Desarrollo, caracterización y aplicaciones de dispositivos analíticos basados en oxidorreductasas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Agrícola y Análisis Instrumental. Fecha de lectura: 23-06-200

Sol-gel derived gold nanoparticles biosensing platform for Escherichia coli detection

A three dimensional polymeric network obtained by the sol-gel process was applied to develop disposable, sensitive, selective and stable electrochemical biosensors for detection and quantification of Escherichia coli. This biosensor is based on the integration of a thiolated capture probe sequence from E. coli and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS), formed onto a screen-printed gold electrode surface. MPTS presents the advantage of forming a 3D polymeric network containing a large number of thiol tail groups distributed throughout its structure that enable both its anchoring onto gold surfaces and the AuNPs incorporation. Moreover, this matrix allows the incorporation of a high amount of capture probe and provides a biocompatible environment that preserves its original recognition capability after its immobilization, which is expected to improve the sensitivity of the final biosensing device. The hybridization event is detected using the ruthenium complex [Ru(NH3)5L] 2+, where L is the ligand [3-(2-phenanthren-9-yl-vinyl)-pyridine], as redox indicator, which interacts preferentially with ds-DNA. With the use of this approach, complementary target sequences of E. coli can be quantified over the range of 21-400 pmol with a detection limit of 6.3 pmol and good reproducibility (RSD = 4.3%, n = 4, C = 10 μM). In addition, this approach is very selective allowing the detection, without the need of a hybridization suppressor, of a single mismatch. It is also quite stable under storage conditions, giving response at least for 25 days. © 2013 Elsevier B.V.This work has been supported by Comunidad Autónoma de Madrid (project No. S2009/PPQ-1642, AVANSENS), Ministerio de Ciencia e Innovación (project No. CTQ2011-28157) and Ministerio de Economía y Competitividad (FIS2012-38866-C05-05).Peer Reviewe

Lactate biosensor based on a bionanocomposite composed of titanium oxide nanoparticles, photocatalytically reduced graphene, and lactate oxidase

El pdf del artículo es la versión post-print.-- et al.We have developed a lactate biosensor based on a bionanocomposite (BNC) composed of titanium dioxide nanoparticles (TiO2-NPs), photocatalytically reduced graphene, and lactate oxidase. Graphene oxide was photochemically reduced (without using any chemical reagents) in the presence of TiO2-NPs to give graphene nanosheets that were characterized by atomic force microscopy, Raman and X-ray photoelectron spectroscopy. The results show the nanosheets to possess few oxygen functionalities only and to be decorated with TiO2-NPs. These nanosheets typically are at least 1 μm long and have a thickness of 4.2 nm. A BNC was obtained by mixing lactate oxidase with the nanosheets and immobilized on the surface of a glassy carbon electrode. The resulting biosensor was applied to the determination of lactate. Compared to a sensor without TiO2-NPs, the sensor exhibits higher sensitivity (6.0 μA mM-1), a better detection limit (0.6 μM), a wider linear response (2.0 μM to 0.40 mM), and better reproducibility (3.2 %). [Figure not available: see fulltext.] © 2013 Springer-Verlag Wien.This work has been supported by Comunidad Autónoma de Madrid (project No. S2009/PPQ-1642, AVANSENS), Ministerio de Ciencia e Innovación (project No. CTQ2011-28157) and Ministerio de Economía y Competitividad (project No. FIS2012-38866-C05-05).Peer Reviewe

Laccase biosensors based on different enzyme immobilization strategies for phenolic compounds determination

Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level. The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at -0.10 V versus Ag/AgCl. However, the performance of biosensors - evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability - depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity. © 2013 Published by Elsevier B.V.This work has been supported by the Comunidad Autónoma de Madrid (Project no. S2009/PPQ-1642, AVANSENS) and Ministerio de Ciencia e Innovación (Projects nos. CTQ2008-05775 and FIS2012-38866-C05-05).Peer Reviewe

Electrocatalytic processes promoted by diamond nanoparticles in enzymatic biosensing devices

We have developed a biosensing platform for lactate determination based on gold electrodes modified with diamond nanoparticles of 4 nm of nominal diameter, employing the enzyme lactate oxidase and (hydroxymethyl)ferrocene (HMF) as redox mediator in solution. This system displays a response towards lactate that is completely different to those typically observed for lactate biosensors based on other nanomaterials, such as graphene, carbon nanotubes, gold nanoparticles or even diamond nanoparticles of greater size. We have observed by cyclic voltammetry that, under certain experimental conditions, an irreversible wave (E = +0.15 V) appears concomitantly with the typical Fe/Fe peaks (E = +0.30 V) of HMF. In this case, the biosensor response to lactate shows simultaneous electrocatalytic peaks at +0.15 V and +0.30 V, indicating the concurrence of different feedback mechanisms. The achievement of a biosensor response to lactate at +0.15 V is very convenient in order to avoid potential interferences. The developed biosensor presents a linear concentration range from 0.02 mM to 1.2 mM, a sensitivity of 6.1 μA mM, a detection limit of 5.3 μM and excellent stability. These analytical properties compare well with those obtained for other lactate-based biosensors that also include nanomaterials and employ HMF as redox mediator.This work has been supported by Ministerio de Economía y Competitividad (Project nos. CTQ2014-53334-C2-1-R, FIS2012-38866-C05-05 and CTQ2015-71955-REDT) and Comunidad Autónoma de Madrid (Project NANOAVANSENS S2013/MIT-3029)