytical chemistry. The well-known advantages of electrochemical biosensors-high sensitivity, fast response, and the possibility of miniaturization, together with the potential for new nanomaterials to improve their design and performance-position them as unique tools to provide a better understanding of the entities of the human microbiome and raise the prospect of huge and important developments in the coming years. This review article compiles recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and disease biomarkers related to different types of human microbiome, with a special focus on the gastrointestinal microbiome. Examples of electrochemical devices applied to real samples are critically discussed, as well as challenges to be faced and where future developments are expected to go.

Development of an Electrochemical CCL5 Chemokine Immunoplatform for Rapid Diagnosis of Multiple Sclerosis.

Serum level of CCL5 chemokine is considered an emerging biomarker for multiple sclerosis (MS). Due to the lack of specific assays for this disease, the development of a point-of-care test for rapid detection of MS could lead to avoiding diagnostics delays. In this paper, we report the first electrochemical immunoplatform for quantification of the CCL5 biomarker at the clinically required levels, able to discriminate between patients diagnosed with MS and healthy individuals. The immunosensing device involves protein capture from biological samples by complexation with biotinylated specific antibodies immobilized onto neutravidin-functionalized microparticles and sandwich assay with anti-CCL5 antibody and IgG labelled with horseradish peroxidase (HRP) for the enzyme-catalyzed amperometric detection of H2O2 using hydroquinone (HQ) as the redox mediator. The method shows excellent analytical performance for clinical application with a wide linear range of concentrations (0.1-300 ng·mL-1 CCL5, R2 = 0.998) and a low detection limit (40 pg·mL-1 CCL5). The biosensing platform was applied to the determination of the CCL5 endogenous content in 100-fold diluted sera both from healthy individuals and patients diagnosed with MS, with no further sample treatment in just two hours. The results were successfully compared with those obtained by the ELISA methodology.

Multimodal/Multifunctional Nanomaterials in (Bio)electrochemistry: Now and in the Coming Decade.

Multifunctional nanomaterials, defined as those able to achieve a combined effect or more than one function through their multiple functionalization or combination with other materials, are gaining increasing attention in the last years in many relevant fields, including cargo targeted delivery, tissue engineering, in vitro and/or in vivo diseases imaging and therapy, as well as in the development of electrochemical (bio)sensors and (bio)sensing strategies with improved performance. This review article aims to provide an updated overview of the important advances and future opportunities exhibited by electrochemical biosensing in connection to multifunctional nanomaterials. Accordingly, representative aspects of recent approaches involving metal, carbon, and silica-based multifunctional nanomaterials are selected and critically discussed, as they are the most widely used multifunctional nanomaterials imparting unique capabilities in (bio)electroanalysis. A brief overview of the main remaining challenges and future perspectives in the field is also provided.

TGF-ß-induced IGFBP-3 is a key paracrine factor from activated pericytes that promotes colorectal cancer cell migration and invasion.

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-ß receptor activation. The prognostic value of a TGF-ß response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-ß1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.

Electrochemical biosensor for the simultaneous determination of rheumatoid factor and anti-cyclic citrullinated peptide antibodies in human serum.

This paper reports a dual electrochemical biosensor involving carboxylated- or neutravidin-functionalized magnetic microbeads and dual screen-printed carbon electrodes for the simultaneous determination of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCPA) autoantibodies used as biomarkers for the detection of rheumatoid arthritis autoimmune disease. Sandwich-type biosensors involving Fc fragments of IgG Fc(IgG) and biotinylated cyclic cytrullinated peptide (CCP-biotin) to form CCP-biotin-Neutr-MBs for the specific immobilization of RF and CCPA, respectively, as well as conjugation with HRP-IgM and HRP-IgG for RF and CCPA, respectively, were prepared. Amperometric detection was performed at -0.20 V vs. Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system upon capturing the bioconjugates onto the corresponding working electrode (WE1 or WE2) of SPCdEs. The dual biosensor exhibits high sensitivity for RF and CCPA with LOD values of 0.8 and 2.5 IU mL-1, respectively. The simultaneous determination can be completed in about two hours using a simple protocol and a sample volume (25 µL) four times smaller than that required by the ELISA method. The dual electrochemical biosensor was used for the determination of both target biomarkers in human serum.

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole.

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H2O2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at -200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL-1 to 50 pg∙mL-1, with a detection limit of 0.92 fg∙mL-1. This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents. Graphical abstract Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

Magnetic multiwalled carbon nanotubes as nanocarrier tags for sensitive determination of fetuin in saliva.

This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label. The determination of HFA was accomplished by measuring the current produced by the electrochemical reduction of benzoquinone at -200 mV upon addition of H2O2 as HRP substrate. The prepared m-MWCNTs were characterized by SEM, TEM, XRD and EDS. All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy and cyclic voltammetry. A linear calibration plot for HFA was found between 20 and 2000 pg/mL with a LOD value of 16 pg/mL. This performance is notably better than that reported for an ELISA kit and a chronoimpedimetric immunosensor. The favorable contribution of m-MWCNTs in comparison with MWCNTs without incorporated magnetic particles to this excellent analytical performance is also highlighted. The immunosensor selectivity against other proteins and potentially interfering compounds was excellent. In addition, the usefulness of the immunosensor was demonstrated by the analysis of HFA in saliva with minimal sample treatment.

Viologen-functionalized single-walled carbon nanotubes as carrier nanotags for electrochemical immunosensing. Application to TGF-ß1 cytokine.

Viologen-SWCNT hybrids are synthesized by aryl-diazonium chemistry in the presence of isoamyl nitrite followed by condensation reaction of the resulting HOOC-Phe-SWCNT with 1-(3-aminoethyl)-4,4'-bipyridinium bromine and N-alkylation with 2-bromoethylamine. The V-Phe-SWCNT hybrids were characterized by using different spectroscopic techniques (FT-IR, Raman, UV-vis), TGA and Kaiser test. Viologen-SWCNTs were used for the preparation of an electrochemical immunosensor for the determination of the transforming growth factor ß1 (TGF-ß1) cytokine considered as a reliable biomarker in several human diseases. The methodology involved preparation of V-Phe-SWCNT(-HRP)-anti-TGF conjugates by covalent linkage of HRP and anti-TGF onto V-Phe-SWCNT hybrids. Biotinylated anti-TGF antibodies were immobilized onto 4-carboxyphenyl-functionalized SPCEs modified with streptavidin and a sandwich type immunoassay was implemented for TGF-ß1 with signal amplification using V-Phe-SWCNT(-HRP)-anti-TGF conjugates as carrier tags. The analytical characteristics exhibited by the as prepared immunosensor (range of linearity between 2.5 and 1000pgmL-1 TGF-ß1; detection limit of 0.95pgmL-1) improve notably those reported with other previous immunosensors or ELISA kits. A great selectivity against other proteins was also found. The prepared immunosensor was validated by determining TGF-ß1 in real saliva samples. Minimal sample treatment was required and the obtained results were in excellent agreement with those obtained by using a commercial ELISA kit.

Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer: Application to the determination of adiponectin cytokine in serum.

An electrochemical immunosensor for adiponectin (APN) using screen printed carbon electrodes (SPCEs) modified with functionalized double-walled carbon nanotubes (DWCNTs) as platforms for immobilization of the specific antibodies is reported. DWCNTs were functionalized by treatment with 4-aminobenzoic acid (HOOC-Phe) in the presence of isoamylnitrite resulting in the formation of 4-carboxyphenyl-DWCNTs. The oriented binding of specific antibodies toward adiponectin was accomplished by using the metallic-complex chelating polymer Mix&Go™. The HOOC-Phe-DWCNTs-modified SPCEs were characterized by cyclic voltammetry and compared with HOOC-Phe-SWCNTs/SPCE. The different variables affecting the performance of the developed immunosensor were optimized. Under the selected conditions, a calibration plot for APN was constructed showing a range of linearity extending between 0.05 and 10.0 µg/mL which is adequate for the determination of the cytokine in real samples. A detection limit of 14.5 ng/mL was achieved. The so prepared immunosensor exhibited a good reproducibility for the APN measurements, excellent storage stability and selectivity, and a much shorter assay time than the available ELISA kits. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing human serum from female or male healthy patients.

A disposable electrochemical immunosensor for the determination of leptin in serum and breast milk.

The preparation of a disposable electrochemical immunosensor for the quantification of the hormone leptin is described in this work. The preparation approach involved immobilization of a specific biotinylated anti-leptin antibody on the surface of streptavidin-functionalized magnetic beads (Strept-MBs) and a sandwich-type immunoassay involving the target analyte, monoclonal anti-leptin, and IgG labeled with alkaline phosphatase (AP-IgG). The electrochemical transduction step was accomplished by trapping the MBs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by means of an Nd magnet and measuring the electrochemical oxidation of the 1-naphthol generated in the AP enzyme reaction upon 1-naphthyl phosphate (1-NPP) additions by differential pulse voltammetry (DPV). A calibration plot with a linear range between 5 and 100 pg mL(-1) as well as a detection limit of 0.5 pg mL(-1) (3sb/m) were achieved. This value is more than 27 times lower than that reported in the only voltammetric immunosensor for leptin described in the literature until now. The usefulness of the immunosensor was demonstrated by analyzing different types of real samples: human serum, infant powdered milk, and breast milk from a nursing mother with two months of breastfeeding.

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors.

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.

A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles.

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.

An electrochemical immunosensor for testosterone using functionalized magnetic beads and screen-printed carbon electrodes.

A disposable electrochemical immunosensor using screen-printed carbon electrodes (SPCEs) and protein A-functionalized magnetic beads (MBs) was developed for the determination of testosterone. Anti-testosterone was immobilized onto MBs and a direct competitive immunoassay involving testosterone labeled with peroxidase (HRP) was performed. The resulting conjugate was trapped on the SPCE with a small magnet. Testosterone determination was carried out by amperometry at -0.2V upon H2O2 additions using hydroquinone (HQ) as the redox mediator. The experimental variables involved in the immunosensor response to testosterone were evaluated. Under the optimized conditions, a calibration plot for testosterone was obtained with a linear range between 5.0×10(-3) and 50 ng/mL (r=0.995). The detection limit was 1.7 pg/mL and the EC50 was 0.25±0.04 ng/mL. These characteristics are notably better than those achieved with other reported immunosensors. Furthermore, anti-testosterone/MBs conjugates were shown to be stable for at least 25 days. A good selectivity was also found against other steroid hormones. The usefulness of the immunosensor was demonstrated by analyzing human serum spiked with 1 and 10 ng/mL testosterone.

Disposable immunosensor for cortisol using functionalized magnetic particles.

A novel electrochemical immunosensor using screen-printed carbon electrodes and functionalized magnetic particles was developed for the determination of cortisol. Anti-cortisol antibody was immobilized onto protein A-modified magnetic particles and a direct competitive immunoassay involving cortisol antigen labeled with alkaline phosphatase (AP) was employed. The resulting conjugate was trapped on the surface of the screen-printed electrode with a small magnet. Cortisol quantification was accomplished by using 1-naphthyl phosphate as AP substrate and differential pulse voltammetry for the detection of the formed 1-naphthol. The variables involved in the preparation of the immunosensor and in the electrochemical detection reaction were optimized. The calibration plot obtained for cortisol exhibited a linear range between 5.0 x 10(-3) and 150 ng mL(-1) (r = 0.993). The limit of detection was 3.5 pg mL(-1), and the EC(50) was 0.19 ng mL(-1). These results demonstrate the high sensitivity achieved with this immunosensor design which compares favourably with other previous reports. The immunosensor also exhibited a high selectivity with respect to other corticosteroid compounds closely related to cortisol. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing certified human sera containing cortisol at two different concentration levels.

Nanostructured progesterone immunosensor using a tyrosinase-colloidal gold-graphite-Teflon biosensor as amperometric transducer.

A novel progesterone immunosensor using a colloidal gold-graphite-Teflon-tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at -0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL(-1) with a slope value of -82.3 nA ng(-1) mL, and a detection limit of 0.43 ng mL(-1). The time needed to reach the steady-state current from the addition of phenyl phosphate was 30-40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL(-1) concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries (n=7) of 98+/-3% and 99+/-3%, respectively, were obtained.