A neural probe to sample brain fluid in nanoliter volumes for quantitative molecular studies

Neural functions operate in tightly controlled conditions that are mediated by multiple electrical and chemical phenomena. Brain disorders such as Parkinson's Disease and Alzheimer's Disease perturb these conditions and cause a loss of neurons, which impairs the motor and cognitive functions. Thus, an access to the concentration of the neurochemicals in the brain and to their temporal evolution would be an asset to understand these diseases better and develop treatments that could stop their progression and even restore neural functions.Among the techniques to measure neurochemicals in the brain, fluid sampling approaches such as microdialysis allow the continuous collection of molecules from the extracellular fluid (ECF), by diffusion in a perfusate that can be retrieved and analyzed offline. However, these experiments can be long and the real concentration of the molecules in the ECF cannot be quantified directly from the samples. This thesis aims to propose a solution to these limitations; thus, a new sampling approach called Droplet on Demand (DoD) and a neural probe for it were developed.The DoD approach is inspired by the theoretical foundations of microdialysis and by the limitations of continuous sampling methods. Thus, it is proposed as a sequential sampling method in steps: a perfusion step to create a pocket of perfusate in the brain, a diffusion step to capture molecules in this pocket, a sampling step to collect the pocket and the molecules in a droplet, and a phase to let the concentration in the ECF equilibrate. This offers a way to collect samples that closely reflect the molecular concentration in the ECF for quantitative molecular studies. Moreover, the DoD can be applied on-demand, to probe the molecules in the ECF at specific experimental times, punctually or repeatedly.The neural probe was fabricated with polyimide and SU-8 technologies to implement the DoD approach during acute experiments in the brain of mice. It integrates microfluidic channels, droplet-sensing electrodes, and a stimulation electrode in its 320 µm wide and 80 µm thick needle. An optical fiber was also added to it for optical stimulation.The concepts of the DoD were illustrated in simulations and confirmed in experiments in agarose brain phantoms in vitro. They showed that the diffusion time allows tuning the concentration of the molecules collected in the samples, with stable characteristics over repeated sampling events. Acute in vivo experiments were also performed in mice and samples of typically 24.4 nL were collected every 1 to 2 minutes, 20 minutes after inserting the probe. The samples were stored in a capillary and an analytical procedure with mass spectrometry was developed to confirm the properties of the DoD for the quantification of glucose in the brain. This also allowed the detection of acetylcholine and glutamate. Finally, a module for chronoamperometric measurement of dopamine (DA) was developed with pyrolyzed carbon electrodes, and proof-of-concept experiments showed that it could be coupled to the outlet of the probe for DA measurement in droplets.By combining the probe and the DoD with the appropriate analytical methods, quantitative molecular studies of neurotransmitters, metabolites, proteins, or even ribonucleic acids (RNAs) could now be envisioned. They would benefit from the electrical, chemical, and optical stimulation features of the probe and from the accurate chemical recording enabled by the DoD approach.