Driving electrochemical reactions at the microscale using CMOS microelectrode arrays

Abstract

Precise control of pH values at electrode interfaces enables the systematic investigation of pH-dependent processes by electrochemical means. In this work, we employed high-density complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) as miniaturized systems to induce and confine electrochemical reactions in areas corresponding to the pitch of single electrodes (17.5 μm). First, we present a strategy for generating localized pH patterns on the surface of the CMOS MEA with unprecedented spatial resolution. Leveraging the versatile routing capabilities of the switch matrix beneath the CMOS MEA, we created arbitrary combinations of anodic and cathodic electrodes and hence pH patterns. Moreover, we utilized the system to produce polymeric surface patterns by additive and subtractive methods. For additive patterning, we controlled the in situ formation of polydopamine at the microelectrode surface through oxidation of free dopamine above a threshold pH > 8.5. For subtractive patterning, we removed cell-adhesive poly-L-lysine from the electrode surface and backfilled the voids with antifouling polymers. Such polymers were chosen to provide a proof-of-concept application of controlling neuronal growth via electrochemically-induced patterns on the CMOS MEA surface. Importantly, our platform is compatible with commercially available high-density MEAs and requires no custom equipment, rendering the findings generalizable and accessible.

Fig. 1. Inducing hydrolysis locally on CMOS MEAs yields spatially confined pH changes. The CMOS MEA enables simultaneous routing of practically any combination of electrodes. A voltage applied between two sets of electrodes forms an anode–cathode pair, at which hydrolysis can be induced. The electrochemical reactions yield the formation of hydroxide ions (OH⁻), which locally increase the pH. At the anode, the counter-reaction decreases the pH by generating hydrogen ions (H⁺). The potential on the cathode was set by an external SMU while the anodic potential was set by an internal DAC.

Fig. 2. Quantifying pH optically on CMOS MEA surfaces using SNARF. A The fluorescence emission spectrum of SNARF is dependent on the pH of the environment. In an acidic environment, the emission maximum lies at 580 nm. This peak shifts to 640 nm in a basic environment. The structural change of the molecule was adapted from ref. . B Fluorescence emission spectra obtained by imaging SNARF solutions with known pH values on the CMOS MEA. The dashed lines indicate the local maxima of the SNARF emission spectra. C Fluorescence intensity obtained from the emission spectra at the dashed lines shown in B for both the red and green windows. D Dividing the fluorescence intensities from the red vs. green windows yields fluorescence ratios that can be mapped to corresponding pH values.

Fig. 3. The spatial extent of the induced pH change can be confined precisely. A Schematic of a routing in which electrodes are connected to an external voltage source, while all other electrodes are left floating. B Resulting fluorescence images with SNARF as the fluorescent reporter using the routing shown in A with an applied voltage of 1.6 V. The induced pH change is diffuse, lacking spatial confinement. The green image shows the fluorescence intensity within a 10 nm window centered around the 580 nm emission peak while the red image shows the fluorescence intensity around the 640 nm peak. C Schematic of a routing where neighboring electrodes are connected to a low-impedance sink, i.e., to a DAC that is set to the mid potential of the chip at 1.65 V. D Applying a voltage of 1.6 V between the two electrode combinations, yields a precisely confined pH change. The reduced fluorescence intensity in the green window and the increase in the red window, demonstrate the fluorescence emission shift of SNARF that occurs when the solution shifts toward a basic environment. E Switching between predefined electrode configurations allows changing the locations of pH induction quickly in sequence. Here, three frames of a simulated game of Snake utilizing FITC as the fluorescent pH dye are shown. The “snake”, “food”, and square boundary comprise the cathode, and all other electrodes form the anode.

Fig. 4. Relationship between applied voltage and induced pH changes on platinum black (PtB) and platinum (Pt) coated microelectrodes. A Increasing the applied voltage in steps of 0.1 V after 1 min yielded large changes in the fluorescence emission within the green and red emission windows when PtB electrodes were used. In comparison, Pt electrodes induced smaller changes. The grey vertical lines indicate time stamps at which the applied voltage was increased. B The fluorescence emission ratio from the data as shown in A yields a curve that relates the applied potential to the induced pH. A large pH range (6–9) can be covered using PtB electrodes, while Pt electrodes are limited in pH range with a maximum pH of around 7 when the highest voltage was applied. Shown here are the mean values and the standard deviation for N = 2 Pt and N = 3 PtB chips. C Temporal behavior of the induced pH change after the application of an external voltage for two PtB CMOS MEAs. The solid and dotted lines indicate data from two different chips. The initial pH was set to 5.8 and a voltage between 1.1–1.3 V was applied. The induced pH changes at 1.3 V showed decay to pH 8 within a 20 min window, while at 1.1 V the induced pH change decayed to a value around pH 6, close to the initial solution pH value. At 1.2 V, a decay to initial pH was observed on one chip (continuous blue line), while a steady pH around 7 was achieved on another chip (dashed blue line). The arrow indicates the formation of an air bubble under the microscope, which yielded the termination of the experiment after 20 min.

Fig. 5. Precise pH control allows additive and subtractive patterning at the single-electrode level. A Additive patterning using polydopamine. Left: Forming polydopamine through direct oxidation of dopamine. At the electrode, dopamine is electrochemically oxidized forming 5,6-dihydroxyindole, which then crosslinks to form polydopamine that deposits on the electrode surface (black coating). Right: Polydopamine formation through oxidation of dopamine in a basic environment. Inducing hydrolysis yields an increased pH at the cathode, which subsequently yields the oxidation of dopamine to 5,6-dihydroxyindole and the formation and deposition of polydopamine. The figure is adapted from previous literature ref. and . B Additive and subtractive patterning on a CMOS MEA that was previously coated with fluorescein (FITC)-labeled poly-l-lysine (PLL). Left: Deposition of polydopamine on the cathode. Right: Delamination of PLL on the anode. C Electrochemically driven subtractive patterning. Applying a large enough positive electric potential at a microelectrode leads to the delamination of previously bound FITC-PLL. The void can subsequently be filled with another molecule, such as an antifouling polymer, PMOXA. D Removal of FITC-PLL and replacement of PMOXA. The PMOXA is rendered fluorescent using rhodamine and hence yields a fluorescence signal on the CMOS MEA. The white (reference) and red (signal) dashed boxes indicate the regions of interest for the analysis shown in E. E Quantitative analysis of the PMOXA addition. The relative fluorescence intensity within the FITC detection range did not change significantly (t-test, p > 0.2) indicating the PLL replacement outside the routed electrodes is negligible. The increase in fluorescence in the PMOXA detection range demonstrates PMOXA adherence to the anodic electrodes (t-test, p < 0.001).

Fig. 6. Neurons following the induced surface pattern on top of the CMOS MEA surface. Cortical neurons were seeded onto a CMOS chip on whose surface PLL was locally removed in the shape of a square. The resulting void was backfilled with PMOXA. The cortical primary rat neurons were rendered green fluorescent using CMFDA and imaged at DIV 14. No soma are visible within the square while axons were able to grow inside the square in between electrodes.