Silicon and Parylene C-Based Microfabricated Push-Pull Perfusion Probes: Developing Robust Tools for In Vivo Neurochemical Sampling Applications

Abstract

Implantable direct sampling neural probes have proven valuable to in vivo neurochemistry studies. They allow access to target brain regions and enable the quantification of the chemical messengers which drive brain activity. Push-pull perfusion and microdialysis are compatible with analytical methods which can simultaneously measure multiple compounds with high selectivity and sensitivity. However, their bulkiness (diameters > 200 µm) can exacerbate tissue damage during insertion and limit spatial resolution. Microfabrication techniques can miniaturize devices with µm-scale features and improve device uniformity, insertion footprint, and spatiotemporal resolution. Si microfabrication methods produced push-pull perfusion of various sampling area designs. Our lab’s previous Si-probe design possessed orifices open on the side of the shank (70 µm thick x 84 µm wide x 6 mm long) which left them vulnerable to clogging from brain tissue debris. We tested five different sampling area designs to determine sampling area geometries best suited to in vivo applications. The fabrication protocol was updated to create the different testable designs. Flow screening in brain phantoms and relative recovery testing was carried out for all designs. Relative recoveries for dopamine (DA) and glutamate (Glu) which were derivatized with benzyl chloride (BzCl) and analyzed through liquid chromatography mass-spectrometry (LC-MS). DA recovery ranged between 51-96%, while Glu recoveries ranged between 85-109%. Two designs (recessed and flat tip) proved suitable for in vivo testing based upon flow screening and relative recovery results. Both collected fractions from live rats at average experiment times of 190 min for 50 and 100 nLmin-1 flow rates. Basal levels of acetylcholine (Ach), DA, Glu, and serotonin (5HT) were detected via LC-MS. Next, Parylene C sampling probes were microfabricated for in vivo applications. Parylene C is softer than Si and known for its biocompatibility and chemical inertness. We devised a fabrication method which allowed us to create testable sampling probes which was simpler than Si fabrication. Their shanks were 45 µm thick x 180 µm wide x 3 mm long. Two different sampling area designs (sheathed and end-on) were evaluate for flow screening robustness in brain analogues and for relative recovery. The sheathed probe proved acceptable for in vivo experiments, and it offered superior sampling flow post implantation with acceptable recovery. Gamma-amino butyric acid (GABA) levels increased by an average of 750% temporal response to nipecotic acid (n = 3 rats). Basal concentrations for Ach, Glu, glutamine (Gln), glycine (Gly), phenylalanine (Phe), and serine (Ser) were also detected. Non-targeted metabolomics analysis identified an average of 107 compounds for n = 3 experiments via LC-MS. The sheathed probes showed promise for further in vivo applications Finally, COMSOL Multiphysics benefited both the Si and Parylene C projects. Simulations (2-D and 3-D) were created in COMSOL Multiphysics for elucidating the effects of sampling area geometries on relative recoveries for both the Si and Parylene C probes. The 3-D simulations provided a more accurate recreation of multidirectional fluid motion with respect to collection efficiency. Both quiescent and stirred solution conditions were modeled. This permitted the testing of sampling area geometry variations to gauge their impact on relative recovery. Further, models created in both COMSOL and MATLAB guided the design process for the Parylene C probes. Mathematical models revealed suitable design parameters for penetrating brain tissue. Such models may elucidate understanding of the nuances of experimental results and project performance of future designs.