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)

Diamond nanoparticles based biosensors for efficient glucose and lactate determination

In this work, we report the modification of a gold electrode with undoped diamond nanoparticles (DNPs) and its applicability to the fabrication of electrochemical biosensing platforms. DNPs were immobilized onto a gold electrode by direct adsorption and the electrochemical behavior of the resulting DNPs/Au platform was studied. Four well-defined peaks were observed corresponding to the DNPs oxidation/reduction at the underlying gold electrode, which demonstrate that, although undoped DNPs have an insulating character, they show electrochemical activity as a consequence of the presence of different functionalities with unsaturated bonding on their surface. In order to develop a DNPs-based biosensing platform, we have selected glucose oxidase (GOx), as a model enzyme. We have performed an exhaustive study of the different steps involved in the biosensing platform preparation (DNPs/Au and GOx/DNPs/Au systems) by atomic force microscopy (AFM), field emission scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV). The glucose biosensor shows a good electrocatalytic response in the presence of (hydroxymethyl)ferrocene as redox mediator. Once the suitability of the prototype system to determine glucose was verified, in a second step, we prepared a similar biosensor, but employing the enzyme lactate oxidase (LOx/DNPs/Au). As far as we know, this is the first electrochemical biosensor for lactate determination that includes DNPs as nanomaterial. A linear concentration range from 0.05mM to 0.7mM, a sensitivity of 4.0μAmM-1 and a detection limit of 15μM were obtained.This work has been supported by Ministerio de Ciencia e Innovacion (Project no. CTQ2011-28157), Ministerio de Economía y Competitividad (Project no. FIS2012-38866-C05-05) and Comunidad Autónoma de Madrid (Project NANOAVANSENS S2013/MIT-3029)