Bioelectrochemistry (v.56, #1-2)
Subject Index to Volume 55 and 56 (III-XII).
Contents of Volume 55 and 56 (XIII-XVII).
Author Index to Volume 55 and 56 (I-II).
Editorial by Tibor Hianik; Dieter Walz (1).
Effect of protein relaxation on electron transfer from the cytochrome subunit to the bacteriochlorophyll dimer in Rps. sulfoviridis reaction centers within mixed adiabatic/nonadiabatic model by A.I. Kotelnikov; J.M. Ortega; E.S. Medvedev; B.L. Psikha; D. Garcia; P. Mathis (3-8).
The broad set of nonexponential electron transfer (ET) kinetics in reaction centers (RC) from Rhodopseudomonas sulfoviridis in temperature range 297–40 K are described within a mixed adiabatic/nonadiabatic model. The key point of the model is the combination of Sumi–Marcus and Rips–Jortner approaches which can be represented by the separate contributions of temperature-independent vibrational (v) and temperature-dependent diffusive (d) coordinates to the preexponential factor, to the free energy of reaction ΔG=ΔG v+ΔG d(T) and to the reorganization energy λ=λ v+λ d(T). The broad distribution of protein dielectric relaxation times along the diffusive coordinate is considered within the Davidson–Cole formalism.
Keywords: Marcus theory; Free energy; Energy of reorganization; Protein dynamics; Dielectric relaxation;
Coupling of electron and proton transport in photosynthetic membranes: molecular mechanism by A Kukushkin; S Poltev; S Khuznetsova (9-12).
Using the method of Modified Neglect of Diatomic Overlap (MNDO), the electronic structure of plastoquinol (PQH2) and plastoquinone (PQ) in neutral, singly (PQ−) and doubly (PQ2−) reduced states is studied. The conformational analysis performed on these molecules shows that in the lowest energy conformation, the angle between the first link of the tail backbone and the ring plane of neutral and singly reduced PQ and plastoquinol is nearly the same and differs by 15° from that of doubly reduced PQ. Nevertheless, for all states of plastoquinone and for plastoquinol, the total energy changes by less than 0.2 eV when the studied angle is varied from 0° to 180°. As in Rhodobacter sphaeroides, the oxygen of the PQ ring is reported to form a hydrogen bond with a nitrogen in the ring of Histidine (His) L 190. The energy of the PQ–His complex was calculated for different redox states of PQ and for several values of the distance between the molecules (N–O distance from 0.2 to 0.5 nm). For every considered complexes, the total energy dependence on the proton position on the line connecting the N and O atoms was determined, to see if the hydrogen bond is formed. It is shown that for only singly reduced PQ this dependence has a symmetric two-well form, i.e. the hydrogen bond is formed. For neutral and doubly reduced PQ, the curve is also two-well but asymmetric, so that the proton is bound to His or to PQ, correspondingly.On the basis of these results, we propose the following scheme of electron–proton coupling. Negatively charged oxygens of PQ form H-bonds with proton donor groups of the surrounding protein and fix PQ in its pocket. While the negative charges of oxygens increase after quinone reduction, protons shift to PQ oxygens and form strong hydrogen bonds with them. Upon second PQ reduction, protons are torn away from surrounding amino acids and form covalent bonds with the quinol. Resulting PQH2 detaches from its binding place and is replaced by a neutral PQ. The lacking protons on amino acids in the QB pocket are replaced by a step-by-step transfer from the stroma bulk through the proton channels.
Keywords: Plastoquinone; Electron and proton transport; Photosystem 2;
Backward electron transport in photosystem 2 reaction center and temperature dependence of delayed luminescence characteristics by D.Z Badretdinov; S.A Kuznetsova; S.V Poltev; A.K Kukushkin (13-16).
The temperature dependence of parameters of light-induced changes in millisecond delayed luminescence (half-width of the maximum, maximal and steady-state luminescence intensity) is studied within the temperature range from −23 to 45 °C in leaf segments of Chinese rose (Hibiscus rosa sinensis). Delayed luminescence (DL) is induced and registered by a homemade setup based on a Lewis–Kasha-type phosphoroscope. The temperature dependence of steady-state luminescence intensity is shown to have two maxima, at −10 and 35 °C. At room temperatures, the steady-state value of luminescence intensity is minimal, and its value correlates with the temperature tolerance of the plant. Depending on cooling and heating regimes, the DL steady-state value vs. temperature curves is found to be different. We suppose this effect to be caused by temperature-induced destructive changes in the structure of photosystem 2 reaction centre and probably by salting out.
Keywords: Delayed luminescence; Cold and heat injury; Tolerant range;
Enzyme-catalysed nitrate reduction—themes and variations as revealed by protein film voltammetry by Julea N. Butt; Lee J. Anderson; Luis M. Rubio; David J. Richardson; Enrique Flores; Antonia Herrero (17-18).
Protein film voltammetry has been used to define the catalytic performance of two nitrate reductases: the respiratory nitrate reductase, NarGH, from Paracoccus pantotrophus and the assimilatory nitrate reductase, NarB, from Synechococcus sp. PCC 7942. NarGH and NarB present distinct ‘fingerprints’ of catalytic activity when viewed in this way. Potentials that provide insufficient driving force for significant rates of nitrate reduction by NarB result in appreciable rates of nitrate reduction by NarGH. However, both enzymes display complex modulations in their rate of substrate reduction when viewed across the electrochemical potential domain.
Keywords: Nitrate reductase; Protein film voltammetry; Molybdenum; Iron–sulphur cluster; Electrochemistry;
Flavin photochemistry in the analysis of electron transfer reactions: role of charged and hydrophobic residues at the carboxyl terminus of ferredoxin–NADP+ reductase in the interaction with its substrates by Merche Faro; John K. Hurley; Milagros Medina; Gordon Tollin; Carlos Gómez-Moreno (19-21).
The enzyme Ferredoxin-NADP+ reductase participates in the reductive side of the photosynthetic chain transferring electrons from reduced Ferredoxin (Fd) (or Flavodoxin (Fld)) to NADP+, a process that yields NADPH that can be used in many biosynthetic dark reactions. The involvement of specific amino acids in the interaction between the two proteins has been studied using site-directed mutagenesis. In the present study, the participation of charged (H299), polar (T302) or hydrophobic (V300) amino acid residues that are in the NADP+-binding domain of the reductase have been examined by analyzing its C-terminal region, which is located close to the active site. Stopped-flow and laser flash photolysis results of the reaction in which these mutant proteins participate show very little differences with respect to the wild-type protein. These results suggest that the NADPH-binding domain of the reductase has little effect on the processes of recognition and electron transfer to (and from) Fd or Fld, according to the recently reported crystallographic structure of the FNR/Fd complex
Keywords: Electron transfer; Ferredoxin; NADP+;
Indirect evidence of direct electron communication between the active site of galactose oxidase and a graphite electrode by Jan Tkac; Igor Vostiar; Peter Gemeiner; Ernest Sturdik (23-25).
Bi-enzymatic biosensor based on galactose oxidase (GalOD) and horseradish peroxidase (HRP) using ferrocene as an efficient mediator was constructed. When a dependence of a working potential on the sensor performance was examined, an unusual behaviour was observed. With increasing of an applied working potential a lower concentration of substrate to attain full linear range was needed. A fully linear dependence from the first substrate addition was observed at and above the working potential of 150 mV. This activation of the biosensor response by an applied working potential very well corresponds with a formal potential of GalOD (156 mV). When a membrane prevented GalOD access to the electrode surface was applied, no activation effect of a working potential on the sensor performance was observed. Thus, it can be assumed that direct electron communication between GalOD and the electrode occurred.
Keywords: Biosensor; Galactose oxidase; Direct electron transfer; Copper oxidase;
Monolayers of a de novo designed 4-α-helix bundle carboprotein and partial structures on Au(111)-surfaces by Jesper Brask; Hainer Wackerbarth; Knud J. Jensen; Jingdong Zhang; Jens U. Nielsen; Jens E.T. Andersen; Jens Ulstrup (27-32).
Mapping of structure and function of proteins adsorbed on solid surfaces is important in many contexts. Electrochemical techniques based on single-crystal metal surfaces and in situ scanning probe microscopies (SPM) have recently opened new perspectives for mapping at the single-molecule level. De novo design of model proteins has evolved in parallel and holds promise for test and control of protein folding and for new tailored protein structural motifs. These two strategies are combined in the present report.We present a synthetic scheme for a new 4-α-helix bundle carboprotein built on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The galactopyranoside with thiol anchor and the thiol anchor alone were prepared for comparison. Voltammetry of the three molecules on Au(111) showed reductive desorption peaks caused by monolayer adsorption via thiolate-Au bonding. In situ STM of the thiol anchor disclosed an ordered adlayer with clear domains and molecular features. This holds promise, broadly for single-molecule voltammetry and the SPM and scanning tunnelling microscopy (STM) of natural and synthetic proteins.
Keywords: 4-α-helix bundle; Carboprotein; De novo design; Single-crystal voltammetry; Au(111)-electrodes; In situ STM;
An impedance study of the adsorption of nucleic acid bases at glassy carbon electrodes by A.M. Oliveira-Brett; C.M.A. Brett; L.A. Silva (33-35).
Electrochemical impedance has been used to study the adsorption at glassy carbon electrodes of guanine, its corresponding nucleoside, guanosine, and adenine. Impedance studies at different concentrations and applied potentials show clearly that all three bases are adsorbed on the electrode, blocking the surface. Irradiating the electrode with low-frequency (20 kHz) ultrasound whilst recording the impedance spectra increased transport of molecules to the electrode surface with cavitation cleaning the surface and removing strongly adsorbed molecules of bases. In this way, sonoelectrochemical experiments enabled the electrode processes to be studied in the absence of adsorption.
Keywords: Guanine; Guanosine; Adenine; Adsorption; Electrochemical impedance; Ultrasound;
Electrochemical impedance spectroscopy of polynucleotide adsorption by L. Strašák; J. Dvořák; S. Hasoň; V. Vetterl (37-41).
The dependence of the impedance of the electrode double layer of mercury electrode on frequency around the potentials of the tensammetric peaks of single-stranded and double-helical polynucleotides and DNA was studied. From the frequency dependence of the impedance of the electrode double layer represented in a complex plane impedance plot, the electric equivalent circuit of the electrode covered with adsorbed DNA layer was determined. It was concluded that the desorption of denatured ssDNA is accompanied by higher dielectric losses than the desorption of native dsDNA. This can be explained by the higher flexibility of ssDNA compared to the dsDNA. The capacitance peak of single-stranded polyadenylic acid (poly A) observed at pH 8 around −1.3 V splits at low frequencies in two peaks.
Keywords: Electrochemical impedance spectroscopy; DNA adsorption; Poly A adsorption; Differential capacitance;
Two-dimensional condensation of nucleic acid components at mercury film and gold electrodes by Stanislav Hasoň; Vladimı́r Vetterl (43-45).
The adsorption of cytidine at the mercury film electrodes and at the Au (111) single crystal electrode has been investigated. Some kinetic aspects such as the influence of pH and temperature on the formation or dissolution of cytidine adlayer on the pyrolytic graphite electrode covered by a mercury film or on the Au (111) have been studied.
Keywords: Pyrolytic graphite electrode; Mercury film electrodes; Gold single crystal; Adsorption; Cytidine adlayers; Two-dimensional phase transition;
A novel method of introducing hydrophobic moieties into oligonucleotides for covalent and non-covalent immobilization on electrode surfaces by T.S. Oretskaya; E.A. Romanova; S.Yu. Andreev; S.I. Antsypovich; C. Tóth; V. Gajdos; T. Hianik (47-51).
An effective method for the introduction of oleylamine-modified cytidine units into predetermined position(s) of the oligodeoxyribonucleotide (ON) chain during automated ON synthesis has been developed. The high yields of the condensation products upon the introduction of the modified units allow the methods suggested to be used for the synthesis of ONs with two hydrophobic substituents. We also suggest a simple method for obtaining ONs with 5′-terminal hydrophobic linker with free thiol group. The functionality of synthesized ON modified by thiol group and that with hydrophobic spacer for the detection DNA hybridization has been approved in conductometric experiments.
Keywords: Oligodeoxyribonucleotide; DNA sensors; Hybridization; Conductivity;
Reduction of lapachones and their reaction with l-cysteine and mercaptoethanol on glassy carbon electrodes by A.M. Oliveira-Brett; M.O.F. Goulart; F.C. Abreu (53-55).
The electrochemical reduction of β-lapachone and its 3-sulphonic salt was studied by cyclic, square wave and differential pulse voltammetry in aqueous media using a glassy carbon electrode. These compounds have a wide range of biological activities, including antibacterial, cytotoxic, antifungal, trypanocidal and anticancer action. The reduction of β-lapachone in the presence of l-cysteine and 2-mercaptoethanol was studied and the results, together with others already published, suggest that the anticancer mechanism of β-lapachones can be explained via interaction with topoisomerase.
Keywords: β-Lapachone; Cytotoxic action; Electrochemical reduction; Topoisomerase;
Catalytic signal of rabbit liver metallothionein on a mercury electrode: a combination of derivative chronopotentiometry with adsorptive transfer stripping by Libuše Trnková; René Kizek; Jan Vacek (57-61).
Constant current chronopotentiometric stripping analysis (CPSA) in combination with adsorptive transfer stripping (AdTS) technique was used to study the rabbit liver metallothionein (MT) on a hanging mercury drop electrode (HMDE). Metallothionein yielded a distinct, sharp chronopotentiometric signal at very negative potentials (about −1.7 V), also known as the “peak H”. The height and potential of this peak were dependent on experimental conditions, such as buffer composition, pH, and the presence oxygen in solutions. The peak H was highest in borate buffer with pH close to the isoelectric point (pI) of MT. The chronopotentiometric results contribute to a deeper understanding of the nature of catalytic hydrogen evolution and demonstrate the usefulness of the peak H in peptide and protein research.
Keywords: Metallothionein; Chronopotentiometric stripping analysis; Adsorptive transfer stripping; Peak H; Catalytic hydrogen evolution; Effect of aeration;
Differential pulse adsorptive stripping voltammetry of osmium-modified peptides by Sabina Billová; René Kizek; Emil Paleček (63-66).
Complexes of osmium tetroxide with nitrogen ligands were developed and used in our laboratory as probes of the DNA structure. Here, we show that the complex of osmium tetroxide with 2,2′-bipyridine (Os,bipy) can be used for modification and electrochemical detection of proteins at neutral pH. Salmon luteinizing hormone (SLH) containing two tryptophan (Trp) residues and human luteinizing hormone (HLH) containing one Trp were modified by Os,bipy and measured by differential pulse adsorptive stripping voltammetry (DPAdSV) at a hanging mercury drop electrode (HMDE). The intensity of the DPAdSV catalytic signals corresponded to the number of Trp residues in the peptide molecule. Decreasing pH of the background electrolyte from 6.6 to 3.8 led to the increase of DPAdSV signals, suggesting that at pH 3.8, the DPAdSV detection limit might be well below 1 ng/ml. Our results suggest that Os,bipy is potentially useful for chemical modification of proteins.
Keywords: Chemical modification of peptides; Osmium tetroxide,2,2′-bipyridine; Luteinizing hormones; Differential pulse adsorptive stripping voltammetry; Mercury electrode;
Mediator-modified electrodes for catalytic NADH oxidation: high rate constants at interesting overpotentials by F.D. Munteanu; N. Mano; A. Kuhn; L. Gorton (67-72).
Carbon paste electrodes were modified with a nitrofluorenone derivative, 2,4,7-trinitro-9-fluorenone, adsorbed on zirconium phosphate (ZP). After electrochemical reduction of the fluorenone derivative, it turns into a very efficient mediator for electrocatalytic NADH oxidation, with a formal potential of about +250 mV vs. Ag/AgCl. The electrochemistry and the electrocatalytic properties of the mediator were investigated with cyclic voltammetry and rotating disk electrode methodology. The second order rate constant with NADH was evaluated and found to be higher than 106 M−1 s−1, thus approaching true diffusion controlled currents for NADH oxidation.
Keywords: NADH oxidation; Fluorenone; Mediator; Carbon paste electrode; Cyclic voltammetry; Rate constant;
Nitric oxide determination by amperometric carbon fiber microelectrode by Jaroslav Katrlı́k; Pavlı́na Zálešáková (73-76).
Nitric oxide (NO) amperometric microsensor was prepared by the modification of bare carbon fiber electrode by Nafion and cellulose acetate (CA). Detection limit, response time, reproducibility and influence of some possible interferences (nitrite, nitrate, arginine) were tested and evaluated. This sensor was used for in vitro determination of NO release from fresh porcine aorta induced by calcium ionophore A23187 (CI).
Keywords: Nitric oxide; NO sensor; Electrode; Electrochemical; Carbon fiber; Endothelium;
The role of hydrogenases in the anaerobic microbiologically influenced corrosion of steels by S. Da Silva; R. Basséguy; A. Bergel (77-79).
The direct electron transfer between 316 L stainless steel and the NAD-dependent hydrogenase from Ralstonia eutropha was studied by spectroelectrochemistry. The presence of hydrogenase and NAD+ clearly increased the quantity of electricity, which was consumed during the electrolysis performed at potential lower than −0.70 V/SCE. The involvement of hydrogenase in the cathodic depolarisation theory was discussed in the light of these results.
Keywords: Hydrogenase; Stainless steel; Direct electron transfer; Corrosion;
Electrochemical sensing of DNA–adriamycin interactions by J.A.P. Piedade; I.R. Fernandes; A.M. Oliveira-Brett (81-83).
Adriamycin, a cancerostatic anthracycline antibiotic, causes considerable death of tumour cells, together with the induction of breaks in DNA single and double strands. The interaction of this compound with DNA was investigated using an electrochemical DNA-biosensor. Adriamycin intercalation in DNA disrupts the double helix and the detection of guanine and 8-oxoguanine could mimic one possible mechanism for the in vivo adriamycin drug action.
Keywords: Adriamycin; DNA-biosensor; Oxidative damage; 8-oxoguanine; Voltammetry;
New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces by E Paleček; M Fojta; F Jelen (85-90).
Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2′-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.
Keywords: DNA sensors; Hybridization; Electrochemical detection; Magnetic beads;
Surfactant bilayers for the direct electrochemical detection of affinity interactions by Mikhail Yu. Vagin; Arkady A. Karyakin; Tibor Hianik (91-93).
Simple methods of preparing the direct affinity sensors are proposed. Due to the self-consistent introduction of a hydrocarbon chain bound with oligonucleotide pentadecathymidylate (dT15) into the hydrophobic region of surfactant bilayer or the adsorption of antibodies on the bilayer surface, the immobilizations of oligonucleotide or antibodies were carried out correspondingly. The responses were detected by impedance spectroscopy. Whereas the specific DNA-coupling caused the decrease of real part of impedance, the antibody–antigen interaction caused the increase of real part. The obtained results give an opportunity for the development of impedimetric affinity sensors for clinical analysis or for the detection of various environmental pollutants.
Keywords: DNA sensor; Surfactants; Affinity interactions; Impedance spectroscopy;
A comparative thermodynamic study for both natural and artificial RNA/DNA–protein binary complexes by V. Spiridonova; T. Rassokhin; A. Golovin; E. Petrova; T. Rohzdestvensky; Yu. Pakhomova; A. Kopylov (95-97).
In recent years, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique has been developed into a fast growing field. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value both for the therapy and biosensors. A comparative study of thermodynamic for both natural and artificial RNA/DNA–protein complexes would establish bases for a specificity of complex formation. In particular, we have shown that aptamers could be used for a direct measuring of thrombin enzymatic activity in a solution.
Keywords: RNA/DNA–protein interactions; Ribosome; SELEX; RNA/DNA aptamers; Thrombin; Enzymatic activity;
A glucose/hydrogen peroxide biofuel cell that uses oxidase and peroxidase as catalysts by composite bulk-modified bioelectrodes based on a solid binding matrix by A. Pizzariello; M. Stred'ansky; S. Miertuš (99-105).
An improved composite bulk-modified bioelectrode setup based on a solid binding matrix (SBM) has been used to develop a glucose/hydrogen peroxide biofuel cell. Fuel is combined through a catalytically promoted reaction with oxygen into and oxidized species and electricity. The present work explores the feasibility of a sugar-feed biofuel cell based on SBM technology. The biofuel cell that utilizes mediators as electron transporters from the glucose oxidation pathway of the enzyme directly to electrodes is considered in this work. The anode was a glucose oxidase (GOx, EC 220.127.116.11)/ferrocene-modified SBM/graphite composite electrode. The cathode was a horseradish peroxidase (HRP, EC 18.104.22.168)/ferrocene-modified SBM/graphite composite electrode. The composite transducer material was layered on a wide polymeric surface to obtain the biomodified electrodic elements, anodes and cathodes and were assembled into a biofuel cell using glucose and H2O2 as the fuel substrate and the oxidizer. The electrochemical properties and the characteristics of single composite bioelectrodes are described. The open-circuit voltage of the cell was 0.22 V, and the power output of the cell was 0.15 μW/cm2 at 0.021 V. The biofuel cell proved to be stable for an extended period of continuous work (30 days). The reproducibility of the biotransducers fabrication was also investigated. In addition, an application of presented biofuel cell, e.g. the use of hydrolyzed corn syrup as renewable biofuels, was discussed.
Keywords: Biofuel cell; Solid binding matrix; Spray-printing technology; Renewable fuels;
Electrochemical studies on reconstituted horseradish peroxidase modified carbon paste electrodes by Shailly Varma (107-111).
Horseradish peroxidase (HRP) is a heme protein that acts specifically on H2O2 as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Teale's method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H2O2 using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 μM H2O2. The rec-HRP paste showed ∼100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 μM was observed.
Keywords: Enzyme electrodes; Horseradish peroxidase covalent coupling enzyme electrochemistry;
Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe by Igor Vostiar; Jan Tkac; Ernest Sturdik; Peter Gemeiner (113-115).
The electropolymerized toluidine blue film deposited on the glassy carbon electrode show amperometrically detectable pH sensitivity. This feature of polytoluidine blue (PTOB) film was used for a construction of an amperometric urea biosensor. We have observed a linear shift of the formal redox potential with increasing pH value between 4 and 8 giving the slope of 81 mVΔ pH−1. Polytoluidine blue film has had a significantly increased stability and higher electrochemical activity compared to the adsorbed monomeric dye. The polytoluidine blue urea biosensor has been operating at a working potential of −200 mV vs. SCE. The sensitivity of the biosensor was 980 nA mM−1 cm−2. The biosensor showed linearity in concentration range up to 0.8 mM with the detection limit of 0.02 mM (S/N=3).
Keywords: Amperometric pH sensing; Biosensor; Polytoluidine blue; Urease;
Modified electrodes for NADH oxidation and dehydrogenase-based biosensors by P.N. Bartlett; E. Simon; C.S. Toh (117-122).
The direct electrochemical oxidation of β-nicotinamide adenine dinucleotide (NADH) at clean electrodes proceeds through a radical cation intermediate at high overpotentials and is subject to rapid fouling. Consequently, there has been a considerable body of work over the last 20 years looking at ways in which to catalyse the reaction using a wide variety of different types of modified electrode. These studies have resulted in a good knowledge of the essential features required for efficient catalysis. In designing modified electrodes for NADH oxidation, it is not only important to identify suitable redox groups, which can catalyse NADH oxidation and can be attached to the electrode surface; it is also important to ensure facile charge transport between the immobilised redox sites in order to ensure that, in multilayer systems, the whole of the redox film contributes to the catalytic oxidation. One way to achieve this is by the use of electronically conducting polymers such as poly(aniline).
Keywords: NADH oxidation; Dehydrogenase; Biosensor; Charge transport; Conducting polymers;
Affinity assembled multilayers for new dehydrogenase biosensors by N Mano; A Kuhn (123-126).
We propose a novel approach, which allows the control of the spatial arrangement of redox mediator, coenzyme and enzyme on the electrode at a molecular level, using essentially electrostatic interactions. The first step consists of adsorbing a monolayer of molecules out of a new family of redox mediators, substituted nitrofluorenones. In a second step, a monolayer of calcium cations is immobilized at the interface. It serves as a bridge between the redox mediator and the subsequently adsorbed coenzyme. The weak interaction between a carboxyl group of the redox mediator and the coenzyme's phosphate groups, revealed by QCM measurements, allows the coenzyme to keep its natural activity in the adsorbed state. In the last step, we use the intrinsic affinity of this monolayer of NAD+ for dehydrogenases to build up a supramolecular sandwich composed of mediator/Ca2+/NAD+/dehydrogenase. This simple modification procedure, which might constitute a versatile approach for the low cost assembly of well-defined biosensors surfaces, has been successfully applied to the enzymatic detection of glucose, glutamate and alcohol.
Keywords: Modified electrodes; Nitrofluorenones; Electrocatalysis; Self-assembled monolayers; Biosensors;
Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity by Jan Tkac; Igor Vostiar; Peter Gemeiner; Ernest Sturdik (127-129).
The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 μA mM−1. The microbial biosensor exhibits a very low detection limit of 0.85 μM and a wide linear range from 2 to 270 μM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R 2=0.998).
Keywords: Amperometric biosensor; Gluconobacter oxydans; Ethanol fermentation; Cellulose acetate membrane;
Impedimetric immunosensor using avidin–biotin for antibody immobilization by O. Ouerghi; A. Touhami; N. Jaffrezic-Renault; C. Martelet; H.Ben Ouada; S. Cosnier (131-133).
The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilisation matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. This immobilization method allows to obtain a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10–80 ng ml−1 of antigen and a detection limit of 10 pg ml−1. Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.
Keywords: Electropolymerization; Biotinylated polypyrrole; Impedance spectroscopy; Immunosensor;
Mechanisms of the interaction of α-helical transmembrane peptides with phospholipid bilayers by Ruthven N.A.H. Lewis; Yuan-Peng Zhang; Feng Liu; Ronald N. McElhaney (135-140).
The synthetic peptide acetyl-K2-G-L24-K2-A-amide (P24) and its analogs have been successfully utilized as models of the hydrophobic transmembrane α-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic α-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P24 adopts a very stable α-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of α-helical transmembrane peptides with phospholipid bilayers are summarized here.
Keywords: α-Helical transmembrane peptide; Phospholipid bilayers; Lipid–protein interactions; Hydrophobic mismatch; Phosphatidylcholine; Phosphatidylethanolamine;
Electric field effects in proteins in membranes by Hans G.L. Coster; Terry C. Chilcott (141-146).
Both the organization and function of protein nanostructures in membranes are related to the substructural properties of the lipid portion of the membrane. Potential differences that are established across the membrane and generate electric fields in these very thin portions are shown to modulate the organizational and functional properties of the protein modules. Many protein modules also have nonisotropic distributions of charged sites, including configurations in which there are regions containing predominantly positive fixed charges, juxtaposed with adjacent regions containing predominantly negative fixed charges. In these double fixed charge regions, very large electric fields can manifest in the ionic depletion layer at the junction of the two fixed charge regions.Consideration is also given to the manner in which the intense electric fields that are established in protein modules, such as proton ATPases, can modulate the chemical reactions that are associated with proton transport and dehydration reactions.
Keywords: Membrane; Electric Field; Bilayer; Water dissociation; Molecular organisation; Fixed charges;
Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane by A.V. Shishkov; A.L. Ksenofontov; E.N. Bogacheva; L.V. Kordyukova; G.A. Badun; A.V. Alekseevsky; V.I. Tsetlin; L.A. Baratova (147-149).
The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10–29 of helix A. They turned out to be low (0.04±0.02 Å−1) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15±0.05 Å−1) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.
Keywords: Bacteriorhodopsin; Transmembrane helix; Tritium stratigraphy; Attenuation coefficient;
DC photoelectric signals from bacteriorhodopsin adsorbed on lipid monolayers and thiol/lipid bilayers supported by mercury by Andrea Dolfi; Francesco Tadini Buoninsegni; Maria Rosa Moncelli; Rolando Guidelli (151-156).
Purple membrane (PM) fragments were adsorbed on a dioleoylphosphatidylcholine (DOPC) monolayer and on a mixed alkanethiol/DOPC bilayer supported by mercury to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). The light-on and light-off capacitive currents on an alkanethiol/DOPC bilayer at pH 6.4 were interpreted on the basis of a simple equivalent circuit. The pH dependence of the biphasic decay kinetics of the light-on currents was analyzed to estimate the pKa values for the transitions releasing protons to, and taking up protons from, the solution. The linear dependence of the stationary light-on current of bR on a DOPC monolayer self-assembled on mercury upon the applied potential was interpreted on the basis of an equivalent circuit.
Keywords: Bacteriorhodopsin; Lipid monolayers; Bilayers; Mercury; Proton transport;
Mixed urease/amphiphile LB films and their application for biosensor development by Aidong Zhang; Yanxia Hou; Nicole Jaffrezic-Renault; Jialiang Wan; Alexey Soldatkin; Jean-Marc Chovelon (157-158).
The optimal conditions for the formation of a stable mixed urease/octadecylamine (ODA) film at the air–water interface are determined. This film is efficiently transferred onto the hydrophobized surface of a pH-ISFET, and the features of the urea enzymatic field-effect transistor (ENFET) are determined: detection limit 0.2 mM, response time 15 s, and dynamic range 0–20 mM.
Keywords: Mixed LB films; Urea ENFET; Urease; Octadecylamine; Behenic acid;
Monolayers and multilayers of chlorophyl a on a mercury electrode by M.R. Moncelli; L. Becucci; A. Dolfi; F. Tadini Buoninsegni; A. Agostiano (159-162).
A novel experimental technique used to investigate chlorophyll films on a hanging mercury drop electrode is described. Two different procedures are employed to prepare self-assembled chlorophyll monolayers and multilayers on the mercury electrode. Upon illuminating the chlorophyll a (Chl)-coated mercury electrode with an appropriate light source, the photocurrents generated by the Chl aggregates are measured under short-circuit conditions in the absence of photoartefacts. The preliminary results obtained by this novel technique are presented.
Keywords: Chlorophyll a; Self-assembled film; Mercury; Photocurrent;
Ionic conductivity of electroporated lipid bilayer membranes by S Kakorin; E Neumann (163-166).
The ionic conductivity of lipid membrane pores has been theoretically analysed in terms of electrostatic interactions of the transported ions with the low-dielectric pore wall for a commonly encountered case of unequal concentrations of electrolyte on the two sides of curved lipid membranes. Theoretical analysis of the data on the conductivity of the electroporated membrane of lipid vesicles (Lecithin 20%) of radius a=90 nm yields the molar energy of interaction of a small monovalent ion with a pore wall w 0=9±1 RT (or w 0=22±kJ mol−1), corresponding to a mean pore radius of r̄ p=0.56±0.05 nm. The proposed theoretical approach provides a tool for the analysis and description of the nonlinear current–voltage dependencies in membrane pores and channels.
Keywords: Lipid vesicles; Electroporation; Electrooptics; Conductometry; LambertW function; Nernst–Planck equation;
Spatial distribution of ion channel activity in biological membranes: the role of noise by P. Babinec; M. Babincová (167-170).
A new approach is proposed to model a collective ion channel dynamics. We have assumed that ion channels create a two-component spatio-temporal interaction field. Every channel at its current spatial location in membrane contributes permanently to this field with its state (open or closed) and coupling strength to other channels. This field is described by a reaction–diffusion equation, the transition of ion channel from closed to open state (and vice versa) is described by a master equation, and migration of channels in membrane is described by a set of Langevin equations coupled by the interaction field. Within this model, we have investigated critical conditions for spatial distribution of ion channel activity.
Keywords: Ion channels; Collective phenomena; Nonlinear dynamics; Noise;
The 40th anniversary of bilayer lipid membrane research by Angelica Ottova; H. Ti Tien (171-173).
This paper presents a historic perspective on the origin of the lipid bilayer concept and its experimental realization. Additionally, current studies in close collaboration with our colleagues on the use of supported BLMs as biosensors and molecular devices are delineated. Further, recent research of others on BLMs (planar lipid bilayers) is referenced.
Keywords: Bilayer lipid membrane; BLMs; Molecular recognition; Ion channels; Membrane reconstitution; Biosenors; Molecular devices; Biotechnology;
Polyelectrolyte-supported lipid membranes by R Kügler; W Knoll (175-178).
In this work, we report the influence of the electrostatic interaction between lipid bilayer membranes and their solid polyelectrolyte multilayer support on the properties of the membrane. All involved sample preparation steps were carried out as convenient adsorption procedures from aqueous solutions. The lipid fluidity within the membrane as well as the surface coverage of the support could be tailored via the electrostatic interaction strength between the lipid bilayer and the supporting polyelectrolyte cushion.
Keywords: Lipid bilayer membranes; Polyelectrolyte multilayers; Self-assembly; Neutron reflectometry; FRAP; Impedance spectroscopy; Surface plasmon spectroscopy;
Electrochemical studies of blocking properties of solid supported tethered lipid membranes on gold by Agnieszka Żebrowska; Paweł Krysiński; Zenon Łotowski (179-184).
The insulating properties of self-assembled thiolipid monolayers and tethered lipid bilayers on polycrystalline gold electrodes were studied by means of cyclic voltammetry (CV). These films were formed by two-step self-assembly processes. Electrochemical measurements of the heterogeneous electron transfer rate constant of different redox couples such as potassium ferrocyanide (K4[Fe(CN)6]) and dopamine (DP) were used to examine the molecular integrity and structural defects and pinholes within the monolayers. We demonstrate by means of cyclic voltammetry that the bilayer lipid membranes tethered to the gold surface are blocking, stable, yet retaining their dynamic properties and can be used as a model of the cell membrane.
Keywords: Cyclic voltammetry; t-BLMs; Potassium ferrocyanide; Dopamine; Membrane models;
Cyclic voltammetry study of glucose and insulin interactions with supported lipid membrane by G. Laputková; J. Sabo (185-188).
The effect of d-glucose and insulin on conducting properties of supported bilayer lipid membranes (s-BLM) modified by anthraquinone-2-sulphonic acid (AQS) at the presence of potassium ferricyanide was studied by means of cyclic voltammetry (CV). Both the oxidation and the reduction current peaks were found to decrease at the presence of glucose in concentration range varying from 10 to 320 mM. The influence of insulin on membrane properties is ambiguous. While the pretreatment of membrane with 20 mU l−1 of insulin evoked slight increase of the current with unchanged course of the dependence of peak current on glucose, the decrease of conductance was observed above 105 mU l−1 of insulin.
Keywords: s-BLM; Glucose; Insulin; Cyclic voltammetry; Biosensor;
DNA–membrane complexes, mitochondria and aging by V.V. Kuvichkin (189-193).
The results of extensive in vitro studies of DNA–lipid complexes allowed us to propose a model for the structure of such complexes and their involvement in the formation of DNA–membrane complexes (DMC). DMC seem to form the basis for such cellular structures as Bayer's junctions and nucleoid of bacteria, the nuclear pores, annulate lamellae and nucleoid of eucaryotes. The role of DMC in gene expression is discussed.Numerical density of mitochondria during cell aging correlates with the density of bacteria in batch culture. It is concluded that aging is caused by the unlimited growth of mitochondria and their subsequent degradation. The role of DMC in mitochondrial DNA damage at aging is discussed. The way of increasing the life span by controlling the density of mitochondria in a cell volume is likewise discussed. DMC formed between any two intracellular membranes can serve the basis for the membrane continuum in a cell.
Keywords: DNA; DNA–membrane complexes; Mitochondria; Aging;
Specific natural DNA-bound lipids in post-genome era. The lipid conception of chromatin organization by V.A. Struchkov; N.B. Strazhevskaya; R.I. Zhdanov (195-198).
Two pools of DNA-bound lipids were isolated from DNA supramolecular complex (SC-DNA): loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNase I). The compositions of the two lipid pools from different sources (rat thymus, liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B and T2 phage) were studied. The possible functions of DNA-bound lipids, especially of cardiolipin and cholesterol, at the attachment of DNA loops to the nuclear matrix, in DNA replicon organization, replication and transcription are discussed.
Keywords: DNA; Chromatin; Cardiolipin; Cholesterol; Functional genomics;
The asymmetric, rectifier-like I–V curve of the Na/K pump transient currents in frog skeletal muscle fibers by Wei Chen; Wenhui Wu (199-202).
The Na/K pump transient currents in skeletal muscle fiber were identified using an improved double Vaseline gap voltage clamp technique. The asymmetric characteristics of the pump current–voltage relationship were studied. The definition of the Na/K pump currents was the ouabain-sensitive currents, where ouabain is a specific Na/K ATPase inhibitor. Membrane potential was held at −90 mV, the membrane resting potential. A series of stimulation pulse-pairs symmetric to the membrane resting potential were applied to the cell membrane. The summation of the currents responding to the two pulses in each pair indicates the asymmetry of the pump currents with respect to the membrane resting potential.The voltage dependence of the Na/K pump transient currents from skeletal muscle is similar to the steady-state I–V curve from either skeletal muscle fibers or cardiac muscles. It is a sigmoidal-shaped, asymmetric curve with respect to the membrane resting potential. This asymmetric, rectifier-like voltage dependence indicates that a symmetric oscillating membrane potential may generate a net, outward pump current. In other words, the Na/K pump molecules may be activated by an oscillating membrane potential.
Keywords: Na/K ATPase; Skeletal muscle; Voltage dependence; Oscillating electric field;
The influence of sugar synthesis and transport rates on bioenergetics and kinetics of higher plant photosynthesis by A Tuleshova; D Badretdinov; A Kukushkin; S Khuznetsova (203-205).
We have obtained the theoretical dependences of stromal metabolite steady-state concentrations on external inorganic phosphate (Pi) concentration. For this purpose, the theoretical model of photosynthesis, earlier described in [Ann. Appl. Biol. 138 (2001) 117], was modified to account for the regulation of starch formation by 3-phosphoglycerate (PGA) to Pi ratio. When the rate constant of starch synthesis is taken to be independent on PGA/Pi ratio, the steady-state concentrations of Calvin cycle metabolites and starch were found to change insignificantly as external Pi concentration increases in wide range. However, as external Pi range exceeds a critical value, the steady-state concentrations of all metabolites change abruptly. If the rate constant of starch synthesis depends on PGA/Pi ratio, the steady-state concentrations of stromal metabolites change qualitatively in the same way with the increase of external Pi. However, no abrupt changes of metabolite and starch steady-state levels at high concentration of Pi are observed. These results testify that the control of starch synthesis by PGA/Pi ratio makes the photosynthetic system more stable in wide range of external Pi concentrations.
Keywords: Starch; Inorganic phosphate; Phosphate translocator; Theoretical model;
Understanding conditions for which biological effects of nonionizing electromagnetic fields can be expected by James C. Weaver (207-209).
Scientific interest in the interaction of nonionizing electromagnetic fields with biological systems is longstanding, but often still controversial. Theories, models and computer simulations have usually emphasized physical interactions with subsystems (e.g. cell membranes) of a biological system. By extending this first necessary physical step to a second step of explicitly and quantitatively considering chemical changes, increased understanding appears possible. In the case of “strong fields”, the role of field-altered chemistry is important to electrochemotherapy [Biochem. Pharmacol. 42, Suppl. (1991) 567] and creation of transdermal microconduits [Bioelectrochem. Bioenerg. 49 (1999) 11; J. Controlled Release 61 (1999) 185; J. Invest. Dermatol. 116 (2001) 40] For “weak fields” (a topic with much more controversy) consideration of chemical change shows that organized multicellular systems can be understood to respond to extremely small electric [Chaos 8 (1998) 576] or magnetic fields [Nature 405 (2000) 707]. In contrast, isolated individual cells interacting via voltage-gated channels [Proc. Natl. Acad. Sci. 92 (1995) 3740; Biophys. J. 75 (1998) 2251; Bioelectromagnetics 20 (1999) 102], or processes without “temperature compensation” [Biophys. J. 76 (1999) 3026], appear implausible. Satisfactory understanding is likely only if experimental and theoretical work is reconciled, which should therefore be emphasized. The interaction of electromagnetic fields with biological systems is of interest because of fundamental scientific curiosity, potential medical benefits and possible human health hazards.
Keywords: Nonionizing; Electromagnetic fields; Chemical changes; Strong fields; Weak fields;
Estimating the subcellular absorption of electric field energy: equations for an ellipsoidal single shell model by D. Wachner; M. Simeonova; J. Gimsa (211-213).
An oriented single shell model is used to describe the absorption of electric field energy for a cell of the general ellipsoidal shape exposed to a homogeneous AC-field. A finite element approach allowed us to derive characteristic equations describing the dependence of the field distribution on the cell geometry, the electric properties of the structural media, membrane and bulk solutions, as well as on the field frequency with a subcellular resolution. Finally, equations were derived for the absorption at certain sites of the model. The model allows for the introduction of frequency-dependent cellular media properties. Experimentally, the new cell parameters can be verified by dielectric single-cell spectroscopy.
Keywords: Power dissipation; Absorption; Finite element model; Dielectric dispersion; Electro-smog;
Cellular absorption of electric field energy: influence of molecular properties of the cytoplasm by M. Simeonova; D. Wachner; J. Gimsa (215-218).
Molecular dispersions may significantly alter the frequency dependence of structural polarizations. Consequently, the molecular properties cannot be neglected when the energy absorption is calculated with a subcellular resolution. Our example presents calculations that explain the absorption in single human red blood cells. The molecular properties of the cytoplasm have been derived from literature data on the impedance of Hb suspensions. The resulting cell properties were then compared to own data obtained by single cell dielectric spectroscopy.
Keywords: Molecular dispersions; Human red blood cells; Hemoglobin; Ellipsoidal single shell model; Single cell dielectric spectroscopy;
Evaluation of viability of retinal photoreceptor cells by using their endogenous electrical field by Eugenia Kovács; Roxana Pologea-Moraru; Basarab Gabriel Hosu (219-221).
The rod photoreceptor cells are electrical dipoles sustained by cell metabolic energy. Polarity of photoreceptor cells is directly connected to the so-called “dark current” which circulate along the living photoreceptors. Since only the living cells in a good functional state display electrical polarity, the orientation of photoreceptors in static electric field reflects their viability as long as it depends on the functionality of molecular mechanisms that maintain the dark current.Studying the rod cells' orientation in static electric field at different times after their isolation is thus an accurate way to evaluate the cell viability/degeneration. Retinal transplant experiments in animals and humans, which are presently in progress, require a quick and reliable viability test of cells/tissue to be transplanted. Checking the orientation pattern of rod photoreceptors in static electric field prior to transplantation is a candidate method for an accurate cell viability test.
Keywords: Cell viability; Photoreceptors polarity; Orientation patterns; Retinal transplant;
The effects of low level microwaves on the fluidity of photoreceptor cell membrane by Roxana Pologea-Moraru; Eugenia Kovacs; Karina Roxana Iliescu; Violeta Calota; Gheorghe Sajin (223-225).
Due to the extensive use of electromagnetic fields in everyday life, more information is required for the detection of mechanisms of interaction and the possible side effects of electromagnetic radiation on the structure and function of the organism.In this paper, we study the effects of low-power microwaves (2.45 GHz) on the membrane fluidity of rod photoreceptor cells. The retina is expected to be very sensitive to microwave irradiation due to the polar character of the photoreceptor cells [Biochim. Biophys. Acta 1273 (1995) 217] as well as to its high water content [Stud. Biophys. 81 (1981) 39].
Keywords: Athermal microwave effects; Photoreceptors; Membrane fluidity;
Effects of static magnetic field on human leukemic cell line HL-60 by J. Sabo; L. Mirossay; L. Horovcak; M. Sarissky; A. Mirossay; J. Mojzis (227-231).
A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.
Keywords: HL-60; Magnetic fields; Chemotherapy; Cytotoxicity;
Clinical evaluation of safety and human tolerance of electrical sensation induced by electric fields with non-invasive electrodes by L Zhang; D.P Rabussay (233-236).
This paper reports the first clinical safety study of human tolerance of electrical sensation using non-invasive, flexible surface-type electrodes and exponentially decaying electric pulses. The study evaluated the effect of electric fields in the absence of a drug and an anesthetic, and was performed in light of potential applications in the field of erectile dysfunction (ED). Twenty impotent patients who had previously received injection or intraurethral therapies were enrolled in the study. Voltage escalations from 50 to 80 V (in 10-V increments) with a single pulse of 3-ms duration were performed with meander-type electrodes placed on the shaft and part of the glans of the penis. The electric fields-induced sensation was assessed via a pain scale from 0 to 10. All 20 patients, who were free to withdraw from the study at any point, completed the voltage escalation study. No clinical safety concerns were apparent and no skin irritation was observed after electric treatment. Our initial study indicates that the pulses in the tested voltage range were well tolerated by most patients. In previous animal experiments under analogous experimental conditions, the application of 50 V has been found effective for transdermal drug delivery into the penis.
Keywords: Electrical sensation; Human; Skin; Electroporation; Impotence; Anesthesia;