Immobilization of HRP Enzyme on Layered Double Hydroxides for Biosensor Application

We present a new biosensor for hydrogen peroxide (H2O2) detection. The biosensor was based on the immobilization of horseradish peroxidase (HRP) enzyme on layered double hydroxides- (LDH-) modified gold surface. The hydrotalcite LDH (Mg2Al) was prepared by coprecipitation in constant pH and in ambient temperature. The immobilization of the peroxidase on layered hybrid materials was realized via electrostatic adsorption autoassembly process. The detection of hydrogen peroxide was successfully observed in PBS buffer with cyclic voltammetry and the chronoamperometry techniques. A limit detection of 9？μM of H2O2 was obtained with a good reproducibility. We investigate the sensitivity of our developed biosensor for H2O2 detection in raw milk. 1. Introduction Horseradish peroxidase (HRP) is a glycoprotein with four lysine residues for conjugation to a labeled molecule. It produces a colored, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. Horseradish peroxidase was often used in conjugates to determine the presence of a molecular target and was also commonly used in techniques such as ELISA and Immunohistochemistry. Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive than other popular alternatives such as alkaline phosphatase. Conductor’s polymers and magnetic nanoparticles were used in the fabrication of various types of HRP-based biosensors [1, 2]. To stabilize the immobilized enzyme in the matrix of film, glutaraldehyde (GA) is usually employed as a bifunctional agent to cross-link enzyme molecules [3–6], but the cross-linking efficiency under standard conditions is not always satisfactory [6, 7], which results in the lower sensitivity and poor stability of the resulting biosensor. It was established that cationic clays and especially anionic ones or layered double hydroxides were considered as a new class of materials with a high trapping potential of molecules of different sizes and that they form hybrid materials. Indeed, these materials present very attractive advantages such as a low cost of purification or synthesis and their biocompatibility. Furthermore, the bi-dimensional structure of LDHs with the general formula: [8, 9] present additional advantages relatively to cationic clays. LDHs can be synthesized using the same protocol, and the obtained materials have a large range of physicochemical properties. A wide varieties of LDHs can be achieved by changing the anionic ion A