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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

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).

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.

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

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.

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.

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L&minus, 1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

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

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.

Construction of artificial allosteric protein switches is one of the central goals of synthetic biology that holds promise to transform the way we detect and quantify substances in vitro and in vivo. An artificial chimeric fusion protein of pyrroloquinoline quinone-dependent glucose dehydrogenase with calmodulin (PQQ-GDH-CaM) was covalently attached to graphene nanosheets produced electrochemically on a carbon fiber electrode. The chimeric PQQ-GDH-CaM represents an artificial allosteric switch activated by association of a calmodulin-binding peptide with the Ca2+-bound calmodulin domain. The activity of the immobilized enzyme was switched between active and inactive states by adding/removing the activating peptide. The peptide-signal switchable features originated from the enzyme 3D-structural variations induced by the conformational (folding/unfolding) changes in the connected calmodulin unit upon formation/dissociation of its complex with the specific peptide. The peptide-activated immobilized PQQ-GDH-CaM enzyme displayed direct (non-mediated) electron transfer to the conducting electrode support upon glucose oxidation. On the contrary, in the absence of the peptide, the inactive form of the enzyme demonstrated very low bioelectrocatalytic activity for glucose oxidation. Since the conformational changes of the PQQ-GDH-CaM depend on the presence of Ca2+ cations and the calmodulin-binding peptide, both of them were used as input signals to control the enzyme activity mimicking a Boolean AND logic gate. The switchable behavior of the enzyme-modified electrode was studied electrochemically and used to assemble a signal-switchable biofuel cell. The use of the peptide as the signaling messenger enables the design of generalizable bioelectronic systems controlled by native and synthetic biochemical signaling systems.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL−1 (20.5 pg of protein in 25 μL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

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

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...

An artificial Ca2+-regulated PQQ glucose dehydrogenase (PQQ-GDH) enzyme was electrically connected to conducting electrodes and semiconductor interfaces. Direct electron transfer from the enzyme to the conducting electrode support was stimulated by the addition of Ca2+ cations resulting in reversible enzyme activation. A signal-switchable biofuel cell and biomolecular release have been realized using the Ca2+-activated enzyme immobilized on conducting electrodes. Interfacing the signal-switchable enzyme with a semiconductor chip allowed electronic read out of the enzyme ON-OFF states. The developed approach based on the signal-regulated PQQ-GDH enables numerous bioelectrochemical/bioelectronic applications of the developed systems in signal-activated biosensors and biofuel cells, as well as in biomolecular computing/logic systems.

An enzyme-based biocatalytic system mimicking operation of a logically reversible Controlled NOT (CNOT) gate has been interfaced with semiconductor electronic transducers. Electrolyte–insulator–semiconductor (EIS) structures have been used to transduce chemical changes produced by the enzyme system to an electronically readable capacitive output signal using field-effect features of the EIS device. Two enzymes, urease and esterase, were immobilized on the insulating interface of EIS structure producing local pH changes performing XOR logic operation controlled by various combinations of the input signals represented by urea and ethyl butyrate. Another EIS transducer was functionalized with esterase only, thus performing Identity (ID) logic operation for the ethyl butyrate input. Both semiconductor devices assembled in parallel operated as a logically reversible CNOT gate. The present system, despite its simplicity, demonstrated for the first time logically reversible function of the enzyme system transduced electronically with the semiconductor devices. The biomolecular realization of a CNOT gate interfaced with semiconductors is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.

Signal-controlled release of DNA from Fe3+-cross-linked alginate hydrogel electrochemically deposited on an electrode surface was studied. The multiple input signals were logically processed with the help of the enzyme biocatalyzed reactions. Boolean logic gates, OR, AND, INH, were realized with the biocatalytic reactions performed by the enzymes entrapped in the alginate film. Hydrogen peroxide produced by the enzymatic reactions resulted in the degradation of the alginate hydrogel and DNA release. The alginate degradation was facilitated by the formation of free radicals in the Fenton-type reaction catalyzed by iron cations cross-linking the alginate hydrogel. The studied approach is versatile and can be adapted to various chemical signals processed by various enzymes with differently implemented Boolean logic. This work illustrates a novel concept of functional integration of biomolecular computing and actuation.

We study the mechanisms involved in the release, triggered by the application of glucose, of insulin entrapped in Fe3+-cross-linked alginate hydrogel particles further stabilized with a polyelectrolyte. Platelet-shaped alginate particles are synthesized containing enzyme glucose oxidase conjugated with silica nanoparticles, which are also entrapped in the hydrogel. Glucose diffuses in from solution, and production of hydrogen peroxide is catalyzed by the enzyme within the hydrogel. We argue that, specifically for the Fe3+-cross-linked systems, the produced hydrogen peroxide is further converted to free radicals via a Fenton-type reaction catalyzed by the iron cations. The activity of free radicals, as well as the reduction of Fe3+ by the enzyme, and other mechanisms contribute to the decrease in density of the hydrogel. As a result, while the particles remain intact, void sizes increase and release of insulin ensues and is followed experimentally. A theoretical description of the involved processes is proposed and utilized to fit the data. It is then used to study the long-time properties of the release process that offers a model for designing new drug-release systems.

Electrodes composed of carbon fibers were modified with graphene nano-sheets in order to increase their surface area and facilitate electrochemical reactions. Electrocatalytic species, such as Meldola's blue (MB) and hemin were immobilized on the graphene surface due to their π-π stacking and then used for electrocatalytic oxidation of NADH and reduction of H2O2, respectively. Further modification of these electrodes with enzymes producing NADH and H2O2 in situ (lactate dehydrogenase, LDH, and lactate oxidase, LOx, respectively), allowed assembling of a biofuel cell operating in the presence of lactate, oxygen and NAD+. The cathode of the biofuel cell required lactate and O2 for its operation, while the anode operated in the presence of lactate and NAD+. Notably, both bioelectrocatalytic electrodes operated in the presence of lactate, one producing H2O2 in the reaction catalyzed by LOx in the presence of O2, second producing NADH in the reaction catalyzed by LDH in the presence of NAD+. Both reactions were performed in the biofuel cell without separation of the cathodic and anodic solutions and with no need of a membrane. The biofuel cell was tested in solutions mimicking human sweat and then in real human sweat samples, demonstrating substantial power release being able to activate electronic devices.

A graphene-functionalized carbon fiber electrode was modified with adsorbed polyethylenimine to introduce amino functionalities and then with trigonelline and 4-carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized pyridine groups produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pKa=8.4). The total charge on the monolayer-modified electrode was positive at the neutral pH and negative at pH > 9. Note that 4-carboxyphenylboronic acid was attached to the electrode surface in molar excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH. Negatively charged fluorescent dye-labeled insulin (insulin-FITC) was loaded on the modified electrode surface at pH 7.0 due to its electrostatic attraction to the positively charged interface. The local pH in close vicinity to the electrode surface was increased to ca. 9–10 due to consumption of H+ ions upon electrochemical reduction of oxygen proceeding at the potential of −1.0 V (vs. Ag/AgCl) applied on the modified electrode. The process resulted in recharging of the electrode surface to the negative value due to the formation of the negative charge on the phenylboronic acid groups, thus resulting in the electrostatic repulsion of insulin-FITC and stimulating its release from the electrode surface. The insulin release was characterized by fluorescence spectroscopy (using the FITC-labeled insulin), by electrochemical measurements on an iridium oxide, IrOx, electrode and by mass spectrometry. The graphene-functionalized carbon fiber electrode demonstrated significant advantages in the signal-stimulated insulin release comparing with the carbon fiber electrode without the graphene species.

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.

Biofuel cells based on electrocatalytic oxidation of NADH and reduction of H2O2 have been prepared using carbon fiber electrodes functionalized with graphene nano-flakes. The electrochemical oxidation of NADH was catalyzed by Meldola's blue (MB), while the reduction of H2O2 was catalyzed by hemin, both catalysts were adsorbed on the graphene flakes due to their π-π staking. In the next set of experiments, the MB- and hemin-electrodes were additionally modified with glucose dehydrogenase (GDH) and glucose oxidase (GOx), respectively. The enzyme catalyzed reactions in the presence of glucose, NAD+ and O2 resulted in the production of NADH and H2O2 in situ. The produced NADH and H2O2 were oxidized and reduced, respectively, at the bioelectrocatalytic electrodes, thus producing voltage and current generated by the biofuel cell. The enzyme-based biofuel cells operated in a human serum solution modelling an implantable device powered from the natural biofluid. Finally, two enzyme-based biofuel cell connected in series and operating in the serum solution produced electrical power sufficient for activation of an electronic watch used as an example device.

Single-stranded DNA molecules were loaded on the mixed poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA)/poly(methacrylic acid) (PMAA) brush covalently attached to an indium tin oxide (ITO) electrode on a flexible support. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAEMA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V (vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase in the vicinity of the electrode surface. The process resulted in the transition to negative charge of the mixed polymer-brush due to dissociation of carboxylic groups of PMAA. This resulted in the electrostatic repulsion and release of the loaded DNA. The developed approach allows cyclic load-release of the DNA with the significantly increased amount of the released DNA comparing with previously reported systems. Further options for the improvements of the system are discussed.

An indium tin oxide (ITO) electrode prepared on a flexible polymeric support was modified with an amino-silane and then functionalized with trigonelline and 4-carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized ammonium group produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pKa=8.4). The total charge on the monolayer-modified electrode was positive at the neutral pH and negative at pH>9 (note that 4-carboxyphenylboronic acid was attached to the electrode surface in excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH). Single-stranded DNA molecules were loaded on the modified electrode at pH 7.0 due to their electrostatic attraction to the positively charged surface. By applying electrolysis at −1.0 V (vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase in the vicinity of the electrode surface. The process resulted in the transition to the total negative charge due to the negative charges formed on the phenylboronic acid species. This resulted in the electrostatic repulsion and release of the loaded DNA. The developed approach allowed the electrochemically-triggered DNA release not only in the aqueous solutions, but also in human serum solution, thus giving promise for future biomedical applications.

An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA-based Identity, AND and XOR logic gates. Single-stranded DNA molecules were loaded on the mixed poly(N,N-di-methylaminoethyl methacrylate) (PDMAEMA)/poly-(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAE-MA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals.

A new bioelectronic system activated by a DNA-signal and releasing another DNA has been designed. The system was composed of two modified electrodes: one electrode functionalized with enzymes was activated with a DNA-signal, while another electrode coated with an alginate film released pre-loaded DNA species, when the first electrode was activated. The sensing electrode was functionalized with pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) producing negative potential in the presence of glucose. This bioelectrocatalytic process was inhibited by the presence of glucose oxidase (GOx) at the external surface of the biomolecular system organized on the electrode surface through a DNA-linker. GOx consumed incoming glucose and did not allow its penetration to the internal layer functionalized with PQQ-GDH, thus leaving the electrode mute. The DNA-signal resulted in the detachment of GOx and activated the process biocatalyzed by PQQ-GDH due to penetration of glucose to the internal layer at the electrode surface. Finally, formation of a negative potential on the sensing electrode resulted in the activation of the second electrode and DNA release from the alginate matrix. The Sense-and-Act system allowed amplification of the DNA-signal by releasing much higher amount of DNA comparing with the applied DNA-signal. The DNA-signal amplification will find applications in various biosensor and biomolecular computing systems.

Graphene nanosheets were produced on the surface of carbon fibers by in situ electrochemical procedure including oxidative and reductive steps to yield first graphene oxide, later converted to graphene. The electrode material composed of graphene-functionalized carbon fibers was characterized by scanning electron microscopy (SEM) and cyclic voltammery demonstrating superior electrochemical kinetics comparing with the original carbon paper. The interfacial electron transfer rate for the reversible redox process of [Fe(CN)6]3−/4− was found ca. 4.5-fold higher after the electrode modification with the graphene nanosheets. The novel electrode material is suggested as a promising conducting interface for bioelectrocatalytic electrodes used in various electrochemical biosensors and biofuel cells, particularly operating in vivo.

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.

This work reports a new sensitive strategy for the determination of tau protein, a hallmark of Alzheimer's disease (AD), involving a sandwich immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) modified with a gold nanoparticles-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM) covalently immobilized onto electrografted p-aminobenzoic acid (p-ABA). The capture antibody (CAb) was immobilized by crosslinking with glutaraldehyde (GA) on the amino groups of the 3D-Au-PAMAM-p-ABA-SPCE, where tau protein was sandwiched with a secondary antibody labeled with horseradish peroxidase (HRP-DAb). Amperometry at −200 mV (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate was used to detect the immunocomplex formation. The great analytical performance of the immunosensor in terms of selectivity and low limit of detection (LOD) (1.7 pg mL−1) allowed the direct determination of the target protein in raw plasma samples and in brain tissue extracts from healthy individuals and post mortem diagnosed AD patients, using a simple and fast protocol.

Stimuli responsive release of biomolecules from surfaces such as gold and carbon is gaining importance for past couple of decades due to its application in biomedical field. Electrochemically induced release of biomolecules from the surfaces is one of such techniques. The simplicity of the electrochemical techniques brings-in a wide opportunity for loading and releasing biomolecules from the electrode surfaces. Here-in we report the loading and electrochemical release of His-tagged proteins from the electrode surfaces such as gold and graphite1. Reversible chelation of His-tagged proteins on Ni-NTA surfaces is studied for decades. Competing ligands such as imidazole and EDTA are used for releasing the His-tagged proteins from the Ni-NTA surfaces. However, switching the metal to Cu(II) instead of Ni(II) brings an excellent opportunity for releasing the His-tagged proteins by the reduction of Cu(II). The affinity of Cu(I) towards complexation with NTA and His-tag is substantially lower than Cu(II) which facilitates the release of the protein. Cu-NTA crafted graphite and gold electrodes were used for the coordinative loading of His-tagged proteins and electrochemical reduction of metal ion for the release of the proteins from the surface. In this study a redox mediator coupled model peptide (ferrocene-hexahistidine) and a model recombinant protein (containing His-tag) “Protein A” were used as examples for loading and electrochemical release. ǂ These authors equally contributed to the work Reference: 1. Bellare, M.; Kadambar, V. K.; Bollella, P.; Gamella, M.; Katz, E. and Melman, A; Electrochemical release of His-tagged proteins by destruction of NTA-Cu(II)-protein complex; Electroanalysis 2019, accepted manuscript.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor involved in tumor growth and metastasis by regulating genes involved in response to hypoxia. This work reports the first magnetic beads (MBs)-based electrochemical immunoassay for the determination of HIF-1α. The design involves a sandwich immunoassay using MBs and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates of carboxylic acid-microsized magnetic particles (HOOC-MBs) modified with a specific antibody were used to selectively capture the target protein which was sandwiched with a biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP). The great analytical performance of the immunoassay in terms of selectivity and low limit of detection (76 pg mL−1) for HIF-1α allows the determination of the endogenous amount of this biomarker in 0.5 μg raw cancer cells lysates cultured in normoxia or induced-hypoxia with CoCl2. In addition, the developed immunosensing approach was employed to analyze HIF-1α in saliva samples with results that agree with those provided by an ELISA kit involving the same immunoreagents.