The primary goal of the proposed work is to take the first steps toward an independent research program that focuses on the development and application of biomedical micro/nanosystems based on independently optimized materials for the study, monitoring, and treatment of neurological conditions, including traumatic brain injury (TBI) and stroke. Specifically, this project concerns the development of a platform technology involving the integration of optimal electrode, enzyme immobilization, and substrate materials for an electrochemical sensor to continuously monitor extracellular glutamate levels with high spatiotemporal resolution, sensitivity, selectivity, stability, and wide linear range. This multi-scale materials system will incorporate a bio-adaptive polymer nanocomposite substrate, gold and graphene electrodes, and polymer nanowires for enzyme immobilization. This combination of materials will provide a sensor with the requisite properties for long-term implantation for neurochemical monitoring during normal activity. Though glutamate is the focus of this work, the methods to develop this sensor can be applied to many different bioanalytes, which will be integrated in later implementations of this biosensor. A means to continuously monitor the neurochemical state outside of the hospital environment will 1) provide enhanced understanding regarding the pathophysiology associated with the long-term effects of TBI at the site of the injury 2) delineate the relationship between neurochemistry and clinical dysfunction, 3) allow for long term monitoring of rehabilitation and drug interventions and 4) allow for the novel drug therapies to be developed with highly controlled delivery.
The first aim toward the overall goal is to provide a stable neural interface with high-sensitivity (>500 nA.M-1.cm-2) to changes in in vivo glutamate concentration through efficient glutamate oxidase immobilization. This with be achieved using microfabrication processes customized for the unique materials set proposed to achieve this goal. A novel stimuli-responsive polymer nanocomposite, poly (vinyl acetate) (PVAc-NC), substrate will be implemented to minimize the inflammatory response to the implant, thus maximizing the stability of the biotic/abiotic interface. PVAc-NC has a high elastic modulus (Edry ~ 4 GPa) in its dry state, permitting needle- like insertion into brain tissue, but displays a three order-of-magnitude reduction in elastic modulus (Ewet ~ 12 MPa) after absorption of physiological fluids, greatly reducing mechanical mismatch with cortical tissue (Ecortex ~ 10 kPa). High glutamate sensitivity and selectivity will be established by coating the gold electrode site with a nanostructured conductive polymer nanowire layer, such as polypyrrole, which will serve to immobilize glutamate oxidase, an enzyme that selectively reacts with glutamate to form several products, including hydrogen peroxide. The hydrogen peroxide will then by oxidized by a potential applied to the electrode via external electronics and the oxidation current will be measured. The magnitude of this current is linearly-related to the concentration of glutamate near the electrode.
The second aim i s to expand the dynamic linear range of the glutamate sensor to ensure linearity between measured current and actual glutamate concentration is maintained through the entire range expected in normal and injured brains. Toward this end, thin-film gold electrodes will be replaced with a carbon-based graphene electrode site. A transfer process will be required to integrate graphene onto PVAc-NC, as PVAc-NC is incompatible with graphene growth temperatures. These electrodes will then be functionalized with glutamate oxidase using methods comparable to those developed toward the first objective, but adapted for use on a graphene-based electrode. The two types of electrochemical sensors will be characterized and calibrated in vitro, then tested in vivo in a rodent model of TBI to assess electrode stability and the correspondence of in vivo behavior with the determined in vitro calibration parameters.
Traumatic brain injury (TBI) and other neurological disorders are important concerns with regard to veterans' health, especially for those veterans involved with recent combat operations. Many of the symptoms and effects of severe neurotrauma persist and progress for years beyond the initial injury, causing permanent severe disability. These sequelae are related to alterations in neurochemical regulation, but are not well understood due to the lack of tools available to monitor neurochemical levels continuously outside of the realm of an intensive care unit in a hospital. The proposed biosensor will continuously monitor neurochemical levels during normal activity with very high spatial and temporal resolution. By augmenting the functionality of well-established sensors for detecting and interpreting electrical neural signals, we will gain new understanding of the long-term effects of neurotrauma, the relationship between behavior and neurochemistry, and develop new ways to treat neurotrauma and regulate neurochemistry.
