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Development and comparison of the first electrochemical bioplatforms for determining anti-centromere B antibodies (CENPB-Abs) developed in magnetic microbead-assisted or integrated formats using His-tag chemistry.

First electrochemical bioplatforms described for the single and simultaneous determination of two immunoglobulin (Ig) subtypes, IgE and IgG4 specific to ovalbumin (OVA), considered as reliable markers for the diagnosis and monitoring of egg allergy.

The study of the human microbiome is a multidisciplinary area that affects from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers for the improvement in the diagnosis and monitoring of diseases (e.g. cancer), health conditions (e.g. obesity) or clinical parameters (e.g. ageing) has aroused great expectations, also in the scope of bio-electroanalytical chemistry. The well-known advantages of electrochemical transduction: high sensitivity, rapid response and possibility of miniaturization, together with the potential of new nanomaterials to improve both the design of electrode platforms and the performance of the resulting electrochemical biosensors make presumably a huge development in the next few years providing a better understanding of human microbiome entities. In this review article, the recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and biomarkers of diseases related to different types of the human microbiome, with a special focus on the gastrointestinal microbiome, are reviewed. Examples of electrochemical devices applied to real samples are critically discussed as well as the challenges and prospects to be faced.

20 p.-9 fig.-2 tab., Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The 5-year survival rate of CRC patients depends on the stage at diagnosis, being higher than 80% when CRC is diagnosed in the early stages but lower than 10% when CRC is diagnosed in advanced stages. Autoantibodies against specific CRC autoantigens (tumor-associated antigens (TAAs)) in the sera of patients have been widely demonstrated to aid in early diagnosis. Thus, we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy. To that end, we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis. A total of 103 proteins were identified as potential autoantigens specific to CRC. After bioinformatics and meta-analysis, we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot (WB) and immunohistochemistry (IHC). We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients, along with an association of nine of these proteins with CRC prognosis. After validation, all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals, either by luminescence Halotag-based beads, or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection. Taken together, our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases; they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care (POC) device for CRC detection with high diagnostic ability., This work was supported by the financial support of the PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program to R.B. The financial support of the PID2019-103899RB-I00 (Ministerio de Ciencia e Innovación) Research Project and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (S2018/NMT-4349) to S.C. are gratefully acknowledged. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. The FPU predoctoral contract to A.M-C. is supported by the Spanish Ministerio de Educación, Cultura y Deporte. G.S-F. is recipient of a predoctoral contract (1193818N) supported by The Flanders Research Foundation (FWO). M.A-N. was supported by a contract of the Programa Operativo de Empleo Juvenily la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. The predoctoral contract from the Spanish Ministerio de Economía y Competitividad (BES-2016-076606, E.P.) and Talento-Contract from Comunidad de Madrid (2019-T2/IND-15965, R.M.T-R.) are also gratefully acknowledged.

The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Sí

An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)-specific phage-derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100 % obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD-diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients’ monitoring follow-up currently pursued. The financial support of RTI2018-096135-B−I00 (Spanish Ministerio de Ciencia, Innovación y Universidades) and PID2019-103899RB−I00 (Spanish Ministerio de Ciencia e Innovación), Research Projects, PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. A. Montero-Calle and P. San Segundo-Acosta acknowledge the support of the FPU predoctoral contracts by the Spanish Ministerio de Educación, Cultura y Deporte. A. Valverde acknowledges a predoctoral contract from Complutense University of Madrid. B. Arévalo acknowledges a predoctoral contract from the Spanish Ministerio de Economía y Competitividad (PRE2019-087596). G.S-F. is a recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). Sí

Potassium ion channels are expressed on the cell membranes, implicated in wide variety of cell functions and intimately linked to cancer cell behaviors. This work reports the first bioplatform described to date allowing simple and rapid detection of ion channel activity and the effect of their inhibitors in cancer cells. The methodology involves interrogation of the channel of interest from cells specifically captured on magnetic immunoconjugates using specific detection antibodies that are labeled with horseradish peroxidase enzyme. The channel activity is reflected by an amperometric signal transduction of the resulting magnetic bioconjugates onto screen-printed carbon electrodes. The bioplatform feasibility was proven for the detection of the Kv channels in U87 human glioblastoma cells and their blocking by scorpion venom KAaH1 and KAaH2 peptides. The obtained results confirm the high sensitivity (detection of 5 U87 cells⋅mL

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at − 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL−1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 μL plasma and 2.5 μg brain tissue extracts).

Due to their widespread use in veterinary medicine, the presence of tetracyclines (TCs) and their metabolites in foodstuffs from treated animal represents a growing concern for human health and nutrition and, therefore, many countries have established maximum levels for their presence in foods. The compliance with these legislations requires sensitive and selective methods for the determination of this antibiotics’ family in different matrices. In this sense, electrochemical sensors and biosensors are competitive methodologies versus other approaches commonly used for this purpose such as chromatography and ELISA methods, mainly in terms of low-cost equipment, minimal sample treatment, reduced turnaround time, and compatibility with multiplexed and point-of-care determinations. With this background in mind, this article reviews in a general but comprehensive way recent contributions of electrochemical (bio)sensors developed for the determination of TCs and applied to TC analysis in environmental samples, food, and biological matrices. The highlighted representative methods show the key role played both by the materials (mostly nanomaterials and polymers) employed to impart surface electrocatalytic properties and significant signal amplification, and by different (bio)receptors to provide electrochemical sensing with the sensitivity and selectivity demanded by the determination of TCs in environmental, clinical, and food samples. Main challenges to overcome and future prospects to turn over all the benefits of these simple, rapid, sensitive, selective, and cost effective electroanalytical (bio)tools in single or multiplexed TCs analysis, even at decentralized settings and after minimal sample treatment, are also pointed out.

Recommendations are given concerning the terminology of methods used in electroanalytical chemistry. Fundamental terms in electrochemistry are reproduced from previous PAC Recommendations, and new and updated material is added for terms in electroanalytical chemistry, classification of electrode systems, and electroanalytical techniques.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS2/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H2O2 system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Financial support was provided by Spanish Ministerio de Ciencia, Innovacion y Universidades (RTI2018-096,135-B-I00), Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00) Research Project, and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Financial support of the PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program was also provided to R. B. A predoctoral contract from the Spanish Ministerio de Ciencia, Innovacion y Universidades (PRE2019-087,596) was provided to B. Arévalo. A FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte was provided to A. M.-C. G. S.-F. is recipient of a predoctoral contract (grant number 1193818 N) supported by The Flanders Research Foundation (FWO). Sí

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle’s colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 μmol analyte per μmol antibody, corresponding to 139 μmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL− 1 , while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL− 1 . In addition, polyaldehydedextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors.

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories. The financial support of PID2019-103899RB-I00 and PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects, PI17CIII/00045 and PI20CIII/00019 Grants from the AESISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. B.A. acknowledges predoctoral contracts from the Spanish Ministerio de Ciencia, Innovación y Universidades (PRE2019-087596). M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. Sí

A disposable electrochemical PCR-free biosensor for the selective detection of a fragment encoding the protein Sin a 1, a 2S albumin considered a diagnostic marker for sensitization to mustard, is reported. The methodology is based on the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sin a 1 allergen coding sequence with appropriately designed RNA probes. Labeling with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with horseradish peroxidase (HRP) was carried out onto the surface of magnetic beads (MBs). Amperometric transduction was undertaken on screen-printed electrodes using H2O2 as enzyme substrate and hydroquinone (HQ) a redox mediator. The electrochemical biosensor allows the simple and fast detection (75 min) of Sin a 1 reaching a limit of detection of 3 pM. The bioplatform was successfully applied to the analysis of the targeted Sin a 1 gene specific region using just 50 ng of non-fragmented denatured genomic DNA extracted from yellow mustard seeds.

Pancreatic ductal adenocarcinoma (PDAC) is the solid tumor with the worst prognosis, representing today the third cause of cancer-related deaths in developed countries and expected to be the second in 2030. Today, CA19-9 remains the only clinically used marker for management of PDAC (FDA-approved as a disease moni- toring marker). This work reports a disposable amperometric immunoplatform for the determination of CA19-9. The immunoplatform skilfully combines the advantages of magnetic microsupports (MBs) for implementation of the immunoassay and amperometric transduction on screen-printed carbon electrodes (SPCEs). The method involves the preparation of sándwich immunocomplexes enzymatically labeled with the enzyme horseradish peroxidase (HRP) on the MBs and uses a detection antibody conjugated to HRP. Once the HRP- labeled sandwich immunocomplexes-bearing MBs were trapped on the SPCE surface, the variation of the catho- dic current was measured in the presence of H2O2 and hydroquinone (HQ), which was directly proportional to the concentration of CA19-9. Under the optimized experimental conditions, the immunoplatform allowed the amperometric determination of CA19-9 standards over the 5.0 to 500 U mL−1 concentration range, with a limit of detection (LOD) value of 1.5 U mL−1 in 1 h. The method exhibits good reproducibility and selectivity and the magnetic immunoconjugates shows a good storage stability. The immunoplatform was applied to the deter- mination of CA19-9 in serum samples of a medium-sized cohort (22 individuals) of healthy subjects and patients diagnosed with PDAC. The obtained results demonstrated the immunoplatform ability to discriminateboth types of individuals within 1 h after sample dilution. The developed immunoplatform represents an improvement in terms of cost, applicability and accessibility compared to the ELISA-based techniques currently used in the clinic.

Glial fibrillary acidic protein (GFAP) is a member of the intermediate filament family of proteins with increased levels in serum and cerebrospinal fluid of patients with Alzheimer disease (AD) and other neurodegenerative diseases (NDs), such as vascular dementia (VD). This work describes the first magnetic microbeads (MBs)-based electrochemical immunoplatform for GFAP determination. The platform design comprises a sandwich immunoassay implemented on the MBs surface and amperometric transduction at single-use screen-printed carbon electrodes (SPCEs). Micro-sized carboxylic acid magnetic particles (COOH-MBs) were modified with a specific capture antibody (CAb) to selectively link the target protein, which was sandwiched with a biotinylated detector antibody (btn-DAb) further conjugated with a streptavidin-peroxidase (Strep-HRP) conjugate. Amperometric transduction was performed at SPCEs upon capturing the magnetic bioconjugates on their surface and through the hydrogen peroxide/hydroquinone (H2O2/HQ) system. The immunoplatform achieved a limit of detection of 67 pg mL(-1) for the amperometric detection of standards and selectivity compatible with clinical applicability to assist in minimally invasive NDs diagnosis and prognosis. The MBs-based immunoplatform was applied with good results to determine the endogenous content of GFAP in protein brain extracts without matrix effect and using just 6.25 ng of sample per determination. Furthermore, the developed methodology was capable of differentiating between healthy subjects and patients diagnosed with VD and AD in only 2 h, providing accurate results in line with those obtained by an ELISA kit that used the same immunoreagents.

The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) is grate fully acknowledged. R.B. acknowledges the financial support of the PI20CIII/00019 grant from the AES-ISCIII program. R.M.T.-R. acknowledges Talento-Contract from Comunidad de Madrid (2019-T2/IND-15965). A.M.-C. ac knowledges a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. V.M. acknowledges the financial support of the COV20_00679 grant of the COVID-19 Research Call (ISCIII-Spanish Ministry of Science and Innovation). Sí

This work reports the first electrochemical bioplatform for the determination of soy traces in food. The bioplatform involves sandwich-type immunoassays using specific antibodies for β-conglycinin and glycinin, which are the main allergenic soy proteins, and carboxylic acid-modified magnetic microbeads. Amperometric detection at −0.20 V (vs. an Ag pseudo-reference electrode) was performed using single or dual screen-printed carbon electrodes and the H2O2/hydroquinone (HQ) system. The measured variation in the cathodic current was directly proportional to the concentration of target allergenic proteins. The developed bioplatforms exhibit a good selectivity and sensitivity providing limits of detection (LOD) values of 0.03 and 0.02 ng mL−1 for β-conglycinin and glycinin, respectively. The determination of both proteins can be carried out in only 1.5 h. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. The results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005 ppm of a food allergen in model cookie extracts., The financial support of PID2019-103899RB-I00 to S.C., (Spanish Ministerio de Ciencia e Innovación) Research Project, and TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to S.C. are gratefully acknowledged. S.B. acknowledges a Juan de la Cierva Incorporación contract (Ref.: IJCI-2017-31345) from the Spanish Ministerio de Economía, Industria y Competitividad.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS

This work reports the first electroanalytical bioplatform to date for the determination of antibodies against aquaporin-4 (AQP4-Abs), whose serum level is considered as relevant biomarker for certain autoimmune diseases. The bioplatform relies on the use of magnetic microparticles modified with the biotinylated protein for the capture of specific antibodies. The captured IgGs are enzymatically labelled with a secondary antibody conjugated to the horseradish peroxidase (HRP) enzyme. Amperometric transduction is performed using the H