Electrochemical Characterization and Bioelectrocatalytic H₂O₂ Sensing of Non‐Symbiotic Hexa‐Coordinated Sugar Beet Hemoglobins

Abstract

The biological role of non-symbiotic plant hemoglobins (Hbs) is not well understood. It may involve sensing and signaling of reactive nitrogen and oxygen species-a property that can be used in electrochemical sensing. Here, we electrochemically studied two novel non-symbiotic Beta vulgaris Hbs: BvHb1.2 and BvHb2 expressed in E. coli. At pH 7, we observed close potentials of their Fe 2 + /3 + hemes, À 349 mV for BvHb1.2 and À 345/À 457 mV vs. Ag/AgCl for the "open" penta-/"closed" hexa-coordinated states of BvHb2. BvHbs bound and bioelec-trocatalytically reduced O 2 and H 2 O 2 at potentials significantly exceeding their Fe 2 + /3 + heme potentials. BvHb2, with the onset of H 2 O 2 reduction at 370 mV, enabled O 2 -interference-free 10 μM H 2 O 2 detection at 0 mV, with a 87 nA μM À 1 cm À 2 sensitivity comparable to some peroxidases. The results underpin broad electrochemical applications of BvHbs in the electroanalysis of reactive species and in electrochemical biotransformations.

Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex

“…However, it can still proceed through the formation of a higher oxidation state formed in the presence of the intermediate product of O 2 reduction – H 2 O 2 - and stabilized by the G4-hemin complex similarly to the peroxidase catalysis and bioelectrocatalysis: , Here, the analogue of peroxidase compound 1, consisting of an oxyferryl iron (Fe 4+ =O) and a porphyrin π cation radical, is presumably formed and then further reduced at the electrode surface in a 2e – /2H + reaction to ferric hemin starting from 0.4 V: This reaction explains the consistently higher potential for the reaction of O 2 reduction (from 0.4 V, Figure A) compared to the G4-unbound hemin (from 0 V, Figure S3). Similarly higher onset potentials for the bioelectrocatalytic O 2 reduction, with two regions of a higher and lower electrocatalytic activity, were observed with some hemoglobins exhibiting both a pseudoperoxidase and oxidase electrocatalytic activity. , …”

“…In the absence of thrombin, the aptamer-conjugated hemin electrocatalyzed O 2 reduction in aerated solutions according to where reaction proceeds via formation of H 2 O 2 as intermediate. The electrocatalytic reactions and , observable even in the presence of trace amounts of hemin, might complicate analysis of thrombin, unless the signals from the G4-hemin complex formed after thrombin binding exhibit either a different or enhanced electrocatalytic pattern in the reaction of O 2 reduction, as was previously shown for hemin-protein and hemin-DNA complexes whose higher oxidation states are stabilized by either protein , or G4 matrices …”

“…Similarly higher onset potentials for the bioelectrocatalytic O 2 reduction, with two regions of a higher and lower electrocatalytic activity, were observed with some hemoglobins exhibiting both a pseudoperoxidase and oxidase electrocatalytic activity. 30,31 Electroanalysis of Thrombin in Serum and CSF. To demonstrate the feasibility of the approach for practical analysis of thrombin, we tested the aptasensor performance in 1% human serum and in artificial CSF (Figure 5).…”

“…The covalent G4-hemin complexes showed improved DNAzyme stability and integrity and allowed not only indicator-free peroxidase DNAzyme activity (no hemin or substrates and mediators present in solution) but also O 2 -dependent oxidase activity of DNAzyme at potentials exceeding those of free hemin . A similar O 2 -dependent bioelectrocatalysis of heme proteins, such as bacterial and plant hemoglobins, was shown to be superior to the peroxidase one, with its onset approaching the potentials of H 2 O 2 bioelectrocatalysis. , Thus, by analogy to heme proteins, it was suggested that the O 2 -dependent electrocatalytic activity of the covalent G4-hemin complexes might allow simpler and faster electrocatalytically amplified analysis of thrombin both in serum and in a cerebrospinal fluid (CSF) (Figure ).…”

Electrocatalytic Oxidation of Water by OH−‐ and H2O‐Capped IrOx Nanoparticles Electrophoretically Deposited on Graphite and Basal Plane HOPG: Effect of the Substrate Electrode**

“…[55] While the atomically flat surface of HOPG is extremely useful as a substrate for AFM studies, [52,56,57] its reactivity is also very attractive for electrochemical applications. [51][52][53][54] Cheap and easily renewable spectroscopic Gr, widely used in electroanalysis of small molecules and proteins due to its high-surface area enabling a higher surface coverage with adsorbates, [14,[58][59][60][61][62] has been already used for fabrication of IrO x nanocomposite electrodes. [31] These electrodes demonstrated the hitherto highest electrocatalytic current densities, of 43 mA cm À 2 at 1 V and pH 7, in OER.…”