A Comparative Study of Different Protein Immobilization Methods for the Construction of an Efficient Nano‐structured Lactate Oxidase‐CNT‐Biosensor

Abstract

The development of portable and robust biosensors that exhibit a superior sensitive response and reasonable long‐term stability has gained significant attention especially for monitoring human health and physical conditions. Since lactate levels in blood are a long established indicator for the physical fitness in humans, our long term goal is to develop a stable enzyme‐based biosensor for monitoring human health. The model protein for this sensor is lactate oxidase (Lox). In order to construct the biosensor, the protein was attached to a carbon nanotube (CNT) electrode. The use of CNT electrodes will help not only to warrant proper immobilization of the biomolecule to the sensor but also to provide the highest sensitivity during sensing. The central hypothesis of this work is that the use of a covalent immobilization method to attach the protein onto the CNT sensor will positively affect the long‐term stability of the sensor. To address this aim, lactate oxidase (Lox) was covalently attached to the CNT electrode via EDC‐carbodiimine coupling to provide a durable attachment. This will avoid the loss of protein via leaching when it is in contact with the analyte solution during analysis thus enhancing the long‐term stability of the sensor. Cyclic voltammetry and amperometric experiments were conducted to study the biosensor response, the sensitivity, and the detection limits. XPS demonstrated the covalent attachment of Lox onto the CNT electrode. Temperature experiments were conducted in order to study the stability of the sensor. Our results, demonstrate that the covalently immobilization of the protein onto the CNT sensor increased significantly the long‐term stability of the biosensor.