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-1β (IL-1β) and factor necrosis tumor α (TNF-α). IL-1β. 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 HO 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-1β 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-1β) 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-1β and TNF-α in human serum spiked at clinically relevant concentration levels and in real saliva samples.

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The first electrochemical immunosensor for TGF-β1 cytokine in human serum based on carbon nanotubes functionalized by click chemistry is reported.

The first amperometric immunosensor for the quantification of TGF-β1, 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-β1 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-β1 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-β1 concentrations in real samples was evaluated by analyzing spiked urine at different pg/mL concentration levels.

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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-β receptor activation. The prognostic value of a TGF-β 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-β1, 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.

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 β1 (TGF-β1) 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-β1 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 TGF-β1; detection limit of 0.95pgmL) 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-β1 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.

The first electrochemical immunosensor for the determination of 8-isoprostane (8-iso prostaglandin F2α, ISO), one of the most reliable biomarkers of lipid peroxidation in the human body and of aging related to Alzheimer´s disease or atherosclerosis is reported in this article. Disposable screen-printed carbon electrodes modified with carboxylated carbon nanohorns (CNHs) were employed as scaffolds for covalent immobilization of a specific anti-ISO antibody). A competitive immunoassay involving ISO and HRPlabeled antigen was designed and the determination of ISO was carried out by amperometry at-200 mV using the H 2 O 2 /hydroquinone (HQ) system. Under the optimized conditions, the immunosensor provides a linear response for ISO (r 2 = 0.998) extending up to 700 pg/mL, which is suitable for the determination of the target compound in human serum. The analytical performance of the immunosensor improves that claimed for ELISA kits in terms of linearity of the calibration plot, precision, with RSD values lower than 1 % , and assay time (1h 30min), and exhibits a low limit of detection, 12 pg/mL, a long storage stability (30 days), and an excellent selectivity against other proteins that may be found in human serum. The analytical utility of the developed immunosensor was demonstrated by determining ISO in two types of human serum samples: lyophilized spiked serum, and real human serum from healthy male and female individuals with good results.