The changes in FAM f luorescence (pH 7 f ixed, 0.5μM solution in KCl 100mM) with the applied voltage were tracked focusing the microscope in the WE during the CV measurements and following the peak emission at 525nm. showing the voltage, the current, and the normalized FAMf luorescence in black, red, and green, respectively. The current curves reported the typical redox peaks corresponding to the proton exchange reactions, f rom which f or each cycle the f aradaic currents could be extracted (quantitative data of the cycles and details of the calculations shown in SI-2, SI-3). We attribute the decrease of FAM f luorescence to the increase of acidity in the cell due to the proton exchange reactions. Figure shows the details in one cycle with the FAM f luorescence (in green) and the calculated charge exchanged, Q (blue curve) as f unction of time extracted f rom the Faradaic currents. Q(t) is expressed as the negative logarithm scale normalized to the Faraday number and the volume of the cell, to which we added a baseline of Q (0) = 10 7 (in units of elementary charge) where the exponent 7 corresponds to a baseline of neutral pH to account for the initial conditions of the electrolyte. In this way the charge is expressed in equivalent units of pH. Figure shows the f luorescence curve or the FAM vs. pH, calibrated with an ISFET pH meter in a solution that was titrated with KOH. We observed a small delay of the f luorescence respect to the Faradaic currents part of which (~1s) can be attributed to the integration time of our spectra, but we did not consider this delay relevant in the overall cycle. We also observed a very good correspondence of the f luorescence obtained in the cell at the equivalent pH calculat ed by Q and the one at pH produced with the external titration. The maximum f luorescence is obtained when no protons were released in the system or when they are reabsorbed by the 4-ATP molecules af ter the reduction cycles that correspond to the moment when the normalized Faradaic currents indicate pH 7. The intensity decreases when the oxidation of the 4-ATP occurs (-0.6 to 0.6V) and then increases during the reduction f ragment of the cycle (0.6 to -0.6V) where the protons were re-adsorbed. This returning to the normal f luorescence before the protons activity is also consistent with our interpretation that the phenomenon is quasi-reversible, and that the adsorption of the protons released in the system is eff icient. In the cycle we detail here, the equivalent pH of the Faradic currents reached values close to 1. An independent calibration of the relation of the Q derived f rom the Faradaic currents with the acidity was observed with the Tautomerization of FAM. Tautomerism is a solvent-dependent chemical process that commonly results in the relocation of a hydrogen atom and aff ects the activity of biomolecules and dyes. Either an acid or a base can catalyze the proton transf er . FAM has diff erent forms (each one having diff erent tautomers that can coexist in water), and the transition f rom one to another can be driven by acidity. In aqueous solution, FAM has diff erent acid/base pairs (cation C, neutral N, monoanion M, dianion D, and trianion T) depending on the pH. Aschi et al. were the f irst to present accurate measures of the pK a values of the diff erent pairs of FAM using the spectrophotometric titration, the chemometric analysis as well as quantum mechanical estimations. Based on their paper, we adapted first the nomenclature o f the diff erent species by considering the global charge of the tautomer with the groups: i) the anion f amily (T (FAM 3-), D (FAMH 2-) and M (FAMH 2 -)) that had a similar absorbance at 470 nm (though T, and D had their maximum at lower f requencies) designated as FAM1, ii) the neut ral tautomer N (FAMH 3 ), which had the lowest absorbance at 470 nm designated as FAM2 and iii) the cation compound C (FAMH 4 + ) that increased the absorbance at 470nm respect to FAM 2 but not respect FAM 3. FAM1 to FAM3 are represented in f ig. 3.