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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.

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

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.

Biocatalytic cascades involving more than one or two enzyme-catalyzed step are inefficient inside alginate hydrogel prepared on an electrode surface. The problem originates from slow diffusion of intermediate products through the hydrogel from one enzyme to another. However, enzyme activity can be improved by surface immobilization. We demonstrate that a complex cascade of four consecutive biocatalytic reactions can be designed, with the enzymes immobilized in an LBL-assembled polymeric layer at the alginate-modified electrode surface. The product, hydrogen peroxide, then induces dissolution of iron-cross-linked alginate, which results in release process of entrapped biomolecular species, here fluorescently marked oligonucleotides, denoted F-DNA. The enzymatic cascade can be viewed as a biocomputing network of concatenated AND gates, activated by combinations of four chemical input signals, which trigger the release of F-DNA. The reactions, and diffusion/release processes were investigated by means of theoretical modeling. A bottleneck reaction step associated with one of the enzymes was observed. The developed system provides a model for biochemical actuation triggered by a biocomputing network of reactions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim National Science Foundation, NSF: CBET-1403208 Russian Science Foundation, RSF: 17-13-01096 This work was supported by National Science Foundation, USA, (award CBET-1403208) and by Russian Science Foundation (project no. 17-13-01096).

We report herein the design of a smart delivery system in which cargo delivery from capped mesoporous silica (MS) nanoparticles is controlled by an integrated enzyme-based control unit . The system consists of Janus-type nanoparticles having opposing Au and MS faces, functionalized with a pH-responsive β-cyclodextrin-based supramolecular nanovalve on the MS surface and two effectors, glucose oxidase and esterase, immobilized on the Au face. The nanodevice behaves as an enzymatic logical OR operator which is selectively fueled by the presence of D-glucose and ethyl butyrate., R.V. acknowledges a Ramon & Cajal contract from the Spanish Ministry of Science and Innovation. Financial support from the Spanish Ministry of Science and Innovation (CTQ2011-24355, CTQ2009-12650, CTQ2009-09351, MAT2012-38429-C04-01) and Comunidad de Madrid (S2009/PPQ-1642, programme AVANSENS) is gratefully acknowledged. The Generalitat Valencia (project PROMETEO/2009/016) is also acknowledged.