Surface Amplification of L-Glutamate Using a Patterned Bienzymatic System for Biosensing Applications

Electronic sensing of neurotransmitter molecules is based on a diffusion-limited process, which requires the immobilization of biological recognition elements as close as possible to the active area of the sensor. Moreover, in many applications, the analyte concentration is very low. In particular, the in situ detection of neurotransmitter release from neurons is challenging due to the limited number of molecules secreted and their fast diffusion and reuptake immediately after release. In this paper, we present a method that allows for the local amplification of L-glutamate directly on the chip surface. Our approach is based on the surface patterning of a bienzymatic system consisting of glutamate oxidase (GLOD) and glutamic-pyruvate transaminase (GPT) that amplifies L-glutamate via a recycling process. The surface chemistry was optimized for maximal enzyme loading. The level of amplification was determined using a colorimetric assay. Coimmobilization of GLOD and GPT yielded at least a doubling of the signal, and increasing the surface concentrations of each enzyme led to amplification levels that approached those obtained in solution. We show that these enzymes can be patterned on substrates using a flip chip bonder for aligned microcontact printing. Furthermore, primary mouse hippocampal neurons were successfully cultured on these patterned surfaces and remained viable for at least five days. The enzymatic pattern was preserved on the substrate surface for the same time period. Lastly, amplification of L-glutamate released from neurons seven days in vitro was detected. Thus, we conclude that this bienzymatic system can ultimately be applied to biosensor surfaces for the in vitro detection of L-glutamate.