Adsorption and catalytic activity of glucose oxidase accumulated on OTCE upon the application of external potential

This article describes the adsorption of glucose oxidase (GOx) onto optically transparent carbon electrodes (OTCE) under the effect of applied potential and the analysis of the enzymatic activity of the resulting GOx/OTCE substrates. In order to avoid electrochemical interferences with the enzyme redox center, control electrochemical experiments were performed using flavin adenine dinucleotide (FAD) and GOx/OTCE substrates. Then, the enzyme adsorption experiments were carried out as a function of the potential applied (ranged from the open circuit potential to +950. mV), the pH solution, the concentration of enzyme, and the ionic strength on the environment. The experimental results demonstrated that an increase in the adsorbed amount of GOx on the OTCE can be achieved when the potential was applied. Although the increase in the adsorbed amount was examined as a function of the potential, a maximum enzymatic activity was observed in the GOx/OTCE substrate achieved at +800. mV. These experiments suggest that although an increase in the amount of enzyme adsorbed can be obtained by the application of an external potential to the electrode, the magnitude of such potential can produce detrimental effects in the conformation of the adsorbed protein and should be carefully considered. As such, the article describes a simple and rational approach to increase the amount of enzyme adsorbed on a surface and can be applied to improve the sensitivity of a variety of biosensors.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados UnidosFil: Torrente, Daniel. University of Texas; Estados UnidosFil: Marucho, Marcelo. University of Texas; Estados UnidosFil: Garcia, Carlos D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados Unido

Dielectric Spectroscopy can Predict the Effect of External AC Fields on the Dynamic Adsorption of Lysozyme

This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular-wave signal with frequencies varying from 0.5 to 2 Hz. The basic experiment, which can be performed in less than 5 min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100 mV and superimposed to +800 mV (applied as a working potential) and varying the frequency in the 0.1–10000 Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1–1.0 Hz), results that are in line with the corresponding dielectric features of the protein.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Guerra, José D. S.. Universidade Federal de Uberlandia; BrasilFil: Garcia, Carlos D.. CLEMSON UNIVERSITY (CLEMSON UNIVERSITY)

Fluorescent patterning of paper through laser engraving

While thermal treatment of paper can lead to the formation of aromatic structures via hydrothermal treatment (low temperature) or pyrolysis (high temperature), neither of these approaches allow patterning the substrates. Somewhere in between these two extremes, a handful of research groups have used CO2 lasers to pattern paper and induce carbonization. However, none of the previously reported papers have focused on the possibility to form fluorescent derivatives via laser-thermal engraving. Exploring this possibility, this article describes the possibility of using a CO2 laser engraver to selectively treat paper, resulting in the formation of fluorescent compounds, similar to those present on the surface of carbon dots. To determine the most relevant variables controlling this process, 3 MM chromatography paper was treated using a standard 30 W CO2 laser engraver. Under selected experimental conditions, a blue fluorescent pattern was observed when the substrate was irradiated with UV light (365 nm). The effect of various experimental conditions (engraving speed, engraving power, and number of engraving steps) was investigated to maximize the fluorescence intensity. Through a comprehensive characterization effort, it was determined that 5-(hydroxymethyl)furfural and a handful of related compounds were formed (varying in amount) under all selected experimental conditions. To illustrate the potential advantages of this strategy, that could complement those applications traditionally developed from carbon dots (sensors, currency marking, etc.), a redox-based optical sensor for sodium hypochlorite was developed.Fil: Clark, Kaylee M.. Clemson University; Estados UnidosFil: Skrajewski, Lauren. Clemson University; Estados UnidosFil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. Clemson University; Estados UnidosFil: Mendes, Letícia F.. Universidade de Sao Paulo; BrasilFil: Bastos, Erick L.. Universidade de Sao Paulo; BrasilFil: Dörr, Felipe A.. Universidade de Sao Paulo; BrasilFil: Sachdeva, Rakesh. Clemson University; Estados UnidosFil: Ogale, Amod A.. Clemson University; Estados UnidosFil: Paixão, Thiago R. L. C.. Universidade de Sao Paulo; BrasilFil: Garcia, Carlos D.. Clemson University; Estados Unido

Comparative behavior of glucose oxidase and oxalate oxidase immobilized in mucin/chitosan hydrogels for biosensors applications

The analytical response of amperometric glucose electrodes was explained in the framework of the properties of hydrogels with different muc/chit ratio at pH = 7.00 used as immobilization matrix for glucose oxidase. The continuous increase in the sensitivity of the electrodes with the amount of chitosan was attributed to a more efficient crosslinking without affecting the enzyme activity. The results were compared with those obtained using the same matrices to immobilize Oxalate Oxidase at pH = 2.85 where it has the highest activity. A maximum sensitivity at muc/chit 70/30 ratio for these oxalate electrodes was observed. Decrease in permeability of negatively charged oxalate with the chitosan content in the matrices, in spite of the high partition coefficient, explains the analytical differences with glucose electrodes. Swelling and viscoelasticity of the hydrogels allowed to asses the importance of a hydrophilic environment for the enzyme in both cases. © 2011 Elsevier Ltd. All rights reserved.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Baruzzi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentin

Del canario al biosensor

Un breve comentario sobre un proyecto en el cual estamos comenzando a trabajar y cuya motivación principal es lograr desarrollar biosensores basados en mecanismos de transducción fotoelectroquímica con aplicación concreta a la detección de marcadores de interés bioquímico.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Baruzzi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentin

Potential-assisted adsorption of bovine serum albumin onto optically transparent carbon electrodes

This article describes the effect of the applied potential on the adsorption of bovine serum albumin (BSA) to optically transparent carbon electrodes (OTCE). To decouple the effect of the applied potential from the high affinity of the protein for the bare surface, the surface of the OTCE was initially saturated with a layer of BSA. Experiments described in the article show that potential values higher than +500 mV induced a secondary adsorption process (not observed at open-circuit potential), yielding significant changes in the thickness (and adsorbed amount) of the BSA layer obtained. Although the process showed a significant dependence on the experimental conditions selected, the application of higher potentials, selection of pH values around the isoelectric point (IEP) of the protein, high concentrations of protein, and low ionic strengths yielded faster kinetics and the accumulation of larger amounts of protein on the substrate. These experiments, obtained around the IEP of the protein, contrast with the traditional hypothesis that enhanced electrostatic interactions between the polarized substrate and the (oppositely charged) protein are solely responsible for the enhanced adsorption. These results suggest that the potential applied to the electrode is able to polarize the adsorbed layer and induce dipole-dipole interactions between the adsorbed and the incoming protein. This mechanism could be responsible for the potential-dependent oversaturation of the surface and could bolster to the development of surfaces with enhanced catalytic activity and implants with improved biocompatibility.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados UnidosFil: Garcia, Carlos D. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados Unido

Spectroscopic ellipsometry as a complementary tool to characterize coatings on PDMS for CE applications

This paper describes the use of spectroscopic ellipsometry to investigate the adsorption process of model polyelectrolytes (PDDAC and PSS) to thin-films of PDMS. A description of the information collected by ellipsometry as well as complementary information obtained by atomic force microscopy and contact angle measurements is discussed. Upon identification of the driving forces and optimum experimental conditions required for the adsorption, multilayer constructs were fabricated (ranging from 1 to 20 nm in thickness) and used to evaluate their effect on the separation of phenolic compounds by capillary electrophoresis. According to the presented results, polyelectrolyte layers of approximately 10 nm thick provided the best conditions for the separation of the selected phenolic compounds.Fil: Benavidez, Tomás Enrique. Clemson University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Garcia, Carlos D.. Clemson University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentin

Spectroscopic and electrochemical characterization of nanostructured optically transparent carbon electrodes

The present paper describes the results related to the optical and electrochemical characterization of thin carbon films fabricated by spin coating and pyrolysis of AZ P4330-RS photoresist. The goal of this paper is to provide comprehensive information allowing for the rational selection of the conditions to fabricate optically transparent carbon electrodes (OTCE) with specific electrooptical properties. According to our results, these electrodes could be appropriate choices as electrochemical transducers to monitor electrophoretic separations. At the core of this manuscript is the development and critical evaluation of a new optical model to calculate the thickness of the OTCE by variable angle spectroscopic ellipsometry. Such data were complemented with topography and roughness (obtained by atomic force microscopy), electrochemical properties (obtained by cyclic voltammetry), electrical properties (obtained by electrochemical impedance spectroscopy), and structural composition (obtained by Raman spectroscopy). Although the described OTCE were used as substrates to investigate the effect of electrode potential on the real-time adsorption of proteins by ellipsometry, these results could enable the development of other biosensors that can be then integrated into various CE platforms.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados UnidosFil: Garcia, Carlos D. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. University of Texas; Estados Unido

Physicochemical properties of a mucin/chitosan matrix used for the development of an oxalate biosensor

Immobilization conditions of biomolecules play an important role in a biosensor performance. In this paper the response of amperometric electrodes for oxalate is related with some physicochemical properties of the matrices where oxalate oxidase was immobilized. Swelling indexes, partition coefficients and permeability of oxalate in mucin/chitosan matrices in a pH = 2.85 succinic acid solution were measured. These properties were also determined in the 70/30 mucin/chitosan matrix in solutions containing succinic acid and sulfate or chloride anions at the same pH value. The results indicate that the sensitivity of the biosensors is mainly determined by the swelling index of the matrix. High slopes of the calibration curves of the electrodes were obtained with highly swollen matrices indicating the importance of a hydrophilic environment for a good enzymatic response.Fil: Benavidez, Tomás Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones en Físicoquímica de Córdoba; Argentina; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Alvarez, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Baruzzi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones en Físicoquímica de Córdoba; Argentina; Argentina. Universidad Nacional de Córdoba; Argentin

Immobilization of glucose oxidase to nanostructured films of polystyrene-block-poly(2-vinylpyridine)

A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to investigate the immobilization of glucose oxidase, a model enzyme for the development of biosensors. According to the results presented, the nanoporous films can provide significant increases in surface area of the substrate and the immobilization of larger amounts of active enzyme. The characterization of the substrate-enzyme interface discussed in the manuscript aims to provide critical information about relationship between the surface (material, geometry, and density of pores), the protein structure, and the immobilization conditions (pH, and protein concentration) required to improve the catalytic activity and stability of the enzymes. A maximum normalized activity of 3300±700Um(-2) was achieved for the nanoporous film of PS-b-P2VP.Fil: Bhakta, Samir A.. University Of Texas At San Antonio; Estados UnidosFil: Benavidez, Tomás Enrique. University Of Texas At San Antonio; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Carlos D.. University Of Texas At San Antonio; Estados Unido