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Local stimulation of tissue can occur naturally in events like immune-mediated inflammation and focal ischemic injuries in brain and is confined to specific regions within tissue, occurring on various timescales. Making chemical measurements at the exact site of stimulation with current technologies is difficult yet important for understanding tissue response. We have developed a microfluidic device capable of local stimulation of brain slices with minimal lateral spread over time and submillimeter, tunable spatial resolution. This device is compatible with electrochemical measurements to monitor signaling at the site of stimulation over time. The PDMS-based device is three layers and contains a culture well, channel layer, and exit port layer for the channels. Channels with exit ports straddling the stimulus channels and ports were specifically fabricated to focus the stimulus over time. We demonstrated that the device is compatible with fast-scan cyclic voltammetry (FSCV) recording of neurotransmitter release. Localized hypoxia in tissue was verified using Image-iT Green Hypoxia Reagent and coupling this device with FSCV enabled measurement of local dopamine changes at the site of focal ischemia for the first time. This work provides a significant advance in knowledge of local neurochemical fluctuations during sustained tissue injury. Overall, the unique capabilities of the device to deliver sustained localized stimulation combined with real-time sensing provide an innovative platform to answer significant biological questions about how tissues respond at the site of controlled, localized injury and chemical stimulation.

Guanosine is an important neuromodulator and neuroprotector in the brain and is involved in many pathological conditions, including ischemia and neuroinflammation. Traditional methods to detect guanosine in the brain, like HPLC, offer low limits of detection and are robust; however, subsecond detection is not possible. Here, we present a method for detecting rapid fluctuations of guanosine concentration in real-time using fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. The optimized waveform scanned from -0.4 V to 1.3 V and back at a rate of 400 V s-1 and application frequency of 10 Hz. Potential limits were chosen to increase selectivity of guanosine over the structurally similar interferent adenosine. Two oxidation peaks were detected with the optimized waveform: the primary oxidation reaction occurred at 1.3 V and the secondary oxidation at 0.8 V. Guanosine detection was stable over time with a limit of detection of 30 ± 10 nM, which permits its use to monitor low nanomolar fluctuations in the brain. To demonstrate the feasibility of the method for in-tissue detection, guanosine was exogenously applied and detected within live rat brain slices. This paper demonstrates the first characterization of guanosine using FSCV, and will be a valuable method for measuring signaling dynamics during guanosine neuromodulation and protection.