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

Abstract

Este trabalho descreve a construção de um filme de multicomponentes via método layer-bylayer (LbL) e a análise cinética da interação da enzima peroxidase de raiz forte (HRP) sobre a camada de ds-DNA de timo de bezerro formada numa superfície de ouro modificada. As técnicas de ressonância de plásmon de superfície (SPR) e a espectroscopia de impedância eletroquímica (EIE) foram usadas para caracterizar a construção do filme sobre a superfície de ouro. A ressonância de plásmon de superfície forneceu informações essenciais para o estudo e caracterização da interação proteína e ácido nucléico. Este método não necessita de marcador e monitora as interações em tempo real. Os estudos cinéticos obtidos por SPR da formação do filme de HRP sobre o ds-DNA apresentaram valores de 24,7 L mol -1 s -1 e 1,2×10 -3 s -1 para k a e k d , respectivamente. A energia livre de Gibbs obtida para o sistema foi de -23,1 kJ mol -1. Os resultados obtidos mostram que a interação das moléculas da enzima sobre o ds-DNA é cinética e termodinamicamente favoráveis e a interação entre as camadas ocorre principalmente pela atração de cargas opostas. 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.Keywords: SPR, EIS, kinetic adsorption, DNA, HRP, protein-DNA interaction IntroductionThe interaction of nucleic acids with protein has been a primary area of interest for therapeutic development and in the study of cellular processes.1-3 Development of devices based on this interaction presents application in the investigations of the toxicity pathways of nucleic acid. Recently, 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 and other charged materials and to achieve high specificity, high sensitivity, rapid response and flexibility of use, it is clear that the research continues to focus ...

Electrochemical Impedance Analysis of Adsorption and Enzyme Kinetics of Calf Intestine Alkaline Phosphatase on SAM-Modified Gold Electrode

“…The plots obtained for different systems are fitted with three commonly used models for adsorption, viz., the Langmuir model, θ ( t ) = [ 1 − e − k t ] the diffusion controlled Langmuir model, θ ( t ) = [ 1 − e − k t 1 / 2 ] and the purely diffusion controlled adsorption model θ ( t ) = k t 1 / 2 where k is the respective rate constants and t is the time. Of the three, the best fit is obtained with the Langmuir model with very low χ 2 values (<0.005) as shown in Figure .…”

“…The plots obtained for different systems are fitted with three commonly used models for adsorption, 16 viz., the Langmuir model, where k is the respective rate constants and t is the time. Of the three, the best fit is obtained with the Langmuir model with very low χ 2 values (<0.005) as shown in Figure 3.…”

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“…The resonance of excited surface plasmons is sensitive to the change in refractive index near the metal surface, and therefore SPR data is commonly recorded as reflectivity versus angle of incidence . SPR spectroscopy has been used to measure the kinetics of multilayer formation as well as the film thickness and refractive index, , which has made it useful for conducting bioassays on LbL films . SPR, like ellipsometry, can also be used to characterize air-dried LbL films, which is useful for studying the influence of pH and polymer molecular weight on film growth. − However, the dry film thickness can decrease ∼70% of that of the hydrated film, which has to be accounted for .…”

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