Effect of Anesthesia Gases on the Oxygen Reduction Reaction

The oxygen reduction reaction (ORR) is of high importance, among others, because of its role in cellular respiration and in the operation of fuel cells. Recently, a possible relation between respiration and general anesthesia has been found. This work aims to explore whether anesthesia related gases affect the ORR. In ORR, oxygen which is in its triplet ground state is reduced to form products that are all in the singlet state. While this process is “in principle” forbidden because of spin conservation, it is known that if the electrons transferred in the ORR are spin-polarized, the reaction occurs efficiently. Here we show, in electrochemical experiments, that the efficiency of the oxygen reduction is reduced by the presence of general anesthetics in solution. We suggest that a spin–orbit coupling to the anesthetics depolarizes the spins. This causes both a reduction in reaction efficiency and a change in the reaction products. The findings may point to a possible relation between ORR efficiency and anesthetic action.


Materials and Methods
Electrochemical instrumentation: The electrochemical measurements were performed using a three-electrode cell in a flat configuration to avoid gas entrapping. The working electrode (WE) was prepared by e-beam evaporation of Ti (10 nm)/Ni (150 nm)/Au (8 nm) on Si (100) wafer with deposition rates of 0.2, 0.5 and 0.2 Å/s, respectively in base pressure of 10 -7 Torr. An Hg/Hg2Cl2/KClsaturated (SCE) and a Pt wire were used as reference electrode (RE) and counter electrode (CE), respectively. It is important to note that the working electrode was static during all measurements and its area (~0.78 cm 2 ) was constant. Before using, the working electrode was first cleaned in boiling acetone for 10 mins and then in boiling ethanol for 10 mins. This was followed by UV/ozone treatment for 10 mins and then immersed in ethanol for 45 mins and finally dried with an N2 gun and used for the experiment. All the electrochemical measurements were performed with a potentiostat (PalmSens4) electrochemical workstation at room temperature, electrochemical data acquisition and post-elaboration were performed with the PSTrace 5.9 software, also from PalmSens.
For oxygen reduction, 7 ml of 0.1 M KOH (pH = 12.6) electrolyte was used in the cell. In a typical experiment, the working electrode was tightened via a silicon o-ring to the bottom of a teflon cell featuring a hole of 0.78 cm 2 area, in an air tight configuration to avoid gass loss. Before performing the experiments, all gases were purged into the electrolyte for 30 mins. For the liquid anesthetics (CHCl3, Halothane and CCl4), oxygen was purged through a closed container with the anesthetic, and the mixed vapors continued to the electrochemical cell. Thus, oxygen was purged through a conical flask which contained the pure liquid inhalational anesthetics, maintaining a constant hydraulic swing of 2.5 cm. In the case of the solid KBr, O2 was purged into a cell containing a 0.1 M KOH aqueous solution with 10 mM KBr concentration. In the case of gas anesthetics, i.e., N2O, N2 and Ar, these were purged in the cell together with oxygen for 30 minutes (to obtain careful solution saturation in the gas mixture), the appropriate "oxygen/anesthetic-gas" dosing ratio was implemented by using a flow meter. Then, Linear scan voltammetry (LSV) measurements, in the 0 to -0.5 V potential range with a potential scan rate of 50 mV/s, were carried out. It should be noted that during the scan, the needle, for gas purging, was taken out from the solution for stable current but it is kept above the solution to avoid change in concentration of gas.
For measurements carried out using ferromagnetic surfaces a magnet was placed just below the S3 surface of the WE (so to induce an out-of-plane magnetic field). The position of magnet and distance between the WE surface and the magnet were constant in all the experiments. A permanent block magnet, 15 mm × 15 mm × 5 mm in size, NdFeB of class N45, with a surface field ~0.5 T, was used for the experiments.

Hydrogen peroxide quantification
The influence of the anaesthetics compounds on the ORR mechanism can be rationalized by measuring the quantity of hydrogen peroxide produced under potentiostatic regime (chronoamperometry) by applying a suitable negative potential (-0.4 V vs. SCE for 30 minutes).
Once again, before the chronoamperometry run, a 0.1 M KOH aqueous solution saturated by pure oxygen (or by the suitable "oxygen/anesthetic-gas" mixture) for 30 mins. The formation of hydrogen peroxide was measured exploiting the reaction with o-tolidine, detected by measuring UV-Vis spectra (after determining the appropriate calibration curve).
During chronoamperometric measurements, the gas needle was put above the surface of the solution so to avoid gas vortices in the solution. For chronoamperometry measurements with the magnetic field, the magnet was placed below the working electrode, following the same procedure

Working Electrode Magnetic characterization
The magnetic property of working electrode (Ti (10 nm)/Ni (150 nm)/Au (8 nm) on Si (100) surface) was measured using a MPMS3 superconducting quantum interference device (SQUID) (LOT-Quantum Design Inc.) with an absolute sensitivity of 10 -8 emu. The magnetic moment of sample of size ~ 4 × 4 mm and weight 22.7 mg was measured at room temperature and in an outof-plane applied magnetic field geometrical configuration. The surface exhibits ferromagnetic behaviour with a coercivity of about 100 Oe and a saturated magnetization at about 0.47 T. The magnetic field of the working electrode was also measured using a 3-axis magnetic field transducer with sensitivity of 50 V/T from Sentron and the value of the surface magnetic field was 0.42 T. Figure S5: The magnetic moment measured by a quantum interference magnetometer (SQUID) as a function of the magnetic field applied for the Ni (150 nm)/Au (8 nm) layer that covers the silicon substrate.