We constructed lactate biosensors by immobilization of lactate oxidase (LOx) onto a single-walled carbon nanotube (SWCNT) electrode. The first step of the sensor construction was the immobilization of oxidized SWCNT onto a platinum electrode modified with 4-aminothiophenol (4-ATP). Two enzyme immobilization methods were used to construct the biosensors, i.e., covalent immobilization using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and physical adsorption. Atomic force microscopy (AFM) experiments confirmed the immobilization of SWCNT during the biosensor construction and X-ray photoelectron spectroscopy (XPS) experiments confirmed covalent immobilization of LOx onto the SWCNT in the first method. The biosensor based on covalent enzyme immobilization showed a sensitivity of 5.8 μA/mM, a linearity up to 0.12 mM of L-lactate, and a detection limit of 4.0 μM. The biosensor based on protein adsorption displayed a sensitivity of 9.4 μA/mM, retaining linearity up to 0.18 mM of L-lactate with a detection limit of 3.0 μM. The difference in the biosensor response can be attributed to protein conformational or dynamical changes during covalent immobilization. The stability of the biosensors was tested at different temperatures and after different storage periods. The thermostability of the biosensors after incubation at 60°C demonstrated that the biosensor with covalently immobilized LOx retained a higher response compare with the adsorbed protein. Long-term stability experiments show a better residual activity of 40% for the covalently immobilized protein compared to 20% of residual activity for the adsorbed protein after 25 d storage. Covalent protein immobilization was superior compared to adsorption in preserving biosensor functionality over extended time period.

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.