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This review delves into the titanic research efforts carried out during the last years on affinity-based wearable electrochemical biosensors, using both natural (antibodies) and biomimetic (aptamers, peptides and molecular imprinted polymers) receptors. The rationale and application of selected representative strategies is critically discussed, ending with realistic and futuristic visions of the technical barriers, challenges and prospects in the development and adoption of these biodevices in daily routines to ensure well-being against known, unknown and unexpected threats.

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

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.

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.

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.

Electrochemical biosensors continue to evolve at an astonishing pace, con-solidating as competitive tools for determining a wide range of targets and relentlessly strengthening their attributes in terms of sensitivity, selectivity, simplicity, response time, and antifouling ability, making them suitable for getting a foothold in real-world applications. The design and exploitation of nanostructured or multifunctional nucleic acid or peptide biomaterials are playing a determinant role in these achievements. With the aim of high-lighting the potential and opportunities of these biomaterials, this perspec-tive article critically discusses and overviews the electrochemical biosensors reported since 2019 involving nanostructured and multifunctional DNA biomaterials, multifunctional aptamers, modern peptides, and CRISPR/Cas systems. The use of these biomaterials as recognition elements, electrode modifiers (acting as linkers or creating scaffolds with antifouling properties), enzyme substrates, and labeling/carrier agents for signal amplification is discussed through rationally and strategically selected examples, concluding with a personal perspective about the challenges to be faced and future lines of action. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project, the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349), and PI20CIII/00019 Grant from the AES-ISCIII program are gratefully acknowledged. Sí

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

Growth arrest-specific 6 (GAS6) protein plays a key role in processes related to proliferation, inflammation, angiogenesis, and atherosclerotic plaque formation. In addition, it has been reported that plasma levels of GAS6 are related to cancer prognosis and other relevant pathologies, such as heart failure or sepsis. We report here the first electrochemical immunoplatform for the determination of GAS6, which has demonstrated to be competitive with other available methodologies in terms of cost, simplicity, and decentralized application. The developed immunoplatform involves a sandwich immunoassay using magnetic microparticles (MBs) and uses amperometric detection at disposable screen-printed carbon electrodes (SPCEs). The MBs were modified with an antibody specific to GAS6 for its selective capture, which is further recognized by a biotinylated secondary antibody subsequently labeled with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection was carried out using the hydroquinone (HQ)/H2O2 system. The developed bioplatform exhibits a great selectivity and low limit of detection (27 pg/mL) that allowed the determination of the GAS6 circulating level in plasma samples from patients suffering heart failure (HF) and diagnosed with pancreatic ductal adenocarcinoma (PDAC), as well as the determination of the target protein in raw secretomes of human colorectal cancer cell lines., This work is part of the POSITION-II project fundedby the ECSEL Joint Undertaking under grant numberEcsel-783132-Position-II-2017-IA;www.position-2.eu,and PCI2018-093067 (Spanish Ministerio de Ciencia eInnovación) to M.P. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Inno-vación) Research Project to S.C., PI17CIII/00045 andPI20CIII/00019 grants from the AES-ISCIII program toR.B. and the TRANSNANOAVANSENS-CM Program fromthe Comunidad de Madrid (Grant S2018/NMT-4349) toS.C., RTI2018-095672-B-I00 (Spanish Ministerio de Cienciae Innovación) to P.G.F.; Fundació la Marató de TV3 project081010toM.B.; researchprojectPI20/00625,fromtheAES-ISCIII/FEDER program, to P.N, are gratefully acknowl-edged. A. Montero-Calle acknowledges the support of theFPU predoctoral contracts by the Spanish Ministerio deEducación, Cultura y Deporte. G.S-F. is recipient of a pre-doctoral contract (grant number 1193818N) supported byThe Flanders Research Foundation (FWO). C. Muñoz-SanMartín acknowledges a predoctoral contract from Complutense University of Madrid. R.M. Torrente-Rodríguezacknowledges a Talento-Contract from Comunidad deMadrid (2019-T2/IND-15965).

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL ) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease., This research was funded by Spanish Ministerio de Ciencia e Innovación. PID2019- 103899RB-I00, Spanish Ministerio de Ciencia, Innovación y Universidades, RTI2018-095672-B-I00, Comunidad de Madrid, S2018/NMT-4349, and AES-ISCIII/FEDER, PI20/00625

The evolution of human diseases involves a highly dynamic and interactive system of multiple layers of molecular markers. Therefore, it is now fully accepted that simultaneous analysis of molecular markers from the same or different omics layers leads to novel strategies for their realistic early detection and monitoring, thus improving disease prevention, treatment, and outcomes. In this regard, features such as versatility for profiling multiple and/or multi-omics biomarkers, simplicity, affordable cost, markedly shorter analysis time and smaller sample amount required for analyses compared to conventional or state-of-the-art methodologies, make electrochemical bioplatforms suitable alternatives for routine determinations in clinical and basic research settings. The great advances demonstrated by electrochemical biosensors in recent years have gone hand in hand with the development of new electrochemical substrates, attractive surface chemistries, bioassay formats and amplification strategies, but also with the production and application of new bioreceptors, such as those provided by HaloTag and Phage Display technologies, which has allowed these devices to incur and demonstrate pioneering applications. With all this in mind, this communication will discuss bioelectroanalytical tools recently developed by us and our partners potentially transferable to the clinic due to their simplicity, cost, testing time and decentralized character, which have shown pioneering applications to decisively aid in personalized diagnostics by targeting methylation events in nucleic acids both globally and regionally and deregulated autoantibody expression. These biotools have shown to be able to determine simultaneously and with monobasic sensitivity the most common methylated bases in nucleic acids: 5-methylcytosine 5-mC), 5-hydroxymethylcytosine (5-hmC) and N6-methyladenine (6mA) in DNA and N6-methyladenosine (m6A) in RNA, both globally and regionally and without adopting other amplification strategies than conventional enzymatic labeling [1,2]. A multiplexed platform was proposed with application to the global determination of m6A in total RNA extracted from colorectal cancer cells with different metastatic potential, and to the determination of global methylations of 5-mC, 5-hmC and 6mA in genomic DNA extracted from matched tumor and healthy tissues of CRC patients. The obtained results demonstrated the clinical potential of the developed multiplexed bioplatform to discriminate the aggressiveness of tumor cells as well as tumor versus healthy tissues (accuracy, sensitivity, and specificity of 87.5, 100 and 75 %, respectively, using Ward's clustering method) in only 45 min from small amounts (no more than 100 ng) of denatured genomic DNA and crude total RNA without fragmentation and amplification. As for the electroanalytical bioplatforms developed for autoantibodies determination, they have shown ability to identify and confirm the clinical potential of previously undescribed specific autoantibody signatures against i) tumor-associated antigens, identified in the circulation or in tumor exosomes, for early and differential diagnosis of a given neoplasm [3,4]; ii) phage-derived and aberrant HaloTagged peptides to diagnose Alzheimer's disease (AD) [5]; and iii) SARS-CoV-2 serum-specific immunoglobulins to identify vulnerable population or with natural or acquired immunizations, allowing early detection of exaggerated immune system responses or epidemiological studies on the strain responsible for the infection and the efficacy of available vaccines against the different variants of the virus. It is exciting and rewarding to think that we are tackling and developing highly versatile technologies with the potential to efficiently provide relevant information on key aspects of known and unexpected diseases, such as their onset, severity, and immune response, enabling their management in a fast, simple, affordable, and personalized way in diverse settings. [1] Povedano et al. Anal. Chem. 92 (2020) 5604−5612. [2] Povedano et al. Anal. Chim. Acta 1182 (2021) 338946. https://doi.org/10.1016/j.aca.2021.338946. [3] Garranzo-Asensio et al. Theranostics 10(7) (2020) 3022–3034. [4] Montero-Calle et al. Engineering (2021) in press. https://doi.org/10.1016/j.eng.2021.04.026. [5] Valverde et al. Analysis & Sensing 1 (2021) 161-165. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación), Research Project, PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program grants from the AES-ISCIII program and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged.

Metastasis is responsible for about 90% of cancer-associated deaths. In the context of solid tumors, the low oxygen concentration in the tumor microenvironment (hypoxia) is one of the key factors contributing to metastasis. Tumor cells adapt to these conditions by overexpressing certain proteins such as programmed death ligand 1 (PD-L1) and hypoxia-inducible factor 1 alpha (HIF-1α). However, the determination of these tumor hypoxia markers that can be used to follow-up tumor progression and improve the efficiency of therapies has been scarcely addressed using electrochemical biosensors. In this work, we report the first electrochemical bioplatform for the determination of PD-L1 as well as the first one allowing its simultaneous determination with HIF-1α. The target proteins were captured and enzymatically labeled on magnetic microbeads and amperometric detection was undertaken on the surface of screen-printed dual carbon electrodes using the hydrogen peroxide/peroxidase/hydroquinone system. Sandwich immunoassays were implemented for both the HIF-1α and PD-L1 sensors and the analytical characteristics were evaluated providing LOD values of 86 and 279 pg mL−1 for the amperometric determination of PD-L1 and HIF-1α standards, respectively. The developed electrochemical immunoplatforms are competitive versus the only electrochemical immunosensor reported for the determination of HIF-1α and the “gold standard” ELISA methodology for the single determination of both proteins in terms of assay time, compatibility with the simultaneous determination of both proteins making their use suitable for untrained users at the point of attention. The dual amperometric immunosensor was applied to the simultaneous determination of HIF-1α and PD-L1 in cancer cell lysates. The analyses lasted only 2 h and just 0.5 μg of the sample was required.

his work reports the first electrochemicalimmunoassay involving magnetic microbeads (MBs) forthe determination of galectin-3 (Gal-3), a -galactosidase-binding lectin that acts as mediator of heart failure (HF).MBs-captured sandwich-type immune complexes andamperometric detection at disposable screen-printed car-bon electrodes were used. The immunoplatform showed adetection limit of 8.3 pgmL 1, good reproducibility, andexcellent selectivity. The endogenous concentration ofGal-3 in human plasma from HF patients was determinedwith results in agreement with those obtained usingELISA. The multiplexing feasibility of the developedimmunoplatform was demonstrated for the simultaneousdetermination of Gal-3 and N-terminal pro-brain natriu-retic peptide (NT-proBNP), This work is part of the POSITION-II project funded bythe ECSEL Joint Undertaking under grant number Ecsel-783132-Position-II-2017-IA; www.position-2.eu, andPID2019-103899RB-I00 (Ministerio de Ciencia e Innova-ción), and of the NANOCARDIOFLEX Project (RetosColaboración RTC-2015-4184-1, cofinanced by the Minis-terio de Economía y Competitividad and FEDER “unamanera de hacer Europa”). The financial support of theRTI2018-096135-B-I00 (Ministerio de Ciencia, Innovacióny Universidades) and PID2019-103899RB-I00 (Ministeriode Ciencia e Innovación) and Research Projects and theTRANSNANOAVANSENS-CM Program from the Co-munidad de Madrid (Grant S2018/NMT-4349) are alsogratefully acknowledged. S.V.P acknowledges the doctor-al fellowship from the Fundación Carolina

Over the past decade, artificial nanomaterials that exhibit properties similar to those of enzymes are gaining attraction in electrochemical biosensing as highly stable and low-cost alternatives to enzymes. This review article discusses the main features of the various nanomaterials (metal oxide, metal, and carbon-based materials) explored so far to mimic different kinds of enzymes. The unprecedented opportunities imparted by these functional nanomaterials or their nanohybrids, mostly providing peroxidase-like activity, in electrochemical affinity biosensing are critically discussed mainly in connection with their use as catalytic labels or electrode surface modifiers by highlighting representative strategies reported in the past 5 years with application in the food, environmental, and biomedical fields. Apart from outlining the pros and cons of nanomaterial-based enzyme mimetics arising from the impressive development they have experienced over the last few years, current challenges and future directions for achieving their widespread use and exploiting their full potential in the development of electrochemical biosensors are discussed. Graphical abstract.

Nowadays, electrochemical biosensors are reliable analytical tools to determine a broad range of molecular analytes because of their simplicity, affordable cost, and compatibility with multiplexed and point-of-care strategies. There is an increasing demand to improve their sensitivity and selectivity, but also to provide electrochemical biosensors with important attributes such as near real-time and continuous monitoring in complex or denaturing media, or in vivo with minimal intervention to make them even more attractive and suitable for getting into the real world. Modification of biosensors surfaces with antibiofouling reagents, smart coupling with nanomaterials, and the advances experienced by folded-based biosensors have endowed bioelectroanalytical platforms with one or more of such attributes. With this background in mind, this review aims to give an updated and general overview of these technologies as well as to discuss the remarkable achievements arising from the development of electrochemical biosensors free of reagents, washing, or calibration steps, and/or with antifouling properties and the ability to perform continuous, real-time, and even in vivo operation in nearly autonomous way. The challenges to be faced and the next features that these devices may offer to continue impacting in fields closely related with essential aspects of people’s safety and health are also commented upon.

Autoantibodies (AAbs) are antibodies produced against our own cells or tissues either providing a first defense against infections or indicating the presence of pathological processes. They are not only able to inform on the evolution of diseases but also to predict some illnesses well in advance. Currently, the evaluation of the number and type of formed AAbs is employed to assess the risk, rate, severity and progression of autoimmune and cancer diseases, and to help find therapies to prevent or mitigate the impact of these illnesses. Conventional methods for the determination of AAbs generally suffer from low sensitivity, time-consuming and laborious methodologies, and need specialized technicians and well-equipped labs. Consequently, seeking new methodologies for the rapid and low-cost screening of AAbs is of great interest for global health because it would shorten the delay between sample collection and diagnosis and improve the introduction of modern diagnostics into the developing world. Electrochemical biosensors are considered as a promising alternative to conventional techniques for the determination of clinical biomarkers due to their simplicity of use, low cost, high sensitivity, multiplexing abilities or compatibility with microfabrication and point of care testing. This review is focused on the critical discussion of selected electrochemical biosensors described to date for the determination of AAbs related to autoimmune diseases and several types of cancer. An overview pointing out future directions in this field is also provided.

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 H

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5-methylcytosines (5-mC) are used for the capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The method is applied in the 45-min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step.

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells. The financial support of the Spanish Ministerio de Economía y Competitividad CTQ2015-64402-C2-1-R and SAF2014-53209-R Research Projects, the PI17CIII/00045 research project from AESI and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT-3029) and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (R.M. Torrente-Rodríguez and E. Povedano) and Universidad Complutense de Madrid (V. Ruiz-Valdepeñas Montiel) are also gratefully acknowledged. Sí

Currently, electrochemical nucleic acid-based biosensing methodologies involving hybridization assays, specific recognition of RNA/DNA and RNA/RNA duplexes, and amplification systems provide an attractive alternative to conventional quantification strategies for the routine determination of relevant nucleic acids at different settings. A particularly relevant objective in the development of such nucleic acid biosensors is the design of as many as possible affordable, quick, and simple methods while keeping the required sensitivity. With this aim in mind, this work reports, for the first time, a thorough comparison between 11 methodologies that involve different assay formats and labeling strategies for targeting the same DNA. The assayed approaches use conventional sandwich and competitive hybridization assays, direct hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes, multienzyme labeling bioreagents, and DNA concatamers. All of them have been implemented on the surface of magnetic beads (MBs) and involve amperometric transduction at screen-printed carbon electrodes (SPCEs). The influence of the formed duplex length and of the labeling strategy have also been evaluated. Results demonstrate that these strategies can provide very sensitive methods without the need for using nanomaterials or polymerase chain reaction (PCR). In addition, the sensitivity can be tailored within several orders of magnitude simply by varying the bioassay format, hybrid length or labeling strategy. This comparative study allowed us to conclude that the use of strategies involving longer hybrids, the use of antibodies with specificity for RNA/DNA heteroduplexes and labeling with bacterial antibody binding proteins conjugated with multiple enzyme molecules, provides the best sensitivity.

Point of care testing (POCT), i.e., medical tests performed wherever the patient is with the possibility to immediately get and send the results to the doctor for clinical decision making, is a social need which nowadays is witnessing rapid growth. Major technological progress, internet, mobile phones available to almost everyone, outstanding developments in the world of sensors, and miniaturization of devices facilitate to make a test without significant medical knowledge, allowing getting the results and the doctor's response in a short time. Electrochemical sensors play a key role in this field due to their ability to be adapted to small devices, whether hand-held or wearable at a low cost. This review focus on new approaches in the world of electrochemical sensors that accomplish the main features that clinical POC devices should comply, such as affordability, disposability, sensitivity, selectivity, stability, flexibility, and ease to handle.

This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin-magnetic microbeads (Strep-MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (-0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA-polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MG...

Proteases are involved in cancer‚ taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts.

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.

Quantum dots (QDs) are a kind of semiconductor nanoparticles with great potential in (bio)sensing for medical diagnosis due not only to their optical properties but also to their applicability in the development of electrochemical (bio)sensors. In this article, the use of metallic and graphene QDs for electrochemical signal amplification, either as signal tags or as electrode surface modifiers to detect clinical markers is reviewed and critically discussed. The advantages and disadvantages arising from the use of these nanoparticles in this context will be outlined together with the still required work to fulfil the characteristics needed to attain suitable electrochemical (bio)sensors with real application in clinical field.

A sensitive and rapid method for the determination of the clinically relevant biomarker human endoglin (CD105) in serum samples is presented, involving a magneto-actuated immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Micro-sized magnetic particles were modified with a specific antibody to selectively capture the target protein which was further sandwiched with a secondary HRP-labeled antibody. The immunocomplexes attached to the magnetic carriers were amperometrically detected at SPCEs using the hydroquinone (HQ)/H2O2/HRP system. The magneto-actuated immunosensing platform was able to detect 5 pmoles of endoglin (in 25μL of sample, 0.2μM) in 30min providing statistically similar results to those obtained using a commercial ELISA kit for the determination of endogenous content of endoglin in human serum samples.

Small RNAs, in particular microRNAs (miRNAs), are important regulators of gene expression and many pathological conditions, which have now been definitively linked to the development of cancer. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis, establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, viral proteins offer interesting alternatives to small RNA biosensing. In this review, an up-to-date critical account of recombinant viral protein-based strategies, involving different readout techniques, for miRNA determination is provided. The variety of viral protein-based strategies for miRNA quantification that have appeared in recent years highlights this growing research field, which has not only several challenges but also great opportunities to develop sensitive, reliable, robust, and cost-effective small RNA biosensing methodologies.

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5′-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

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-β 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.