New nanostructured electrochemical biosensors based on three-dimensional (3-mercaptopropyl)-trimethoxysilane network.

The design and characterization of a new nanostructured organic-inorganic hybrid material and its application to L-lactic acid determination are described. This material is based on the integration of the enzyme lactate oxidase (LOx) and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold 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 provides a biocompatible environment that preserves the catalytic activity of LOx after its immobilization and allows the incorporation of a high amount of enzyme, which is expected to improve the sensitivity of the final biosensing device. Characterization of the designed biosensing platform was performed using quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the conjunction of these techniques, information about (i) the kinetic of LOx adsorption process in real time, (ii) the amount of LOx incorporated into the network, and (iii) the morphological characteristics at the nanometre level of the designed biosensing material was obtained. This information is very useful on the development of successful biosensing devices. Finally, the response of the biosensor to L-lactic acid was evaluated. The biosensor responds linearly to L-lactic acid in the range of 50 µM to 0.25 mM, with a sensitivity of 3.4 µA mM(-1) and a detection limit of 4.0 µM.

Carbon nanotubes/pentacyaneferrate-modified chitosan nanocomposites platforms for reagentless glucose biosensing.

The design, characterization and applicability of a nanostructured biosensor platform are described. The biosensor is developed through the immobilization of three components: a polymeric chitosan network previously modified with a redox mediator (denoted as PCF-Pyr-Ch), an enzyme (glucose oxidase, chosen as a model) and carbon nanotubes onto a solid glassy carbon electrode (C). In order to assess the influence of the nanomaterial in the performance of the resulting analytical device, a second biosensor, free of carbon nanotubes, is developed. The characterization of both biosensing platforms was performed in aqueous phosphate buffer solutions using atomic force microscopy technique. In the presence of glucose, both systems exhibit a clear electrocatalytic activity, and glucose could be amperometrically determined at +0.35 V versus Ag/AgCl. The performance of both biosensors was evaluated in terms of sensitivity, detection limit and linear response range. Finally, the enhancement of the analytical response induced by the presence of carbon nanotubes was evaluated.

One-step covalent microcontact printing approach to produce patterns of lactate oxidase.

A comparative study of three different strategies to pattern lactate oxidase (LOx) onto bare gold substrates by microcontact printing (muCP) is presented. The quality of the resulting patterns in terms of homogeneity, compactness and stability has been evaluated by atomic force microscopy in both air and aqueous conditions. The following approaches have been tested: (i) LOx was directly stamped to a bare gold surface; (ii) LOx was previously covalently bonded to a thiolated molecule, dithiodipropionic acid di(N-succinimidyl ester) (DTSP), and this conjugate (LOx/DTSP) was transferred from an elastomeric stamp to a bare gold substrate; (iii) formation of a LOx/DTSP micropattern on a bare gold surface (as described in approach ii) was followed by exposure to a solution containing hexadecylmercaptane (HDM). In all cases, the catalytic activity of the final LOx patterns has been assessed by electrochemical measurements. From comparison of the three strategies, it can be concluded that the third one gives rise to LOx patterns that present a high stability and compactness, also offering the advantage of reducing the number of microcontact printing steps to one.

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 4nm 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)=+0.15V) appears concomitantly with the typical Fe(II)/Fe(III) peaks (E(0)=+0.30V) of HMF. In this case, the biosensor response to lactate shows simultaneous electrocatalytic peaks at +0.15V and +0.30V, indicating the concurrence of different feedback mechanisms. The achievement of a biosensor response to lactate at +0.15V is very convenient in order to avoid potential interferences. The developed biosensor presents a linear concentration range from 0.02mM to 1.2mM, a sensitivity of 6.1µAmM(-1), a detection limit of 5.3µM 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.

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 0.05 mM to 0.7 mM, a sensitivity of 4.0 µA mM(-1) and a detection limit of 15 µM were obtained.

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.