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An efficient way was proposed for probing the folding/unfolding event of bovine hemoglobin (Hb) through adsorptive-transfer voltammetry. Hb molecules in native and pre-unfolded in different urea conditions for 23 h were adsorbed onto the montmorillonite clay modified glassy carbon electrode (Hb/clay/GCE and uHb/clay/GCE, respectively). Cyclic voltammograms of Hb/clay/GCE and uHb/clay/GCE showed that the unfolding of Hb caused great change in the direct electron transfer between the heme irons within Hb and electrode surface, which was facilitated on clay film. From the amount of the electroactive Hb (W(Hbe)) and the adsorbed Hb (Γ) on clay per unit mass, the minimal electroactive portion (MEP) of the adsorbed Hb was calculated to assess the unfolding state of Hb. With the increase of urea concentration, MEP showed a sigmoid curve. Thermodynamic parameters related to the unfolding event of Hb were also obtained based on the linear free energy model (LEM), including the free energy of folding in water (ΔG(U)(water)), the slope of the Santoro-Bolen equation (m), and the urea concentration required in for achieving half of the total change (S(m)) in the unfolding curves. This work gave the first try for investigating protein unfolding at nano-materials modified electrode using adsorptive-transfer voltammetry, which improved the sensitivity of analysis and avoided the disadvantages involved in the existing electrochemical methods for protein unfolding. The proposed method will benefit the electrochemical studies of protein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The direct electron transfer of myoglobin (Mb) was realized by immobilizing Mb onto ionic liquid (1-butyl-3-methyl imidazolium tetrafluoraborate, [bmim][BF(4)])-clay composite film modified glassy carbon electrode. A pair of well-defined redox peaks of Mb with a formal potential (E(o)') of -0.297 V (vs. Ag/AgCl) was observed in 0.1M phosphate buffer solution (pH 6.0). The ionic liquid-clay composite film showed good biocompatibility and an obvious promotion capability for the direct electron transfer between Mb and electrode. The electron transfer rate constant (k(s)) of Mb was calculated to be (3.58+/-0.12)s(-1). UV-vis spectrum suggested that Mb retained its native conformation in the ionic liquid-clay system. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process, in ionic liquid and clay during the drying process of the modification, and the ionic liquid played the key role for promotion of the direct electron transfer between Mb and the ionic liquid-clay composite film modified electrode. The biocatalytic activity of Mb in the composite film was exemplified by the reduction of hydrogen peroxide. Under the optimal conditions, the reduction peak currents of Mb increased linearly with the concentration of H(2)O(2) in the range of 3.90 x 10(-6) to 2.59 x 10(-4)M, with a detection limit of 7.33 x 10(-7)M. The kinetic parameter I(max) and the apparent Michaelis constant (K(m)) for the electrocatalytic reactions were 3.87 x 10(-8)A and 17.6 microM, respectively. The proposed method would be valuable for the construction of a new third-generation H(2)O(2) sensor.

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.

Dendritic MoO3/Ag with good crystallinity has been prepared through an operando method. In the hydrogen evolution reaction (HER), this catalyst, which was designed to utilize the best properties of each component material, showed a high catalytic activity. After the precursor was drop-cast onto a glassy carbon electrode, a reductive potential was applied to the coated electrode, and H2 evolution occurred within the range of potentials. The overpotential required to evolve H2 at the benchmark rate decreased progressively with subsequent voltammetric cycles, until a steady state was reached at which only 145 mV of overpotential was required to pass −10 mA cm−2 of current density. During the electrocatalysis, the precursor was converted to catalyst through an operando method. This operando-synthesized MoO3/Ag catalyst has a low Tafel slope (43 mV dec−1), low overpotential (145 mV), and excellent durability for HER. It has the potential to be a promising material for HER. [ABSTRACT FROM AUTHOR]

Abstract: A novel electrochemical site marker competitive method is proposed for the study of the binding site and the binding mode between bovine serum albumin (BSA) and alizarin red S (ARS). Two known site-selective markers for BSA, bilirubin (BR, for site I) and diazepam (DIA, for site II), were selected to bind to the two potential binding sites of BSA (site I and site II). The resulting BR–BSA or DIA–BSA complex was added into an ARS solution for binding site competition studies. From the current response of ARS after site competition, binding site I of BSA was identified as the binding site of BSA to ARS. Further investigation of the binding events by electrochemistry, fluorescence, and molecular docking techniques revealed that ARS was buried in the hydrophobic cavity of subdomain IIA, mainly through five hydrogen bonds between ARS and residues ARG(193), ARG(255), LYS(220), ARG(197), and ALA(289) of BSA. These efforts will aid in a better understanding of the toxicological action of active components in anthraquinone dyes. [Copyright &y& Elsevier]

The contribution of the chloride anion (Cl−) and guanidinium cation (Gdn+) to the denaturing efficiency of guanidine hydrochloride (GdnCl) upon the unfolding of hemoglobin (Hb) was investigated electrochemically. Hb was entrapped in a didodecyldimethylammonium bromide (DDAB)-film-modified glassy carbon electrode and unfolded by the components of GdnCl. The changes of the direct electrochemical behaviors of Hb, including peak current (Ip), formal potential (Eo′), and peak-to-peak separation (ΔE), were utilized to characterize different unfolded states of Hb. UV−vis, circular dichroism, and fluorescence spectroscopy were also used to verify the structural information of Hb during the unfolding process. The results indicated that the denaturing efficiency of GdnCl was contributed to by Gdn+ and Cl− in synchronization, and the portions of such contributions were concentration-dependent. In addition, the presence of Gdn+, Cl−, or GdnCl can enhance the reversibility of the redox reaction of Hb to the same degree. The method provides not only an easy way to better understand the conformational changes of Hb but also a strategy to control its conformation. [ABSTRACT FROM AUTHOR]

Abstract: The electrochemical behaviors of hemoglobin (Hb) were studied by adsorbing Hb on the gold electrodes modified with self-assembled monolayers (SAMs) of different terminal groups and alkyl chain length. Specifically, through adsorbing the Hb molecules onto the SAMs of 3-mercaptopropanoic acid (MPA), 1-propanethiol (PT) and cysteamine hydrochloride (cys), the influences of the terminal groups of alkanethiols on the electron transfer of Hb were examined. A quasi-reversible redox process was observed when Hb was adsorbed on the MPA-modified electrode. However, an irreversible reduction process and no redox response were shown when Hb molecules were adsorbed on the SAMs of PT and cys-modified electrodes, respectively. The dependence of the direct electrochemical response of Hb on the alkyl chain length of alkanethiols (n) was further investigated on the SAMs of HS(CH2) n COOH (n =1, 2, 5 and 10). When n was 1, Hb showed a weak current response. When n were 2 and 5, quasi-reversible redox processes with nearly similar electron transfer rate constants (k s) of 0.49 and 0.47s−1 were obtained, respectively. Increasing the number of methylene groups within carboxyl alkanethiol to 10 resulted in a significant decrease of the electron transfer rate and current response of Hb. The observations indicated that the SAM of MPA can provide suitable properties to keep Hb in a favorable adsorption state for direct electron transfer (DET). Furthermore, the direct electrochemistry of the Hb adsorbed on the SAM of MPA was studied in detail. [Copyright &y& Elsevier]

Abstract: A biocompatible nanohybrid material (clay/AuCS) based on clay, chitosan and gold nanoparticles was explored. The material could provide a favorable microenvironment for proteins to realize the direct electron transfer on glassy carbon electrodes (GCE). Myoglobin (Mb), as a model protein to investigate the nanohybrid, was immobilized between the clay/AuCS film and another clay layer. Mb in the system exhibited a pair of well-defined and quasi-reversible redox peaks at −0.160V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), corresponding to its heme FeIII/FeII redox couples. UV–vis spectrum suggested that Mb retained its native conformation in the system. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation–exfoliation–restacking process among Mb, AuCS and clay during the modified film drying. Excellent biocatalytic activity of Mb in the modified system was exemplified by the reduction of hydrogen peroxide and nitrite. The linear range of H2O2 determination was from 3.9×10−5 to 3.0×10−3 M with a detection limit of 7.5μM based on the signal to noise ratio of 3. The kinetic parameters such as α (charge transfer coefficient), k s (electron transfer rate constant) and K m (Michaelis–Menten constant) were evaluated to be 0.55, 2.66±0.15s−1 and 5.10mM, respectively. [Copyright &y& Elsevier]