Síntesis y caracterización de ligandos hidroxilados de base de Schiff: aplicación al desarrollo de sensores y biosensores

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de . Fecha de lectura: 03-07-0

Spectroelectrochemical operando method for monitoring a phenothiazine electrografting process on amide functionalized C-nanodots/Au hybrid electrodes

Phenothiazine derivatives are extensively explored dye molecules, which present interesting electrochemical and optical properties. In recent years, the possibility of transforming some phenothiazines in their aryl diazonium salt derivatives has been proved, what allows them to be electrochemically reduced and electrografted onto conductive surfaces. This is a smart way to modify these surfaces and enable them with specific functionalities. In order to better comprehend the electrografting process and consequently have a higher control of it, in this work we have carried out an exhaustive study by operando UV–Vis spectroelectrochemistry of the electrografting of a phenothiazine aryl diazonium salt onto amide carbon nanodots. As a model of phenothiazine dye we have chosen Azure A. The electrografting onto carbon nanodots has been stablished by comparison with the results obtained on bare gold electrodes in this novel study. The presence of carbon dots improves the reversibility of the electrochemical process as derived from the results obtained by operando UV–Vis spectroelectrochemistry. In addition, to asses that the electrochemical process studied corresponds to the electrografting, the results have been compared to those obtained for the simple Azure A adsorption. This study shows the advantages of obtaining simultaneously the electrochemical and the spectroscopic evolution of an electron-transfer process in a single experiment, in a particular electrochemical reaction. This work could be the starting point for the study of the electrografting on other nanomaterialsFunding from the Spanish Ministerio de Ciencia, Innovación y Universidades (project: CTQ2017-84309-C2-1-R) and Comunidad Autónoma de Madrid (NANOAVANSENS Program) is acknowledged. IMDEA Nanociencia acknowledges support from the 'Severo Ochoa' Programme for Centres of Excellence in R&D (Ministerio de Ciencia, Innovación y Universidades, Grant SEV-2016-0686

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

Insulin sensor based on nanoparticle-decorated multiwalled carbon nanotubes modified electrodes

Insulin sensors based on glassy carbon electrodes modified with nafion-multiwalled carbon nanotubes decorated with nickel hydroxide nanoparticles (Ni(OH)<inf>2</inf>NPs/Nafion-MWCNTs/GC), were prepared by electrochemical deposition of Ni(OH)<inf>2</inf>NPs from a dinuclear paddle-wheel Ni monothiocarboxylate complex on the MWCNTs/GC surface. The size and distribution of the Ni(OH)<inf>2</inf>NPs/Nafion-MWCNTs were characterized by transmission electron microscopy (TEM). The results show that Ni(OH)<inf>2</inf> nanoparticles were electrodeposited on the surface of carbon nanotubes. Moreover, the electrochemical behavior of the modified electrodes in aqueous alkaline solutions of insulin was studied by cyclic voltammetry and chronoamperometry. It was found that the as-prepared nanoparticles have excellent electrocatalytic activity towards insulin oxidation due to their special properties, reducing the overpotential and improving the electrochemical behavior, compared to the bare GC electrode. Amperometry was used to evaluate the analytical performance of modified electrode in the determination of insulin. Excellent analytical features, including high sensitivity (5.0 A mol cm<sup>-2</sup> μM<sup>-1</sup>), low detection limit (85 nM) and wide dynamic range (up to 10.00 μM), were achieved under optimum conditions. Moreover, these insulin sensors show good repeatability and a high stability after successive potential cycling. Common substances such as ascorbic acid, uric acid and acetaminophen do not interfere. Finally, the developed sensors have been applied to the determination of insulin in pharmaceuticals and in human plasma. Efficient recoveries for pharmaceuticals and human plasma demonstrate that the proposed methodology can be satisfactorily applied to these types of samplesThe authors acknowledge Ministerio de Economía y Competitividad (project No. CTQ2014-53334-C2-1-R and MAT2013-46753-C2-1-P) and Comunidad de Madrid (NANOAVANSENS Program) for financial support. E.M.P. gratefully acknowledges the FPU-2010 Grant from the Ministerio de Educació

Sensitive glyphosate electrochemiluminescence immunosensor based on electrografted carbon nanodots

A novel electrochemiluminescence (ECL) immunosensor based on electrografted carbon nanodots (CND) is developed for the sensitive determination of glyphosate in soy milk and tea. Nitrogen rich CND were synthesized by microwave radiations using mild conditions and following the principles of green chemistry. L-Arginine and 3,3′-diamino-N-methyldipropylamine were selected as precursors. CND were exhaustively characterized as well as the resulting nanostructured electrodes after CND electrografting. The high stability of CND nanostructured electrode together with the high electrical conductivity and the improvement of the electrochemiluminescent properties from the luminophore [Ru(bpy)3]2+ makes it an excellent electrochemiluminescence detection platform for biosensing assays. The application to biosensors was assessed by combination with an immunoassay based on magnetic nanoparticles, in which anti-glyphosate-IgG coupled magnetic particles (MP-Ab) was used as recognition element of the analyte, glyphosate. The developed ECL immunosensor was successfully applied for the detection of glyphosate in a wide linear range from 28.9 to 200 pg/mL, a sensitivity of 3.38 × 10−3 mL/pg and a detection limit of 8.66 pg/mL. The immunosensor response is stable and reproducible and it has been applied to the determination of glyphosate in tea and soy milk, with results that agree with those provided by an ELISA kit involving the same immunoreagentsThis work has been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (CTQ2017-84309-C2-1-R; RED2018-102412-T) and Comunidad Autónoma de Madrid (P2018/NMT-4349 TRANSNANOAVANSENS Program and 2017-T1/BIO-5435 Atracción de Talento Project

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

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

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

Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples

Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N,N′-Bis(3,4-dihydroxybenzylidene) -1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS–AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS–AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs assemblies of controlled size. The ligand—in addition to its reduction action—provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of l-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS–AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 μM lactate and a sensitivity of 5.1 ± 0.1 μA·mM−1. The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit

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