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  • br Materials and methods br Results br Conclusions


    Materials and methods
    Conclusions We developed a glutamate biosensor with chitosan as a matrix for the immobilization of the enzyme glutamate oxidase on the surface of a platinum electrode. Our miniaturized biosensor of 50 µm in diameter can be applied for monitoring glutamate in vivo and in Captopril slices. The biosensor exhibited high sensitivity, while rejecting interferences, with a fast response time (~2 s) and a linear range of 5–150 μM. Biosensors were stable for 7 days when stored dry at + 4 °C and had good reproducibility within a batch and between batches. Stimulated glutamate release was successfully measured both in brain slices and in vivo. This biosensor has the potential to directly monitor glutamate in vivo with minimal tissue damage. Future studies can test the long-term operational and storage stability of the sensor and its viability in long-term in vivo applications. Small variations of length between sensors due to hand fabrication could be improved in future with a mass fabrication approach.
    Acknowledgments This work was funded by NIH (National Institue of Health, USA) under grants R01NS076875 and R01EB026497 to BJV.
    Introduction Cyclopropenes have become a popular choice as a bioorthogonal reagent due to their small size, inertness to cellular nucleophiles, and ability to be genetically encoded [1], [2], [3]. They are employed as bioorthogonal reaction partners for 1,3-dipoles [4], tetrazines [5], [6], 1,2,4-triazines [7], and o-quinones [8]. Their small size allows minimum perturbation of the biological system under study; therefore, they have been appended to a number of biomolecules (e.g., glycans [9], proteins [10], nucleic acids [11], and lipids [5], [12]). Recently, photocaged cyclopropenes that allow spatiotemporal control over their ligations with tetrazines have been reported [13], [14], [15]. Additionally, cyclopropenes are important synthetic intermediates [16], [17] and find use for polymerization[18] and unnatural amino acid synthesis [19]. Surprisingly, neurotransmitters, an important class of biomolecules, have been largely excluded from the advances made by biorthogonal reagents. In this report, we sought to generate cyclopropene-bearing glutamate neurotransmitters that are stable and straightforward to synthesize. We focused on the primarily excitatory amino acid neurotransmitter glutamate, which is released from the pre-synaptic nerve terminals and binds to the metabotropic or ionotropic glutamate receptors at the post-synaptic sites in the brain. Synthetic glutamate analogs have helped improve understanding of the pharmacology of glutamate-receptor binding, glutamate receptor function and diversity, and the mechanism of glutamate recycling. For example, N-Methyl-d-aspartate (NMDA), kainic acid (KA), and 2-amino-(3-hydroxyl-5-methyl-4-isoxazol-4-yl) propionic acid (AMPA) were critical for identifying and distinguishing the ionotropic glutamate receptors, whereas L-2-amino-4-phosphonobutyrate (L-AP4) and 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) were used for studying the pharmacology of the metabotropic glutamate receptors (Fig. 1) [20], [21], [22]. More recently, conformationally restricted analogs based on cyclopropanes such as l-CCG or DCG have been identified, which, interestingly, possess biological activity as each diastereomer [23], [24]. The high biological activity of a structurally diverse array of cyclopropane-based glutamate analogs intrigued us, and, in part, inspired us to develop cyclopropene-containing glutamate analogs. Such cyclopropene-containing glutamate analogs will be conformationally restricted, like their cyclopropane counterparts, but they will have the additional advantage of acting as reactants for bioorthogonal chemistry through deployment of pro-fluorescent tetrazines [25] or tetrazoles [26]. Additionally, cyclopropene-glutamates can act as substrates for unnatural peptide synthesis.