An optical micrograph of a single electrode after the complete chip fabrication process is shown in the inset of Fig. . As can be seen from the image, the sensing and protonator IDEs contain 20 microbands each. The optical image also convincingly illustrates the lack of any electrical short circuits between these two electrodes, thereby attesting to the high reproducibility of the lift-off process. The gold microbands are 1 µm wide with a separation of 1 µm. The sensors were strategically placed 2 mm away from the gold CE to avoid any interference from electrolysis products or local pH changes at the counter electrode with the sensor readings. The chip incorporates a platinum pseudo RE situated near the CE. Each electrode, including the protonator/sensing, reference, and counter electrodes, is linked to a potentiostat via a gold pattern that ends with circular and rectangular pin outs for external connections. To verify the operation of the interdigitated electrodes, CV characterization was used in the presence of 1 mM FCA for all the six electrodes in a chip as shown in Fig. . The resultant CVs displayed a diffusion-limited behavior indicating a large microelectrode which arose from the radial diffusion mass transport profiles surrounding to the IDEs, leading to an overall time-independent behavior (Wahl et al., 2014). All the sensors exhibited excellent intra-chip reproducibility, as a RSD was found to be 0.09% from the limiting current values that obtained with forward scans at 0.2 V vs. Pt. The generation of a ~12 nA current serves as confirmation of electron transfer occurring at the sensors IDEs which further demonstrates the effectiveness of the on-chip RE and CE for electrochemical measurements. The sensors were subsequently characterized using the Generator-Collector mode, with a representative voltammogram depicted in Fig. . The generator comb IDEs initially oxidized the FCA, forming FCA + species which then diffused across the gap to the collector electrode, where they were reduced back to FCA. This phenomenon known as redox cycling, leading to an increase in the measured currents. The generator-collector cyclic voltammograms exhibited a quasi steady-state, time independent behavior commonly associated with ultra-microelectrodes (Wahl et al., 2018). The sensor's collection efficiency, calculated as the ratio of the collector to the generator currents, was found to be approximately 83%. This implies that about 83% of the protons generated at a protonator IDEs would diffuse to the working IDEs, thereby adjusting the pH as required. Fig. presented the optimization of o-PD deposition concentration with a 0.5 mM glucose addition to the modified sensor electrodes. Three concentrations of o-PD (5, 10 and 15 mM) were individually prepared and it can be seen from the figure that the 10 mM o-PD concentration exhibits better response compared to the others. This could be attributed to the fact that at lower concentrations of o-PD, there are fewer molecules available for the electrochemical oxidation or reduction of H2O2. On the other hand, at excessive concentrations, o-PD molecules may saturate the active sites on the electrode surface, leaving no room for the oxidation or reduction of the H2O2. This could limit the electrocatalytic activity and thus lower the sensor response. The optimum pH for GOx was determined across a pH range of 4 to 8 at 2 mM glucose concentration. GOx demonstrated high activity within the pH range of 5.0 to 6.0 (Fig. ), with the optimum pH observed at 5.5. It was noted that the enzyme activity diminished when the pH deviated above or below 5.5. Consequently, as discussed above it is essential to ensure that all samples are at pH 5.5. Following each stage of the fabrication process, specific techniques were utilized to meticulously analyze the modifications in surface properties. Since in this work, H2O2 produced enzymatically at the time of interaction of glucose to GOx, thus CVs were carried out in 0.1 mM H2O2 to understand the surface properties of the electrodes (Fig. ). An increase in the oxidation current approximately 20 nA was recorded between the Pt-B (blue curve) modified surface and bare Au (red curve) surface at 0.2 V vs. Pt, indicating the enhanced electroactive area of the Pt-B modification. Following the deposition of o-PD/β-CD (green curve), a distinct decrease in both the oxidation and reduction currents is evident, which can be ascribed to the formation of the o-PD layer (Wang et al., 2017). This observation supports the idea that H2O2 can permeate the o-PD/β-CD layer to reach the sensor electrode. A comparable approach was employed to acquire the voltammograms in 1 mM FCA to understand the impact of including GOx surface modifications, as illustrated in Fig. and the redox currents further decreased as expected. EIS was utilized to evaluate the change in charge transfer resistance following each electrodeposition. Fig. exhibits the corresponding EIS measurements and show excellent agreement with CV data. Nyquist plots may exhibit a semi-circular region along with a linear part. In the higher frequency range, the semicircular area is associated with the electron transfer limited process. The diameter of this semi-circle is known as charge transfer resistance, denoted as Rct. The decrease in Rct after Pt-modification indicates lower diffusion resistance and increased conductivity. The Rct typically decreased from 3.5 MΩ at the bare Au surface to 0.04 MΩ (inset), after the enhanced surface area Pt-B electrodeposition. The equivalent circuit used to fit the EIS data is shown in the Fig. : inset. Upon the deposition of o-PD/β-CD/GOx, using the same parameters, a complete semi-circle could not be achieved for an electrochemical fit. Nevertheless, a distinct change was observed in impedance behavior, i.e. a very large increase in impedance. A diffusion-controlled Warburg effect was not observable, as the electrodes remained diffusionally independent.