Electrochemical immunoplatform for the determination of multiple salivary biomarkers of oral diseases related to microbiome dysbiosis.

Several diseases of the oral cavity are related to compositional and functional shifts in the oral microbiome. The analysis of saliva is an attractive alternative for the diagnosis and prognosis of these diseases. Samples can be obtained by no invasive procedures and processing is relatively simple. However, sensitive and selective analytical methods are needed to make the diagnosis as specific as possible. In this work, four salivary biomarkers of oral diseases: interleukin-6 (IL-6), receptor activator of NF-kB ligand (RANKL), protein arginine deiminase 4 (PAD4) and the corresponding antibody (anti-PAD-4) were selected as targets for their simultaneous determination using an electrochemical immunosensing platform. Sandwich-type amperometric immunoassays were implemented using horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) for application to the analysis of saliva of six volunteers. The developed method provides excellent sensitivity, selectivity, and wide linear ranges with LOD values of 0.09 pg mL-1 (IL-6), 0.10 pg mL-1 (RANKL); 0.09 ng mL-1(PAD4) and 14.5 ng mL-1 (anti-PAD4) and allows the accurate analysis of saliva without matrix effects, using 25 µL of raw sample. The developed methodology is competitive with commercial ELISA kits available only for a single biomarker determination, while the assay for the four biomarkers can be completed in less than two hours.

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.

Carbon-Based Enzyme Mimetics for Electrochemical Biosensing.

Natural enzymes are used as special reagents for the preparation of electrochemical (bio)sensors due to their ability to catalyze processes, improving the selectivity of detection. However, some drawbacks, such as denaturation in harsh experimental conditions and their rapid de- gradation, as well as the high cost and difficulties in recycling them, restrict their practical applications. Nowadays, the use of artificial enzymes, mostly based on nanomaterials, mimicking the functions of natural products, has been growing. These so-called nanozymes present several advantages over natural enzymes, such as enhanced stability, low cost, easy production, and rapid activity. These outstanding features are responsible for their widespread use in areas such as catalysis, energy, imaging, sensing, or biomedicine. These materials can be divided into two main groups: metal and carbon-based nanozymes. The latter provides additional advantages compared to metal nanozymes, i.e., stable and tuneable activity and good biocompatibility, mimicking enzyme activities such as those of peroxidase, catalase, oxidase, superoxide dismutase, nuclease, or phosphatase. In this review article, we have focused on the use of carbon-based nanozymes for the preparation of electrochemical (bio)sensors. The main features of the most recent applications have been revised and illustrated with examples selected from the literature over the last four years (since 2020).

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.

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.

Enzymatic/Immunoassay Dual-Biomarker Sensing Chip: Towards Decentralized Insulin/Glucose Detection.

Performing bioassay formats based on enzyme and antibody recognition reactions with a single detection chip remains an unmet challenge owing to the different requirements of such bioassays. Herein, we describe a dual-marker biosensor chip, integrating enzyme and antibody-based assays for simultaneous electrochemical measurements of insulin (I) and glucose (G). Simultaneous G/I sensing has been realized by addressing key fabrication and operational challenges associated with the different assay requirements and surface chemistry. The I immunosensor relies on a peroxidase-labeled sandwich immunoassay, while G is monitored through reaction with glucose oxidase. The dual diabetes biomarker chip offers selective and reproducible detection of picomolar I and millimolar G concentrations in a single microliter sample droplet within less than 30 min, including direct measurements in whole blood and saliva samples. The resulting integrated enzymatic-immunoassay biosensor chip opens a new realm in point-of-care multiplexed biomarker detection.

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.