# Non-Invasive Hydration Monitoring with a Graphene Dual Sweat Sensor
## 2 Materials and Methods
### Sensor Fabrication  
The sensors utilized in this study were supplied by the Center for Advanced 2D Materials (CA2 DM) at the National University of Singapore (NUS). The electrodes were manufactured using a screen-printing technique on a flexible, high-resistance polyethylene terephthalate (PET) substrate. The sensor comprises two graphene-based working electrodes, each with an area of 2.3 mm\({}^{2}\), sharing an Ag/AgCl quasi-reference electrode (RE) and a graphene counter electrode (CE). A dielectric coating was applied as an insulating layer.  
To verify the initial variability of the electrode surface prior to functionalization, cyclic voltammetry was conducted from \(-\)0.15 V to 0.75 V for five cycles with 2 mM of potassium ferricyanide in 0.1 M of hydrochloric acid and 0.1 M of potassium chloride. The cyclic voltammetry profile of the graphene dual-sensor, as seen in Figure 1b, displays an average peak current of 31.3 \(\mu\)A, with a standard deviation of 0.8 \(\mu\)A across five different sensors. This reproducibility indicates that the base graphene surfaces exhibit similar and stable baseline electrochemical properties, which decreases the sensor-to-sensor variability post-functionalization.  
The sodium ion-selective membrane was prepared by mixing 1 wt.% of sodium ionophore, 65.5 wt.% of DOS, 0.5 wt.% of NaTFPB, and 33 wt.% of PVC. The potassium ion-selective membrane was prepared by mixing 2 wt.% of valinomycin, 65 wt.% of DOS, 0.5 wt.% of NaTPB, and 32.5 wt.% of PVC. The sodium and potassium ion-selective membranes were then prepared by dissolving 150 mg of the mixture in 1 mL of THF. The mixture was stirred with a magnetic stirrer (1000 rpm for 30 min) and sonicated (20 min), resulting in a clear and transparent solution. The potassium and sodium ion-selective membranes were drop-cast on the working electrodes. The working electrodes were covered with 2 \(\mu\)L of the membrane cocktail and left to dry overnight at room temperature to ensure complete solvent evaporation.  
To enhance the functionality of the reference electrode, a multi-layer configuration was implemented. The first layer consisted of a mixture of 1 M NaCl dissolved in a 5 wt.% Nafion solution. The second layer was formulated by dissolving 79 mg of PVB and 50 mg of NaCl in 1 mL of methanol. Finally, the third layer was composed of a 5% Nafion solution. To construct these layers, 2 \(\mu\)L of the first, 4 \(\mu\)L of the second, and 1 \(\mu\)L of the third layer were drop-casted onto the reference electrode. Each layer was subjected to a drying process at 40 \({}^{\circ}\)C for 15 min.  
The sensors were then conditioned in 10 mM of NaCl solution for 30 min before testing. Sodium and potassium standard solutions were prepared by dissolving NaCl or KCl salt in ultrapure water and diluted further to obtain solutions of different concentrations.  
The performance of the reference electrode with the multi-layer junction was evaluated against a single junction commercial Ag/AgCl reference in concentrations of NaCl solution ranging from 0.1 mM to 1 M. To verify the electrode stability, the signal was measured for over 100 min.