On-chip microelectrode array and in situ transient calibration for measurement of transient concentration gradients near surfaces of 2D cell cultures

•Developed on-chip MEA to address gradient measurements in 2D cell culture setups.•Developed in situ transient calibration for minimizing sensitivity variability in MEAs.•Assessed reliability of dynamic gradient measurements using large sink electrode that mimics cellular uptake.•Measured gradients from uptake intervals as short as 150 ms, and up to 2000 s.•Measured physiological dynamic gradient due to H2O2 uptake by human astrocytes. Amperometric microelectrode arrays (MEAs) interrogate the concentration at multiple positions simultaneously and with sufficient sampling rates, thus being able to capture fast transient gradients. However, sensitivity variability issues in amperometric MEAs degrade the reliability of the measurements, particularly at the small concentration scales found in physiological studies. This paper describes the development of on-chip platinum amperometric MEAs and in situ transient calibration for reliable measurement of physiological transient concentration gradients. The designed MEA geometry facilitates positioning near a 2D cell culture setup, and the proposed in situ transient calibration minimizes the effects of sensitivity variability, thus allowing for calculation of gradients based on concentration differences between closely spaced electrodes. The effectiveness of the MEA and the in situ transient calibration was evaluated by measuring controllably-generated gradients, and then calculating the difference between experimental and simulated data using normalized time analysis. Gradients generated by periodic uptake intervals as fast as 150 ms followed by recovery intervals of 60 s were measured over a spatial range of 70 μm, with spatial resolution of 35 μm, and sampling time and measurement time of 10 ms. Transient gradients of hydrogen peroxide were also measured above the surface of a 2D cell culture of human astrocytes, thus demonstrating the approach in actual physiological measurements.