Unlocking Glutamate Dynamics: The Potential of the Glutamate-Binding Protein from Corynebacterium glutamicum as a Fluorescence Probe in Cerebrospinal Fluid Analysis

L-Glutamate (L-Glu) is the primary excitatory neurotransmitter in the mammalian central nervous system. In neurobiology, monitoring L-Glu accumulation is critical to track the neurodegeneration process, since excessive extracellular levels are caused by acute events like stroke, brain injuries, or malignant tumors. Conventional analytical methods are widely used to quantify L-Glu. Recently, alternative approaches based on biosensors have been developed for monitoring L-Glu in biological systems. In this work, we explored the use of the glutamate-binding protein (GluB), isolated from Corynebacterium glutamicum , as a specific probe to design a fluorescence biosensor for L-Glu detection. For this purpose, one of the three cysteine residues of GluB was labeled with the environment-sensitive fluorescence dye acrylodan. The stability of the labeled protein was assessed through thermal stability experiments. The binding of L-Glu to the GluB-acrylodan was investigated by steady-state and time-resolved fluorescence measurements. The results indicate the capability of the biosensor to effectively intercept the concentration values of L-Glu that are characteristic of acute clinical emergencies and tumor microenvironments before they can reach extreme neurotoxic levels. To explore the clinical potential application of the assay, GluB-acrylodan was tested through time-resolved fluorescence measurements in artificial cerebrospinal fluid (aCSF). The results showed a LoD of 310 µM, a threshold value that is compatible with the pathological concentrations of L-Glu, confirming that GluB-acrylodan is a promising analytical tool in neurobiological research, clinical monitoring and cellular excitotoxic events.