A platinum (Pt) sEEG depth electrode (Spencer probe depth electrode, RD10R-SP07X-00, length: 390 mm, diameter: 0.86 mm, electrode length: 2.29 mm, electrode spacing: 7 mm, 10 cylindrical shank electrodes) was supplied courtesy of Glasgow University and Queen Elizabeth University Hospital, Glasgow (UK) and manufactured by Ad-Tech Medical, WI (USA). A Pt macro disc electrode, 1.6 mm dia., was purchased from ALS Co., Ltd. (Japan). A Pt foil counter electrode and an aqueous Ag/AgCl reference electrode filled with 3 M KCl were purchased from Metrohm, Herisau (Switzerland). Deionised (DI) water from an Elga LabWater PURELAB Chorus 2 lab water system was used to make all solutions. H2SO4 and H2O2 were purchased from Fisher Scientific (UK). PBS tablets, hexaammineruthenium(III) chloride, potassium ferricyanide, potassium ferrocyanide, gelatin powder, glutaraldehyde solution, dopamine hydrochloride, D-(+)-glucose, sodium L-glutamate, glucose oxidase from Aspergillus niger, and L-glutamate oxidase from Streptomyces sp. were purchased from Merck Sigma-Aldrich (UK). Chloroplatinic acid hexahydrate and lead acetate trihydrate were supplied courtesy of the Institute of Photonics, University of Strathclyde (UK).

Macro disc electrodes were manually polished using 1, 0.3, and 0.05 μm alumina particle and water slurry on a microfibre pad in a figure eight pattern for 60 repeats. Following each polishing particle size, the electrodes were rinsed with DI water and briefly placed in an ultrasonic cleaner in a beaker of DI water for 20 s then rinsed again. Electrodes were then electrochemically cleaned by applying 10 CV cycles in 0.1 M H2SO4 except where noted. Cleaning CV parameters: potential window −0.4 to +1.8 V, step 0.01 V, scan rate 0.1 V/s.

Phosphate buffer saline (PBS) solution, used as a biological buffer and electrolyte, was made at 1× concentration using PBS tablets in DI water as per the manufacturer’s instructions (1× is typically defined as a solution containing NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 1.8 mM, and is pH 7.4 at 25 °C). Solutions of 1 mM hexaammineruthenium(III) (HexRu) in 1× PBS and 1 mM ferricyanide and ferrocyanide (FF-Cy) in 1× PBS were used in all characterisation experiments. Experiments were performed in a 3-electrode cell with a working, counter, and reference electrode in electrolyte solution in an open beaker unless otherwise noted. Room temperature and standard atmospheric conditions were used except where noted. HexRu solutions were purged with argon for 10 min before use. The CV parameters used were 10 mV E-step, 3 scans, and 100 mV/s unless otherwise noted, and the potential window is shown in each experiment. The DPV parameters used were 10 mV E-step, 25 mV E-pulse, 0.05 s t-pulse, and 50 mV/s scan rate. The SWV parameters used were 10 mV E-step, 100 mV amplitude, and 20 Hz frequency. Electrochemical impedance spectroscopy (EIS) measurements were either taken at E1/2 for HexRu or at OCP measured for 10 s for FF-Cy, max freq. 100 kHz to min freq. 5 Hz in 44 steps, and 10 mV amplitude sine wave stimulation. EIS data was fitted to a Randles equivalent circuit model for extraction of parameter values.

Chronoamperometric (CA) measurements were taken with the working electrode at +0.7 V vs the Ag/AgCl reference electrode. CA measurements to characterise sensitivity were performed by stepwise additions of a stock concentration of analyte to a known volume and concentration, briefly stirred, allowed to settle for 100 s, and then the working electrode current value was extracted from the final data point before the next addition. For example, starting from a baseline of 10 min in 20 ml of 1× PBS, 20 μl of 100 mM glucose was then added, stirred, and allowed to settle for 6 repeated steps. This replicates the real-time nature of a recording in vivo and allows evaluation of the rise and settling time. Dopamine (DA) current measurements were extracted from the measured CVs at a potential of +0.25 V as this was approximately where the largest change was observed.

Electrodeposition of Pt black (also referred to in the literature as electroplating or platinization) was performed by applying a pulsed current rectangular waveform followed by a constant current waveform. The applied current of both the pulsed waveform and the constant current waveform was −0.3 mA. The pulsed waveform had a 1 s period and a 60% on duty cycle at the selected current and was 0 mA between the pulses. Sixty cycles of the pulsed waveform were applied followed by 60 s of constant current. The plating bath solution consisted of 7 mM chloroplatinic acid and 1 mM lead acetate in deionised water.

Dip coating solutions of 10 U/μl GOx, 0.5 U/μl L-gluOx, 1% w/v gelatin, and 1% glutaraldehyde all in 1× PBS were used unless otherwise noted. Functionalisation of the electrode and coating of the enzyme was performed by repeated dip coating in solutions of the selected enzyme, then gelatin, and then glutaraldehyde sequentially. Each dip coated solution was left to dry at room temperature for 3 min before the next was applied and this was repeated 5 times for a total of 15 dipped layers applied. The electrode was then left to finish crosslinking and dry for 16 h at room temperature before use. The dry crosslinked enzyme layer was rinsed, rehydrated, and stabilised in 1× PBS for 30 min before testing and use to wash out any free protein or crosslinker and minimise leaching or changes in the layer whilst recording measurements.

A PalmSens 4 potentiostat with MUX8-R2 multiplexer and PSTrace 5.9 software (PalmSens, Houten, Netherlands) was used for electrochemical measurements, data acquisition, baseline correction, and peak current measurements. OriginPro 2021b software (OriginLab, MA, USA) was used for all other data analysis and presentation. AFM images were acquired using an Asylum Oxford Instruments MFP-3D AFM with a Tap300Al-G probe, 40 N/m, 300 kHz, in AC air topography (tapping) mode.