Kinetic studies of HRP adsorption on ds-DNA immobilized on gold electrode surface by EIS and SPR

Danielle C M Ferreira,Lauro T Kubota,Renata K Mendes

doi:10.1590/s0103-50532010000900008

Danielle C M Ferreira, Lauro T Kubota + Show 1 more

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https://doi.org/10.1590/s0103-50532010000900008

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Abstract

This paper describes the construction of a multicomponent film via layer-by-layer (LbL) method and the kinetic analysis of the interaction between horseradish peroxidase (HRP) enzyme on calf thymus double-stranded DNA layer on a modified gold surface. Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the film on the gold surface. Surface plasmon resonance provided essential information for the study and characterization of protein and nucleic acid interaction and this method is label-free and monitors the interactions in real time. The kinetic studies determined by SPR of the horseradish peroxidase film formation on ds-DNA layer showed values of 24.7 L mol-1 s-1 and 1.2×10-3 s-1 for k a and k d, respectively. The Gibbs free energy obtained for the system was -23.1 kJ mol-1. The results obtained show that the interaction of the enzymes molecules on ds-DNA is kinetically and thermodynamically favourable and the interaction among the layers probably occurs mainly by attraction of opposite charges.

Highlights

The interaction of nucleic acids with protein has been a primary area of interest for therapeutic development and in the study of cellular processes.[1,2,3] Development of devices based on this interaction presents application in the investigations of the toxicity pathways of nucleic acid
Randle’s equivalent circuit was adopted to model the physiochemical process occurring at the gold electrode surface, which is often used to model the interfacial phenomena: Rs(CPE[RctW]) where Rs is the resistance of the solution, resistance of charge transfer (Rct) is the resistance of chargetransfer, W is the Warburg impedance and CPE is a double layer capacitance for constant phase element.[38]
We have shown by using electrochemical impedance spectroscopy and surface plasmon resonance that peroxidase enzyme adsorbed on the ds-DNA layer on modified gold electrode

Summary

Introduction

The interaction of nucleic acids with protein has been a primary area of interest for therapeutic development and in the study of cellular processes.[1,2,3] Development of devices based on this interaction presents application in the investigations of the toxicity pathways of nucleic acid. Electrochemical sensors have been developed for the rapid detection of DNA damage.[4,5,6,7] Such devices are being successfully used in food industry, environmental fields and clinical chemistry. Biosensors for such analysis can be prepared with multicomponent films of polyions. The organized multilayer architectures have attracted great interest because of their broad range of applications in the fields of biosensors, nonlinear optical devices and surface modification.[12,13,18,19,20,21]

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