Direct Covalent Immobilization of new Nitrogen-doped Carbon Nanodots by Electrografting for Sensing Applications

This paper reports a facile strategy to covalently immobilize nanosized carbon dots (CD) onto carbon conductive surfaces for sensing applications. The carbon nanodots designed with surface amine groups (N-CD) can be electrografted onto carbon electrodes and, thus, easily covalently immobilized on these conductive surfaces. They have been synthetized by a carbonization method microwave-assisted using preselected low cost and biocompatible precursors, such as D-fructose as primary carbon source and urea as N-donor reagent to obtain peripheral enriched nitrogen CD. The synthetized nanomaterial has been characterized by different techniques, that confirm the presence of size-regular amorphous structures with blue fluorescence when are irradiated with UV light. The highly stable immobilization of N-CD onto the electrode surfaces by electrografting provides hybrid electrodes with greater relative surface area and improved electron transfer properties, demonstrating to be a great promise for electrochemical sensing. Because of its good electrical conductivity, electrical properties, abundant edges sites and high catalytic activity, N-CD immobilized on carbon electrodes efficient amplify the electrochemiluminiscence (ECL) signal from the luminophore [Ru(bpy)3]2+ in a taurine sensor. A linear concentration range from 126 to 1000 μM, a sensitivity of 7.40ⅹ10-4 μM-1 and a detection limit of 37.8 μM were determined for the taurine sensorThis work has been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades through projects CTQ2017-84309-C2-1-R and RED2018-102412-T, and Comunidad Autónoma de Madrid (S2018/NMT-4349 TRANSNANOAVANSENS-CM Program and 2017-T1/BIO-5435 Atracción de Talento Project). The authors thank Professor Hector Abruña the critical review of this wor

Bifunctional carbon nanodots for highly sensitive HER2 determination based on electrochemiluminescence

Early detection of breast cancer increases the chances of achieving adequate and successful treatment as soon as possible. In this work, a promising disposable electrochemiluminescent immunosensor has been developed for simple, efficient detection of the HER2 protein, a breast cancer biomarker. Nitrogen-rich carbon nanodots were synthesized with two functions: to provide functional groups for covalent immobilization of HER2 antibodies and to act as co-reactants in the electrochemiluminescent process. The proposed immunosensor responded linearly to HER2 concentration over a wide range, showing a detection limit of 20.4 pg mL−1. The reliability of this biosensor was confirmed by analyzing HER2 in the presence of another tumor biomarker (CEA), as well as various proteins and sugars. In addition, this proposed strategy presented good stability and applicability in the analysis of human serum samples, showing great potential for applications in the early diagnosis of breast cancerThis work has been supported by the Spanish Ministry of Science, Innovation and Universities (CTQ2017-84309-C2-1-R; RED2018- 102412-T) and by the Autonomous Community of Madrid (Talent Attraction Project 2017-T1/BIO-5435 and the P2018/NMT4349 TRANSNANOAVANSENS Program

Synergistic enhancement of electrochemiluminescence through hybridization of α-Ge nanolayers and gold nanoparticles for highly sensitive detection of tyramine

This work presents a novel approach for detecting biogenic amine tyramine using a sensitive and disposable electrochemiluminescent sensor. The sensor is fabricated by modifying a screen-printed carbon electrode surface with two nanomaterials, α-Ge nanolayers and AuNP, which synergistically enhance the electrochemiluminescence response. The sensor was characterized using various techniques such as SEM-EDX, EIS, Raman, and AFM. The principle of the biosensor relays on the fact that tyramine molecule acts as an analyte and co-reactant, which interacts with the luminophore [Ru(bpy)3] 2+ on the sensor surface. The proposed sensor shows a linear response to tyramine concentration, with a detection limit of 2.28 µM. The sensor successfully detected tyramine in avocado samples, demonstrating its potential for practical applicationsThis work has been supported the Comunidad Autonoma ´ de Madrid (2021-5A/BIO-20943 Talent Attraction Project, SI3/PJI/2021-00341 and S2018/NMT-4349 TRANSNANOAVANSENS-CM Program) and by the Spanish Ministerio de Ciencia e Innovacion ´ (PID2020-116728RBI00, PDC2021-120782-C21, PID2019-106268GB-C32 and TED2021- 129738B-I00). This work has also been supported by the Spanish MINECO (PID2019-106268GB-C32, CEX2018-000805-M and PDC2021- 120782-C21). We acknowledge the support from the “(MAD2D-CM)- UAM” project funded by Comunidad de Madrid, by the Recovery, Transformation and Resilience Plan, and by NextGenerationEU from the European Unio

Azure A embedded in carbon dots as NADH electrocatalyst: Development of a glutamate electrochemical biosensor

Carbon nanodots modified with azure A (AA-CDs) have been synthesized and applied as redox mediator of bioelectrocatalytic reactions. A deep characterization of AA-CDs nanomaterial has been carried out, proving the covalent attachment of azure A molecules into the carbon dots nanostructure. Disposable screen-printed carbon electrodes (SPCE) have been modified with AA-CDs, through the action of chitosan polymer (Chit-AA-CDs/SPCE). The Chit-AA-CDs/SPCE electrocatalytic activity towards the oxidation of NADH has been proved, obtaining excellent results regarding the low oxidation potential achieved (−0.15 V vs. Ag) and low detection and quantification limits (LOD and LOQ) for NADH, 16 and 53 µM, respectively. The developed electrochemical platform has been applied for the construction of a glutamate biosensor by immobilizing L-glutamic dehydrogenase (GLDH/Chit-AA-CDs/SPCE). The morphology of GLDH/Chit-AA-CDs/SPCE platform was analysed by AFM at each different step of the electrode modification process. The resulting biosensing platform is capable of detect NADH enzymatically generated by GLDH in the presence of glutamate and NAD+. Good analytical parameters were obtained for glutamate analysis using GLDH/Chit-AA-CDs/SPCE, as LOD and LOQ of 3.3 and 11 µM, respectively. The biosensor has been successfully applied to the analysis of food and biological samplesThis work has been supported by the Spanish Ministerio de Ciencia e Innovacion (PID2020–116728RB-I00) and Comunidad Autonoma de Madrid (SI3/PJI/2021–00341, P2018/NMT-4349 TRANSNANOAVANSENS Program

Carbon nanodots modified-electrode for peroxide-free cholesterol biosensing and biofuel cell design

The determination of cholesterol is greatly important because high concentrations of this biomarker are associated to heart disease. Moreover, cholesterol can be used as a fuel in enzymatic fuel cells operating under physiological conditions. Here, we present a cholesterol biosensor and a peroxide-free biofuel cell based on the electrocatalytic oxidation of the NADH generated during the enzymatic reaction of cholesterol dehydrogenase (ChDH) as an alternative to the H2O2 biosensing strategies used with cholesterol oxidase-bioelectrodes. Azure A functionalized-carbon nanodots were used as NADH oxidation electrocatalysts and for ChDH covalent immobilization. The biosensor responded linearly to cholesterol concentrations up to 1.7 mM with good sensitivity (4.50 mA cm−2 M−1) and at a low potential. The ChDH bioelectrode was combined with an O2-reducing bilirubin oxidase cathode to produce electrical energy using cholesterol as fuel and O2 as oxidant. Furthermore, the resulting enzymatic fuel cell was tested in human serum naturally containing free cholesterolA.L.DL. and M.P. thank MCIU/AEI/FEDER, EU for funding project RTI2018–095090-B-I00. M.B. acknowledges funding from the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement No. 713366. This work was also supported by Talent Attraction Project from CAM (SI3/PJI/ 2021–00341 and 2021–5A/BIO-20943), Spanish Ministerio de Ciencia e Innovacion (PID2020–116728RB-I00) and TRANSNANOAVANSENSCAM Program (S2018/NMT-4349

Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)3]2+ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the populationThe authors wish to express their sincere thanks to the Spanish Ministerio de Ciencia e Innovacion (MICINN) (PID2020-116728RB-I00 and PID2020-115204RB-I00) and the Comunidad Autonoma ´ de Madrid (S2018/NMT-4349 TRANSNANOAVANSENS-CM Program, SI3/PJI/ 2021–00341 and 2021-5A/BIO-20943 Talent Attraction Project) for the financial support. C. Toyos-Rodríguez acknowledges the MICINN for the award of a FPI Grant (PRE2018-084953). A. de la Escosura-Muniz ˜ also thanks the MICINN for the research founding by a “Ramon ´ y Cajal” contract (RyC-2016-20299

Pathogen sensing device based on 2D MoS2/graphene heterostructure

In this work we propose a new methodology for selective and sensitive pathogen detection based on a 2D layered heterostructured biosensing platform. As a proof of concept, we have chosen SARS-CoV-2 virus because the availability of new methods to detect this virus is still a great deal of interest. The prepared platform is based on the covalent immobilization of molybdenum disulphide functionalized with a diazonium salt (f-MoS2) onto graphene screen-printed electrodes (GPH SPE) by electrografting of the diazonium salt. This chemistry-based method generates an improved heterostructured biosensing platform for aptamer immobilization and aptasensor development. Electrochemical impedance spectroscopy (EIS) is used to obtain the signal response of the device, proving the ability of the sensor platform to detect the virus. SARS-CoV-2 spike RBD recombinant protein (SARS-CoV-2 S1 protein) has been detected and quantified with a low detection limit of 2.10 fg/mL. The selectivity of the developed biosensor has been confirmed after detecting the S1 protein even in presence of other interfering proteins. Moreover, the ability of the device to detect SARS-CoV-2 S1 protein has been also tested in nasopharyngeal swab samplesThis work has been financially supported by the Spanish Ministry of Economy and Competitiveness (PID2020-116728RB-I00, PID2020- 116661RB-I00, CTQ2015-71955-REDT (ELECTROBIONET)) and Community of Madrid (TRANSNANOAVANSENS, S2018/NMT-4349, and PhotoArt P2018/NMT-4367). E. Enebral thank the financial support of “Nanotecnología para detección del SARS-CoV-2 y sus variantes. NANOCOV” project. IMDEA Nanociencia receives support from the “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant CEX2020-001039-S). We also thank the Spanish Ministry of Universities for supporting Laura Gutiérrez-Galvez with the Formación del Profesorado Universitario (FPU) grant (FPU19/06309

A MoS2 platform and thionine-carbon nanodots for sensitive and selective detection of pathogens

This work focuses on the combination of molybdenum disulfide (MoS2) and à la carte functionalized carbon nanodots (CNDs) for the development of DNA biosensors for selective and sensitive detection of pathogens. MoS2 flakes prepared through liquid-phase exfoliation, serves as platform for thiolated DNA probe immobilization, while thionine functionalized carbon nanodots (Thi-CNDs) are used as electrochemical indicator of the hybridization event. Spectroscopic and electrochemical studies confirmed the interaction of Thi-CNDs with DNA. As an illustration of the pathogen biosensor functioning, DNA sequences from InIA gen of Listeria monocytogenes bacteria and open reading frame sequence (ORF1ab) of SARS-CoV-2 virus were detected and quantified with a detection limit of 67.0 fM and 1.01 pM, respectively. Given the paradigmatic selectivity of the DNA hybridization, this approach allows pathogen detection in the presence of other pathogens, demonstrated by the detection of Listeria monocytogenes in presence of Escherichia coli. We note that this design is in principle amenable to any pathogen for which the DNA has been sequenced, including other viruses and bacteria. As example of the application of the method in real samples it has been used to directly detect Listeria monocytogenes in cultures without any DNA Polymerase Chain Reaction (PCR) amplification processAuthors thank the financial support from the Comunidad de Madrid (NANOAVANSENS, S2013/MIT-3029, MAD2D-CM Program, S2013/ MIT-3007 and 2017-T1/BIO-5435), Ministerio de Economía, Industria y Competitividad (CTQ 2015-71955-REDT (ELECTROBIONET), CTQ2014-53334-C2-1-R. and MAT 2015-71879-P). EMP acknowledges the European Research Council (ERC-PoC-842606), MINECO (CTQ 2017- 86060-P), Comunidad de Madrid (MAD2D-CM S2013/MIT-3007). IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, Grant SEV2016–0686). RdC acknowledges support from UAM, Banco Santander, Fundacion ´ IMDEA (convocatoria CRUE–CSIC–SANTANDER, fondo supera 2020, project with reference 10.01.03.02.41). Authors also acknowledge BAT unit of CIA

Papel de los nucleótidos en el control de la estabilidad de los microtúbulos de cerebro

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica y Biología Molecular. Fecha de lectura: 22-06-198

Fluorescent enzymatic assay for direct total polyphenol determination in food-related samples

A direct and simple fluorescent assay for the total polyphenol determination based on the bioconjugate formed between the laccase enzyme (TvL from Trametes versicolor) and carbon nanodots (CD) is developed. One of the most used reactions for the determination of phenols is based on the enzymatic reaction of their oxidation to quinones. In this work, CD has been biofunctionalized with TvL (TvL-CD) and employed as a fluorescent label to follow the enzymatic reaction. The bioconjugate was formed and characterized by spectroscopy and microscopy. The optimal TvL-CD ratio was established. The reaction between the bioconjugate and a phenolic compound such as gallic acid (GA) was followed by monitoring the fluorescence bioconjugate decrease due to the quenching effect of the quinones generated in the enzymatic reaction. These studies confirm that bioconjugation does not inhibit the enzymatic activity and the fluorescence decrease during the enzymatic reaction is mainly due to an electron transfer processes. Based on these results, a new method for the quantitative determination of polyphenols measured as GA concentration is developed. The detection and quantification limit was found to be 7.4 and 25 μM, respectively. Subsequently, the method has been applied to the direct determination of GA in wine, juice, and rice leaf extractsThis research was supported by the Spanish Ministerio de Ciencia e Innovacion ´ (PID2020-116728RB-I00) and Comunidad Autonoma ´ de Madrid (S2018/NMT-4349 TRANSNANOAVANSENS-CM Program, 2017-T1/BIO-5435, 2021-5A/BIO-20943 Talent Attraction Project and SI3/PJI/2021–00341). The authors acknowledge the assistance of R. Wanemacher from IMDEA-Nanociencia to interpret the 3D fluorescence spectr