Sondas de Muestreo Microfabricadas de Parylene-C para Monitorización Neuroquímica In Vivo

Monitoring chemistry in the central nervous system in vivo is an important route to better understand chemical signals and metabolism underlying brain function. Sampling from the brain extracellular space allows for in depth and multiplexed analysis of this compartment. Miniaturized sampling devices prepared by using microfabrication are emerging as tools that allow high spatial resolution measurements with low invasiveness. In this work, we describe a microfabrication method for Parylene-C-based push-pull sampling probes. Parylene-C is an attractive material because its biocompatibility and flexibility may allow for chronic measurements. Probes with 3 mm long shanks and 45 × 166 μm cross sections were prepared and tested for suitability for in vivo experiments. Two designs, one with push-pull orifices on the probe tip and the other with the orifices recessed ∼40 mm within a sheath of Parylene-C, were investigated. The sheathed probes successfully flowed in tofu (n = 5) and ex vivo brain tissue (n = 5) for at least 3 h at 100 nL/min. In contrast, only 60% (n = 5) of the end-on probes flowed successfully in tofu. In vitro recoveries for the sheathed and design were 66 ± 2 and 91 ± 7%, respectively, in stirred solution. The sheathed probes were tested by sampling from the cortex of anesthetized rats. In 13 of 19 experiments, push-pull fractions were successfully collected. Of the six failures, all had a sheath shallower than the 30 μm design depth, suggesting that this depth is necessary for reliable sampling flow. Basal concentrations of 20 neurotransmitters and metabolites determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS) were comparable with concentrations recorded using microdialysis sampling in prior work. Infusion of 2.0 mM nipecotic acid through the probe resulted in a significant increase in GABA with little effect on other compounds. Untargeted metabolomics analysis by LC-MS/MS identified 141 metabolites within the push-pull perfusates. The results demonstrate the feasibility of using Parylene-C push-pull probes with a sheathed tip design for multiplexed and temporally resolved monitoring of brain chemistry in vivo.