Extended Data Fig. 1: Nitroxyl (HNO) and nitric oxide (NO) biosensors calibration.
From: Discovery of endogenous nitroxyl as a new redox player in Arabidopsis thaliana
a, Mechanism of specific electrochemical detection of HNO by cobalt porphyrin (CoP) covalently bonded to a gold surface. The developed sensor is an electrochemical HNO sensing device based on the covalent bonding of a cobalt porphyrin to a gold surface. A surface effect modulates the redox potentials allowing discrimination between HNO and NO. The electrode potential is set at a value of 0.8 V, where the porphyrin is stable as Co(III)P, observing a basal current. The reaction with HNO produces the Co(III)PNO¯ complex, which under the conditions described above is oxidized to Co(III)PNO. Due to the lability of the resulting Co(III)PNO complex, the system rapidly returns to Co(III)P, allowing the catalytic cycle to begin again. Since each electrode is only covered with a surface concentration, the total amount of trapped HNO is very small. Also, it is important to note that since a small amount of current can be detected, the ‘real’ amount of azanone reacting with the electrode is generally negligible compared to the total amount of HNO in the system (less than 1 %), and does not significantly perturb the production and consumption of HNO, making it a powerful tool in mechanism and kinetics studies where azanone plays a key role.11,12 b-e, HNO biosensor calibration; b, Current response after adding different Angeli’s salt (AS) concentrations to a stabilized sensor’s baseline. One microliter of an AS stock solution was added to a stabilized sensor’s baseline. A few seconds after, the signal starts to grow, and after 15 min, it begins to decrease. No signal was detected after adding SNAP, a nitric oxide donor. c, Current response after pretreatment of 10 min of different AS concentrations. One microliter of an AS stock solution was added to 1 ml of buffer and then was waited for 10 min. Next, the measurement with the sensor was started. In this case, as expected, the signal only decreased. d, Δcurrent vs. AS concentration. e, Δcurrent vs. HNO concentration. The HNO concentration in the Co(P) electrode reaction solution was estimated by the following kinetic analysis11,12: \(Donor\,\left( {AS/PA} \right)\mathop{\longrightarrow}\limits^{{k_d{\it{v}}_d}}HNO\) (1) \(2HNO\mathop{\longrightarrow}\limits^{{k_{h,}{\it{v}}_{\it{h}}}}N_2O + H_2O\) (2) Where reaction (1) represents the donor decomposition characterized by a first-order rate constant kd, and (2) represents bimolecular HNO dimerization, characterized by rate constant kh. Reaction (1) was considered an irreversible reaction because its inverse rate is negligible concerning reactions (2) at the initial rate condition. Reaction (2) is irreversible. Calculating [HNO], all the HNO produced through reaction (1) must be consumed in reaction (2), therefore both rates must be equal: vd = vh. The donor decomposition obeys first-order kinetics, characterized by kd(kd(AS) = 8 ×10−4 s−1)14,58 while the dimerization is a second-order rate reaction characterized by kh (kh = 8 ×106 M−1s−1)2,59,60, thus \(k_{d(AS)}\left[ {AS} \right] = k_h\left[ {HNO} \right]^2\)(3) therefore [HNO] can be estimated as: \(\left[ {HNO} \right] = \sqrt {\frac{{k_d\left[ {Donor} \right]}}{{k_h}}}\)(4) f, Micrograph of Au tip microelectrode fabricated by electrochemical etching under constant potential of 1.3 V vs. Ag/AgCl in 2.8 M KCl. The gold wire served as working electrode, platinum ring was applied as counter electrode and Ag/AgCl (3 M KCl) was a reference electrode. The applied constant potential to the Au working electrode was 1.3 V. Supporting electrolyte was 2.8 M KCl. The gold wire was immersed into the solution to ca. 1 mm in depth. The effect of electrochemical etching was assessed microscopically on each microelectrode fabrication. g-h, NO sensor calibration. g, Response of the sensor to increasing concentration of NO, generated in situ from the reaction of iodide with nitrite in acid solution41. No signal was detected after adding a nitroxyl donor. h, Relationship between NO concentration and electrode current measured. Data are presented as mean ± s.d. of three biologically independent replicates (n = 3).
