Glutamate oxidase biosensor based on mixed ceria and titania nanoparticles for the detection of glutamate in hypoxic environments

• Ceria nanoparticles enable operation of a glutamate oxidase microbiosensor in hypoxic conditions.
• The biosensor is fabricated by co-immobilizing glutamate oxidase with oxygen-rich ceria/titania nanoparticles dispersed in chitosan matrix on a Pt microelectrode.
• The biosensor design is a promising tool for the in vivo detection of glutamate in oxygen depleted conditions.

We report on the design and development of a glutamate oxidase (GmOx) microelectrode for measuring l-glutamic acid (GluA) in oxygen-depleted conditions, which is based on the oxygen storage and release capacity of cerium oxides. To fabricate the biosensor, a nanocomposite of oxygen-rich ceria and titania nanoparticles dispersed within a semi-permeable chitosan membrane was co-immobilized with the enzyme GmOx on the surface of a Pt microelectrode. The oxygen delivery capacity of the ceria nanoparticles embedded in a biocompatible chitosan matrix facilitated enzyme stabilization and operation in oxygen free conditions. GluA was measured by amperometry at a working potential of 0.6 V vs Ag/AgCl. Detection limits of 0.594 µM and 0.493 µM and a sensitivity of 793 pA/µM (RSD 3.49%, n=5) and 395 pA/µM (RSD 2.48%, n=5) were recorded in oxygenated and deoxygenated conditions, with response times of 2 s and 5 s, respectively. The biosensor had good operational stability and selectivity against common interfering substances. Operation of the biosensor was tested in cerebrospinal fluid. Preliminary in vivo recording in Sprague-Dawley rats to monitor GluA in the cortex during cerebral ischemia and reperfusion demonstrate a potential application of the biosensor in hypoxic conditions. This method provides a solution to ensure functionality of oxidoreductase enzymes in oxygen-free environments.