Bioelectrochemistry (v.65, #1)

Contents (v).

Indirect electrochemical reduction of nicotinamide coenzymes by K. Vuorilehto; S. Lütz; C. Wandrey (1-7).
Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2′-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1–10 mM NAD(P)+ and 2.5–200 μM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)+ was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.
Keywords: Coenzyme reduction; Indirect reduction; Mediator; NAD; NADP; Rhodium;

Effects of palmitic acid and cholesterol on proton transport across black lipid membranes by K. Brunaldi; M.A. Miranda; F. Abdulkader; R. Curi; J. Procopio (9-13).
We studied the effect of palmitic acid (PA) and cholesterol (∼17 wt.%) on proton translocation across asolectin (charged) and diphytanoylphosphatidylcholine (DPhPC, neutral) black lipid membranes (BLMs). Potential difference (PD), short circuit current (SCC), and conductance (G total) were measured with a digital electrometer. Membranes were exposed to pH gradients (0.4–2.0 units), followed by PA addition to bath (symmetrically, 40–65 μM). The membrane conductive pathway was subdivided into an unspecific and a proton-related routes. A computer program estimated the conductances (G un and G H) of the two pathways from the measured parameters. No significant differences in proton selectivity were found between DPhPC membranes and DPhPC/cholesterol membranes. By contrast, cholesterol incorporation into asolectin increases membranes selectivity to proton. Cholesterol dramatically reduced G un reflecting, probably, its ability of inducing order in lipid chains. In asolectin membranes, PA increases proton selectivity, probably by acting as a proton shuttle according to the model proposed by Kamp and et al. [Biochemistry 34 (1995) 11928]. Cholesterol incorporation into asolectin membranes eliminates the PA-induced increase in proton selectivity. In DPhPC and DPhPC/cholesterol membranes, PA does not affect proton selectivity. These results are discussed in terms of the presence of cardiolipin (CL) in asolectin, cholesterol/PA interactions, and cholesterol order-inducing effects on acyl-chains.
Keywords: Black lipid membrane; Palmitic acid; Cholesterol; Proton; Conductance; Permeability;

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of platinum electrode modified with colloidal gold and polyvinyl butyral (PVB) as matrices to detect hepatitis B surface antigen (HBsAg) via electrochemical impedance spectroscopy (EIS). The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that K4[Fe(CN)6]/K3[Fe(CN)6] reactions on the platinum electrode surface were blocked due to the procedures of self-assembly of HBsAb-Au-PVB. The binding of a specific HBsAb to HBsAg recognition layer could be detected by measurements of the impedance change. A new strategy was introduced for improving the sensitivity of impedance measurements via the large specific surface area and high surface free energy of Au nanoparticles and the encapsulated effect of polyvinyl butyral. The results showed that this strategy caused dramatic improvement of the detection sensitivity of HBsAg and had good linear response to detect HBsAg in the range of 20–160 ng·ml−1 with a detection limit of 7.8 ng·ml−1. Moreover, the studied immunosensor exhibited high sensitivity and long-term stability.
Keywords: Hepatitis B; Electrochemical impedance spectroscopy; Colloidal gold; Polyvinyl butyral;

Polyamidoamine (PAMAM) dendrimers impact on activity of acetylcholinesterase was studied. It has been shown that dendrimers induce a biphasic effect: depending on their concentrations they increase or decrease enzyme activity. It may be due to two types of interactions: direct—between dendrimers and the enzyme; indirect—via a modification of the physical state of membrane phospholipids affecting the acetylcholinesterase.
Keywords: Acetylcholinesterase; Erythrocyte membrane; Erythrocyte ghosts; Michaelis–Menten constant; Dendrimer;

The electroporative effect on the stratum corneum (SC) is highly localized. However, the fractional area for the transport of small ions and larger ionic species differs considerably during and after high voltage (HV) application. Electroporation of SC creates new aqueous pathways, accessible for small ions, such as Cl and Na+ ions. The pores are distributed across the skin surface yielding a fractional area for current flow during electroporation of up to 0.1%. An increased permeability after high voltage application persists within a fractional area on the order of 10−3%. The permeabilization of SC for larger, charged molecules (M>200 g/mol) involves Joule heating and a phase transition of the long chain sphingolipids within local transport regions (LTR). The transport area for these molecules (≈10−3%) changes only negligibly after high voltage application.
Keywords: Transdermal drug delivery; Fractional area; Molecular transport; Stratum corneum; Electroporation;

A novel strategy for fabricating horseradish peroxidase (HRP)-based H2O2 sensor has been developed by combining the merits of carbon sol–gel supporting matrix and nano-scaled particulate gold (nano-Au) mediator. The thiol functional group-derived carbon ceramic electrode (CCE) was first constructed using (3-mercaptopropyl) trimethoxy silane as sol–gel monomer. Then, the stable nano-Au monolayer was obtained through covalent linkage between nano-Au and thiol group on the surface of CCE. The experimental results showed that nano-Au monolayer formed not only could steadily immobilize HRP but also efficiently retain its bioactivity. Hydrogen peroxide was detected with the aid of hydroquinone mediator to transfer electrons between the electrode and HRP. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as the operating potential, mediator concentration and pH of background electrolyte were explored for optimum analytical performance of the enzyme electrode. The biosensor had a fast response of less than 8 s with linear range of 1.22×10−5 to 1.10×10−3mol l−1 and a detection limit of 6.1×10−6mol l−1. The sensitivity of the sensor for H2O2 was 0.29 A l mol−1 cm−2. The activation energy for enzyme reaction was calculated to be 10.1 kJ mol−1. The enzyme electrode retained 75% of its initial activity after 5 weeks storage in phosphate buffer at pH 7.
Keywords: Amperometric biosensor; Horseradish peroxidase; Nano-Au monolayer; Hydrogen peroxide;

4-Amino-2-mercaptopyrimidine self-assembled monolayer (AMP SAMs/Au) was prepared on a gold electrode. The AMP SAMs/Au was characterized by using attenuated total reflection-fourier transform infrared (ATR-FTIR) and A.C. Impedance. The electrochemical behavior of brucine on AMP SAMs/Au was studied by cyclic voltammetry (CV) and square wave adsorptive stripping voltammetry (SWASV). The modified electrode showed an excellent electrocatalytic activity for the redox of brucine. The catalytic current increased linearly with the concentration of brucine in the range of 4.0×10−7 to 2.0×10−4 mol l−1 by square wave voltammetry response. The detection limit was 6.0×10−8 mol l−1.
Keywords: Brucine; 4-amino-2mercaptopyrimidine; Self-assembled monolayer (SAMs); Square wave adsorptive stripping voltammetry (SWASV);

Previous potentiometric attempts to determine the formal potential (E0) of key intracellular redox buffer glutathione resulted in contradictory values. We have developed a spectroelectrochemical method using direct reduction on metal oxide electrodes. Disulfide absorbance at 258 nm was used to titrate glutathione in the thin layer cell reversibly. At conditions close to physiological ([GSH]=0.001–0.005 mol/l, pH=7.34; I=0.1 mol/l; T=298.15 K), we have measured glutathione E 0′=−0.22±0.02 V (NHE), corroborating the results of equilibrium measurements.
Keywords: Glutathione; Formal potential; Spectroelectrochemistry; Metal oxide electrode; Redox equilibrium;

Study on the enhancement of catalytic activity for hemoglobin by quinhydrone in poly(o-aminophenol) film by Wenyan Tao; Yingju Liu; Dawei Pan; Lihua Nie; Shouzhuo Yao (51-58).
Hemoglobin (Hb) and quinhydrone (QHQ) were incorporated in poly(o-aminophenol) [o-AP, POAP] film by electropolymerization of o-aminophenol in a weak acid solution containing Hb and QHQ. The nonconducting polymer film was found to be nearly rigid by piezoelectric quartz crystal (PQC) impedance. Therefore, the thickness of the Hb–QHQ–POAP film was estimated as about 104±10 nm by quartz crystal microbalance (QCM). The QHQ mediation effects on the biomacromolecule Hb entrapped in the POAP film were investigated by using cyclic voltammetry, amperometric technique and kinetic study. Cyclic voltammograms showed that the redox peaks in the Hb–QHQ–POAP film are much more reversible than those in the Hb–POAP film. The response current of the Hb–QHQ–POAP film to H2O2 was almost twice than that of the Hb–POAP film. The Michaelis–Menten constant and the activation energy of Hb in the Hb–QHQ–POAP film are 7.47 mM and 13.91 kJ/mol, respectively, both are smaller than that in the Hb–POAP film. These results showed that the immobilized Hb in POAP film exhibited higher catalytic activity to H2O2 due to the mediation of QHQ.
Keywords: Hemoglobin; Poly(o-aminophenol); Quinhydrone; Hydrogen peroxide; Piezoelectric quartz crystal impedance;

Strong static magnetic field effects on yeast proliferation and distribution by Masakazu Iwasaka; Masateru Ikehata; Junji Miyakoshi; Shoogo Ueno (59-68).
The present study focuses on the effects of gradient magnetic fields on the behavior of yeast, such as its proliferation and mass distribution, and evaluates the effects of magnetism on materials in the yeast culture system. Yeast, Saccharomyces cerevisiae, was incubated in a liquid medium under magnetic fields (flux density B=14 T). When yeast in a tube was exposed to 9–14 T magnetic fields with a maximum flux density gradient of dB/dx=94 T/m, where x is the space coordinate, the rate of yeast proliferation under the magnetic fields decreased after 16 h of incubation compared to that of the control group. The physical properties of the yeast culture system were investigated to discover the mechanism responsible for the observed deceleration in yeast proliferation under magnetic fields. Gas pressure inside the yeast culture flask was compared with and without exposure to a magnetic field. The results suggested that the gas pressure inside a flask with 6 T, 60 T/m slowly increased in comparison to the pressure inside a control tube. Due to the diamagnetism of water (medium solution) and yeast, the liquid surface distinctly inclined under gradient magnetic fields, and the hydrostatic force in suspension was strengthened by the diamagnetic forces. In addition, magnetophoresis of the yeast cells in the medium solution exhibited localization of the yeast sedimentation pattern. The roles of magnetically changed gas-transport processes, hydrostatic pressures acting on the yeast, and changes in the distribution of the yeast sedimentation, as well as the possible effects of magnetic fields on yeast respiratory systems in the observed disturbance of the proliferation are discussed.
Keywords: Magnetic field; Yeast proliferation; Yeast distribution; Magnetic force; Diamagnetism; Moses effect;

The electrode reaction of glutathione (GSH) at the hanging mercury drop electrode is studied by means of square-wave voltammetry (SWV). At potentials more positive than −0.350 V (vs. Ag/AgCl (3 mol/l KCl)) the oxidation of the mercury electrode in the presence of GSH leads to creation of a sparingly soluble mercury–GSH complex that deposits onto the electrode surface. Under cathodic potential scan, the deposited complex acts as a reducible reactant, giving raise to a well-defined cathodic stripping reversible SW voltammetric response. The electrode reaction can be described by the scheme: Hg(SG)2(s)+e+2H(aq) +=Hg(l)+2GSH(aq). Thus, the electrode reaction provides information on both thermodynamics and kinetics of the chemical interactions of GSH with mercury. An experimental methodology for measuring the kinetics of the electrode reactions, based on the property known as “quasireversible maximum”, is developed. The standard redox rate constant is 5.09, 5.75 and 5.22 cm s−1 in a phosphate buffer at pH 5.6, 7.0 and 8.5, respectively, with a precision of ±10%. The high rate of the electrode reaction reflects the strong affinity of GSH towards chemical interaction with mercury. The electrode reaction is particularly sensitive to the presence of heavy metal ions such as Cu2+, Cd2+, and Zn2+. The rate of the electrode reaction decreases significantly in the presence of these ions due to simultaneous interactions of GSH with the respective ion and mercury.
Keywords: Glutathione; Square-wave voltammetry; Redox kinetics; Quasireversible maximum;

Electrochemical cleavage of DNA in the presence of copper–sulfosalicylic acid complex by Yan-Ling Wang; Yun-Chun Liu; Zhou-Sheng Yang; Guang-Chao Zhao (77-81).
Electrochemical cleavage of DNA in the presence of copper–sulfosalicylic acid [Cu(ssal)2 2+] complex was studied. The cleavage was observed in a certain potential region where redox cycling of Cu(ssal)2 2+/Cu(ssal)2 + took place. Cu(ssal)2 2+ complex mediate generation of reactive oxygen species from O2 by the Fenton reaction, these radicals are capable of damaging DNA. The cleaved DNA fragments were separated by high-performance liquid chromatography (HPLC). The experimental results indicated that the method for electrochemical cleavage of DNA by Cu(ssal)2 2+ complex was simple and efficient.
Keywords: Electrochemical; DNA cleavage; Cu(ssal)2 2+ complex; HPLC;

Deactivation of bilirubin oxidase by a product of the reaction of urate and O2 by Chan Kang; Hyosul Shin; Yongchao Zhang; Adam Heller (83-88).
The “wired” bilirubin oxidase (BOD) bioelectrocatalyst is superior to pure platinum as an electrocatalyst of the four-electron electroreduction of O2 to water. Not only is its overpotential for O2 reduction lower, but unlike platinum, it is not affected by organic compounds like glucose. The “wired” BOD-coated carbon cathode operates for >1 week at 37 °C in a glucose-containing physiological buffer solution. One of its key applications would be in a glucose–O2 biofuel cell, which would operate in living tissues. The cathode is, however, short-lived in serum, losing its electrocatalytic activity in a few hours. Here we show that the damaging serum component is a product of the reaction of urate and dissolved oxygen. Exclusion of urate, by application of Nafion™ film on the cathode, improves the stability in serum.
Keywords: Bilirubin oxidase; Urate; O2;