Bioelectrochemistry (v.79, #1)

Contents (v-vi).

A carbon-coated nickel magnetic nanoparticles modified glassy carbon electrode (C–Ni/GCE) was fabricated. The carbon-coated nickel magnetic nanoparticles were characterized with transmission electron microscopy (TEM). The electrochemical behaviors of norepinephrine (NE) were investigated on the modified electrode by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The carbon-coated nickel magnetic nanoparticles showed excellent electrocatalytic activity for the electrochemical redox of NE. NE exhibited two couples of well-defined redox peaks on C–Ni/GCE over the potential range from − 0.4 to 0.8 V in phosphate buffer solution (PBS) (pH = 7.0). The redox mechanism for NE was proposed. DPV response of NE on the C–Ni/GCE showed that the catalytic oxidative peak current was linear with the square root concentration of NE in the range of 2.0 × 10− 7 to 8.0 × 10− 5  M, with a detection limit of 6.0 × 10− 8  M. The C–Ni/GCE showed good sensitivity, selectivity and stability for the determination of NE.
Keywords: Carbon-coated nickel; Magnetic nanoparticles; Norepinephrine; Electrochemical determination;

Interaction of surface-attached haemoglobin with hydrophobic anions monitored by on-line acoustic wave detector by Jonathan S. Ellis; Steven Q. Xu; Xiaomeng Wang; Grégoire Herzog; Damien W.M. Arrigan; Michael Thompson (6-10).
The behaviour of proteins on surfaces and at interfaces is an important field with applications in drug development, clinical diagnostics and studies of device biocompatibility. A key factor is the conformation of surface-bound proteins, which can affect chemical signalling and drug binding. A recent study of the interactions of haemoglobin with hydrophobic anions at a liquid–liquid interface has shown that a pH- and orientation-dependent conformational change occurs in the haemoglobin molecule upon interaction with these anions. To corroborate these results, we use an acoustic wave detector to study binding of solution-phase hydrophobic anions to surface-adhered haemoglobin. The orientation of protein is controlled by thiol chemistry, which generates hydrophilic and hydrophobic surfaces. Tetraphenylborate-based anions are introduced to the haemoglobin coated surface via an on-line flow-injection system to monitor the signal in real-time. Changes in the acoustic properties of the surface, measured piezoelectrically, are related to interactions between the protein and the anions. Signal strength is proportional to the degree of interaction between the salts and the haemoglobin, which in turn, is influenced by its conformation.
Keywords: Acoustic wave biosensor; QCM; Protein adsorption; Protein–small molecule interactions; Haemoglobin;

Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor by A. Ramanavicius; A. Finkelsteinas; H. Cesiulis; A. Ramanaviciene (11-16).
Polypyrrole (Ppy) has been shown as a matrix for label-free electrochemical immunosensor based on electrochemical impedance spectroscopy (EIS) measurements. The immunosensing system model presented here was based on bovine leukemia virus (BLV) protein (gp51) entrapped within electrochemically-synthesized polypyrrole (Ppy/gp51). This Ppy/gp51 layer interacted with antibodies against gp51 (anti-gp51-Ab) that are present in significant concentration in the blood serum of BLV infected cattle. After this interaction protein complex (Ppy/gp51/anti-gp51-Ab) was formed. The horseradish peroxidase (HRP) labeled secondary antibodies (Ab⁎) against anti-gp51-Ab were applied as agents interacting with Ppy/gp51/anti-gp51-Ab and forming the large protein complex (Ppy/gp51/anti-gp51-Ab/Ab⁎). The EIS study was performed for electrodes modified with different Ppy layers described here and an optimal equivalent circuit was adopted for evaluation of EIS spectra, it was a major outcome of this study.
Keywords: Conducting polymers; Polypyrrole; EIS immunosensor; Nanotechnology; NanoBioTechnology;

Electrochemical and AFM characterization on gold and carbon electrodes of a high redox potential laccase from Fusarium proliferatum by K. González Arzola; Y. Gimeno; M.C. Arévalo; M.A. Falcón; A. Hernández Creus (17-24).
The redox potential of the T1 copper site of laccase from Fusarium proliferatum was determined by titration to be about 510 mV vs. SCE (750 mV vs. NHE), which makes it a high redox potential enzyme. Anaerobic electron transfer reactions between laccase and carbon and gold electrodes were detected, both in solution and when the enzyme was adsorbed on these surfaces. In solution, a single high-potential signal (660 mV vs. SCE) was recorded at the carbon surfaces, attributable to the T1 copper site of the enzyme. However, a well-defined oxidative process at about 660 mV and an anodic wave at 350 mV vs. SCE were recorded at the gold electrode, respectively associated with the T1 and T2 copper sites. Laccase-modified carbon electrodes behaved analogously when the enzyme was in solution, unlike laccase adsorbed on gold, which showed only a low-potential signal. Laccase molecules were successfully imaged by AFM; obtaining a thick compact stable film on Au(111), and large aggregates forming a complex network of small branches leaving voids on the HOPG surface. Laccase-modified carbon electrodes retained significant enzymatic activity, efficiently oxidising violuric acid and reducing molecular oxygen. Explanations are proposed for how protein-film organisation affects the electrode function.
Keywords: Laccase; Redox potential; Catalytic activity; Oxygen electroreduction; AFM;

Improvements in the extraction of cell electric properties from their electrorotation spectrum by Damien Voyer; Marie Frénéa-Robin; Franois Buret; Laurent Nicolas (25-30).
In this paper, we propose a new approach to perform cell dielectric characterization from their electrorotation spectrum. At first, a variance analysis is carried out to quantify the dispersion in electrorotation spectra due to the different parameters involved. On this basis, the impact of each parameter is emphasized by weighing the spectrum with an appropriate frequency-dependent coefficient: this technique enables to minimize the coupling effects which deteriorate the accuracy of parameter extraction. In addition, the Nelder–Mead simplex algorithm used in the identification procedure is modified to account for bounded intervals in which the unknown parameters are expected to vary. Both these techniques have proven to give increased confidence levels compared to previous work reported in the literature.
Keywords: Electrorotation; Dielectric parameters; Variance analysis; Nelder–Mead simplex;

Electrochemical DNA biosensor for the detection of specific gene related to Trichoderma harzianum species by Shafiquzzaman Siddiquee; Nor Azah Yusof; Abu Bakar Salleh; Fatimah Abu Bakar; Lee Yook Heng (31-36).
A new electrochemical biosensor is described for voltammetric detection of gene sequence related to Trichoderma harzianum. The sensor involves immobilization of a 20 base single-stranded probe (ssDNA), which is complementary to a specific gene sequence related to T. harzianum on a gold electrode through specific adsorption. The DNA probe was used to determine the amount of target gene in solution using methylene blue (MB) as the electrochemical indicator. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the electrode. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE and the probe-modified AuE which localized to the affinity of MB. Control experiments with the non-complementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. DNA biosensor also able to detect microorganism at the species levels without nucleic acid amplification. The redox current was linearly related to the concentration of target oligonucleotide DNA, ranged from 1–20 ppm. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time.
Keywords: Trichoderma harzianum; Electrochemical labeled probe; DNA; Hybridization;

In this article, a simple strategy of electroless deposition for gold nanoparticle (Au NP) modification on the conductive substrate is developed. The morphology of Au NP modified electrode could be controlled to some extent by choosing different solution concentrations, deposition times, etc. The Au NP modification increased the electrode surface area largely, and the surface area after Au NP modification on the polyelectrolyte multilayer (PEM) assembled electrode was about 3.3 times that of the planar gold electrode. The enhancement of DNA immobilization and hybridization on the Au NP modified electrode were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) with the use of Ru(NH3)6 3+ as an electrochemical redox indicator. With this approach, the sensitivity of Au NP modified PEM electrode for target DNA could reach 1 × 10− 11  M. Compared with that of planar gold electrode, the detection limit was increased to be about 3 orders of magnitude.
Keywords: Electroless deposition; Gold nanoparticles; DNA hybridization; Electrochemistry;

In this work, studies on the effects produced by atrazine, terbutryn or diuron onto spinach photosynthetic materials were performed by observing changes in fluorescence emission and in electron transfer activities of the bio-samples in the presence of such herbicides; chloroplasts, thylakoids, Photosystem II-enriched thylakoids (BBYs) and isolated Photosystem II (PSII) were employed. This approach evidenced differences in the herbicide–photosynthetic material interactions going up–down from chloroplasts to proteins. Rapid emission increments were detected for chloroplasts and thylakoids, in particular in the presence of terbutryn; no remarkable emission increment was recorded when BBYs or PSII were used for this assay. The dependences of the chloroplast and thylakoid emission intensities upon herbicide concentration were investigated with responses even at concentrations below 10− 7  M. The influence of lowering the temperature was also tested, and the stabilizing effects on the resistances of the bio-samples against herbicides were recorded. Furthermore, Hill Reaction-based colorimetric assays were performed to monitor the electron transfer activities of the bio-samples in the presence of herbicides, after brief incubations. As a result, chloroplasts and thylakoids resulted to be sensitive tools in responding to concentrations even lower than 10− 7  M of most herbicides; nevertheless, an interesting sensitivity to herbicides was also observed for PSII.
Keywords: Photosynthetic materials; Herbicides; Optical assays; Fluorescence emission;

Nanostructured polypyrrole-coated anode for sun-powered microbial fuel cells by Yongjin Zou; John Pisciotta; Ilia V. Baskakov (50-56).
Sun-powered or photosynthetic microbial fuel cells (PMFCs) offer a novel approach for producing electrical power in a CO2-free self-sustainable manner in the absence of organic fuel. Recent discovery that cyanobacteria display electrogenic activity under illumination emphasized the need to develop improved anode materials capable of harvesting electrons directly from photosynthetic cultures. Here, we showed that nanostructured electrically conductive polymer polypyrrole substantially improved the efficiency of electron collection from photosynthetic biofilm in PMFCs. Nanostructured fibrillar polypyrrole showed better performance than granular polypyrrole. Cyclic voltammetry and impedance spectroscopy analyses revealed that better performance of nanostructured anode materials was due to the substantial improvement in electrochemical properties including higher redox current and lower interface electron-transfer resistance. At loading density of 3 mg/cm2, coating of anode with fibrillar polypyrrole resulted in a 450% increase in the power density compared to those reported in our previous studies on PMFCs that used the same photosynthetic culture.
Keywords: Microbial fuel cells; Polypyrrole; Photosynthetic; Self-sustainable; Bioelectricity; Cyanobacteria; Electrochemistry; Electrogenic;

The electrochemical oxidation of 3,4-dihydroxyphenylacetic acid (DOPAC) on a carbon fiber microelectrode (CF) and a glassy carbon macroelectrode (GC) in glacial acetic acid solutions was investigated using voltammetric techniques. Voltammograms recorded at these electrodes show well-defined single waves or peaks. The proposed mechanism of the anodic oxidation of DOPAC consists of two successive one-electron one-proton steps. The loss of the first electron proceeds irreversibly and determines the overall rate of the electrode process. This stage is accompanied by the generation of an unstable phenoxyl radical in position 4 of the aromatic ring. The second stage of the electrode reaction produces substituted orto-quinone as the final product of the electrode process of DOPAC. DOPAC exhibits more antioxidative power than synthetic BHT and can be useful in food protection against reactive oxygen species. The results presented can help to explain biochemical and antioxidative properties of DOPAC in a living cell and can be useful in determination of this compound in real samples.
Keywords: 3,4-dihydroxyphenylacetic acid; Electrochemical oxidation; Voltammetry; Microelectrode; Acetic acid; Antioxidant;

It is the first report about the usage of Rhodotorula mucilaginosa as a biomaterial to construct a microbial biosensor based on carbon paste for determination of copper. Cu(II) was preconcentrated electrode surface at open circuit and then detected with electrochemical techniques, including Cyclic Voltammetry (CV) and Differential Pulse Stripping Voltammetry (DPSV). Some parameters such as pH of preconcentration solution, preconcentration time, scan rate and effect of interfering heavy metal ions were carried out for optimum responses. The best defined cathodic peak was obtained at pH 5 with 0.05 M NaNO3 and a scan rate of 100 mV/s. The linear range for the developed microbial biosensor was found in the range of 1.0 × 10− 7 and 1.0 × 10− 5  M (0.0064 and 0.64 mg/L) at the response time of 15 min (R 2  = 0.98). The easy fabrication, sensitivity, low cost and fast response time showed the advantages of the biosensor to conventional techniques.
Keywords: Microbial biosensor; Carbon paste electrode; Copper; Rhodotorula mucilaginosa; Voltammetry;

Microbial fuel cell (MFC) holds a great promise to harvest electricity directly from a wide range of ready degradable organic matters and enhance degradation of some recalcitrant contaminants. Glucose, acetate sodium and ethanol were separately examined as co-substrates for simultaneous bioelectricity generation and Congo red degradation in a proton exchange membrane (PEM) air-cathode single-chamber MFC. The batch test results showed that more than 98% decolorization at the dye concentration of 300 mg/L were achieved within 36 h for all tested co-substrates during electricity generation. The decolorization rate was different with the co-substrates used. The fastest decolorization rate was achieved with glucose followed by ethanol and sodium acetate. Accumulated intermediates were observed during Congo red degradation which was demonstrated by UV–Visible spectra and high performance liquid chromatography (HPLC). Electricity generation was sustained and not significantly affected by the Congo red degradation. Glucose, acetate sodium and ethanol produced maximum power densities of 103 mW/m2, 85.9 mW/m2 and 63.2 mW/m2, respectively, and the maximum voltage output decreased by only 7% to 15%. Our results demonstrated the feasibility of using various co-substrates for simultaneous decolorization of Congo red and bioelectricity generation in the MFC and showed that glucose was the preferred co-substrate.
Keywords: Microbial fuel cell; Congo red; Co-substrate; Decolorization; Electricity generation;

Electrochemical oxidation of amphetamine-like drugs and application to electroanalysis of ecstasy in human serum by E.M.P.J. Garrido; J.M.P.J. Garrido; N. Milhazes; F. Borges; A.M. Oliveira-Brett (77-83).
Amphetamine and amphetamine-like drugs are popular recreational drugs of abuse because they are powerful stimulants of the central nervous system. Due to a dramatic increase in the abuse of methylenedioxylated derivatives, individually and/or in a mixture, and to the incoherent and contradictory interpretation of the electrochemical data available on this subject, a comprehensive study of the redox properties of amphetamine-like drugs was accomplished. The oxidative behaviour of amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) was studied in different buffer systems by cyclic, differential pulse and square-wave voltammetry using a glassy carbon electrode. A quantitative electroanalytical method was developed and successfully applied to the determination of MDMA in seized samples and in human serum. Validation parameters, such as sensitivity, precision and accuracy, were evaluated. The results found using the developed electroanalytical methodology enabled to gather some information about the content and amount of MDMA present in ecstasy tablets found in Portugal. Moreover, the data found in this study outlook the possibility of using the voltammetric methods to investigate the potential harmful effects of interaction between drugs such as MDMA and methamphetamine and other substances often used together in ecstasy tablets.
Keywords: Amphetamines; Ecstasy; Electrochemistry; Seizure samples; Biological fluids;

Platinum nanoparticles (nano-Pt) and poly(o-aminophenol) (POAP) were fabricated onto the glassy carbon electrode(GCE) to form the nano-Pt/POAP/GCE for the electrochemical determination of l-cysteine. The POAP film was obtained through electrochemical polymerization of o-aminophenol on GCE. The nano-Pt was electrochemically deposited onto the surface of the activated POAP/GCE The resultant nano-Pt/POAP/GCE was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), and showed excellent electrochemical response to l-cysteine at low oxidative potential in Britton–Robinson (BR) buffer solution (pH = 3.0), with good stability and sensitivity, and featured with a low detection limit (0.08 μM, signal/noise = 3) and wide linear range (0.4 μM–6.3 mM).The mechanism for the electrochemical oxidation of l-cysteine on the nano-Pt/POAP/GCE was also investigated.
Keywords: Poly(o-aminophenol); Platinum nanoparticles; l-cysteine;

The effects of the electro-photodynamic in vitro treatment on human lung adenocarcinoma cells by Jolanta Saczko; Mariola Nowak; Nina Skolucka; Julita Kulbacka; Malgorzata Kotulska (90-94).
The effectiveness of the photodynamic therapy (PDT), a low-invasive and targeted therapy of cancer, could be intensified by increasing the intracellular transport of a photosensitizer. The electroporation is used to generate non-specific transient nanopores that facilitate local drug delivery into cells. Photodynamic therapy assisted by electroporation was tested in vitro on the human lung carcinoma cell line A549 by determining the mitochondrial cell function using the MTT assay. The photodynamic activity of the electro-photodynamic treatment (EPDT) with the hematoporphyrin derivative was evaluated in relation to the photodynamic method alone. The experiments show significantly increased efficiency of EPDT, which allows reducing drug doses and exposure time of the cells to the drug in standard PDT. The results have been confronted with the model based on van't Hoff equation. This showed that the growth fractions of cells after EPDT depend on the electric field according to the same relation as in case of electroporation alone.
Keywords: Electroporation; Electrochemotherapy; Photodynamic therapy; Lung cancer; Van't Hoff equation;

Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death by Franck M. André; Mikhail A. Rassokhin; Angela M. Bowman; Andrei G. Pakhomov (95-100).
It has been widely accepted that nanosecond electric pulses (nsEP) are distinguished from micro- and millisecond duration pulses by their ability to cause intracellular effects and cell death with reduced effects on the cell plasma membrane. However, we found that nsEP-induced cell death is most likely mediated by the plasma membrane disruption. We showed that nsEP can cause long-lasting (minutes) increase in plasma membrane electrical conductance and disrupt electrolyte balance, followed by water uptake, cell swelling and blebbing. These effects of plasma membrane permeabilization could be blocked by Gd3+ in a dose-dependent manner, with a threshold at sub-micromolar concentrations. Consequently, Gd3+ protected cells from nsEP-induced cell death, thereby pointing to plasma membrane permeabilization as a likely primary mechanism of lethal cell damage.
Keywords: Permeabilization; Plasma membrane; Cell death; Nanosecond electric pulses; Gadolinium;

Scanning electrochemical microscopy activity mapping of electrodes modified with laccase encapsulated in sol–gel processed matrix by Wojciech Nogala; Katarzyna Szot; Malte Burchardt; Martin Jönsson-Niedziolka; Jerzy Rogalski; Gunther Wittstock; Marcin Opallo (101-107).
Electrodes modified with sol–gel encapsulated laccase (isolated from Cerrena unicolor) exhibiting mediated or mediatorless bioelectrocatalytic dioxygen reduction activity were inspected using confocal laser scanning microscopy, atomic force microscopy and scanning electrochemical microscopy. Potential-driven leaching of the redox mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) from carbon ceramic electrodes covered by hydrophilic silicate-encapsulated laccase was detected during electrocatalytic action. Strongly non-homogeneous lateral distribution of the activity towards dioxygen reduction was found by redox competition mode of scanning electrochemical microscopy using a similar electrode with syringaldazine as redox mediator. Hydrogen peroxide formation at these electrodes is detected at potentials lower than 0.05 V. It is ascribed to the electrochemical oxygen reduction at the carbon material while laccase-catalyzed oxygen reduction occurs below 0.35 V without hydrogen peroxide formation. The scanning electrochemical microscopy images of electrodes consisting of single-walled carbon nanotubes non-covalently modified with pyrenesulfonate and laccase encapsulated in a sol–gel processed silicate film confirm direct electron transfer electrocatalysis in redox competition mode experiments and show that the enzyme is evenly distributed in the composite film. In conclusion scanning electrochemical microscopy proved to be useful for mapping of enzyme activity on different materials.
Keywords: Laccase; Dioxygen reduction; Carbon nanotubes; Carbon ceramic electrode; Scanning electrochemical microscopy;

Maltose biosensing based on co-immobilization of α-glucosidase and pyranose oxidase by Dilek Odaci; Azmi Telefoncu; Suna Timur (108-113).
A new bi-enzymatic system was designed by co-immobilization of α-glucosidase (AG) and pyranose oxidase (PyOx) for maltose analysis. The immobilization was carried out by cross-linking enzyme mixture, chitosan (CHIT) and carbon nanotube (CNT) via glutaraldehyde. The structure of biosensor including enzyme, CHIT, glutaraldehyde and CNT amount together with operational conditions like pH, temperature and applied potential were optimized. Then analytical characterization was performed. A fast linear response of the biosensor was observed for maltose in the concentration range from 0.25 to 2.0 mM at 35 °C and pH 6.0. The effect of CNT addition into the immobilization matrix was also investigated. The linear relationships between sensor response (y; μA/cm2 ) and substrate concentration (x; mM) were defined by the equations of y  = 0.844x  + 0.029 (R 2  = 0.999) and y  = 0.882x  + 0.0625 (R 2  = 0.996) for AG/PyOx/CHIT and AG/PyOx/CHIT–CNT biosensors, respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free. Finally, for the sample application, maltose was analyzed in beer samples. As a result, it has been found that; complex matrix of natural beer samples had no influence on the biosensing response. Also the results were in good agreement with those obtained by spectrophotometric measurements.
Keywords: Maltose; α-glucosidase; Pyranose oxidase; Carbon nanotube; Chitosan;

Plasma membrane permeabilization by trains of ultrashort electric pulses by Bennett L. Ibey; Dustin G. Mixon; Jason A. Payne; Angela Bowman; Karl Sickendick; Gerald J. Wilmink; W. Patrick Roach; Andrei G. Pakhomov (114-121).
Ultrashort electric pulses (USEP) cause long-lasting increase of cell membrane electrical conductance, and that a single USEP increased cell membrane electrical conductance proportionally to the absorbed dose (AD) with a threshold of about 10 mJ/g. The present study extends quantification of the membrane permeabilization effect to multiple USEP and employed a more accurate protocol that identified USEP effect as the difference between post- and pre-exposure conductance values (Δg) in individual cells. We showed that Δg can be increased by either increasing the number of pulses at a constant E-field, or by increasing the E-field at a constant number of pulses. For 60-ns pulses, an E-field threshold of 6 kV/cm for a single pulse was lowered to less than 1.7 kV/cm by applying 100-pulse or longer trains. However, the reduction of the E-field threshold was only achieved at the expense of a higher AD compared to a single pulse exposure. Furthermore, the effect of multiple pulses was not fully determined by AD, suggesting that cells permeabilized by the first pulse(s) in the train become less vulnerable to subsequent pulses. This explanation was corroborated by a model that treated multiple-pulse exposures as a series of single-pulse exposures and assumed an exponential decline of cell susceptibility to USEP as Δg increased after each pulse during the course of the train.
Keywords: Ultrashort electric pulses; Electroporation; Patch clamp; Membrane;

The paper is a new approach which aims to evaluate the relation between surface aspects (wettability and roughness) of materials based on titanium with native passive TiO2 as untreated samples and TiO2 nanotubes as treated discs respectively, their electrochemical stability in artificial saliva, and fibroblast cell behavior.Ti/TiO2 modified electrodes as nanotubes with 120 nm as diameter were obtained using an electrochemical method as anodizing and surface analysis as SEM, AFM and contact angle measurements were performed to obtain topographical features and wettability.The TiO2 nanotube structured oxide films electrochemical growth increases the stability of titanium surfaces. The electrochemical behavior of the Ti/TiO2 nanotube surface was evaluated by corrosion parameters obtained from Tafel plots and electrical parameters for proposed circuits from electrochemical impedance spectroscopy were analyzed.The cell results indicated a slight preference in terms of cell survival and adhesion for nanostructure TiO2 with a more hydrophilic character and the electrochemical data revealed that such features are connected with better stability in artificial saliva. The roughness seems to be not conclusive for this case.
Keywords: Gingival fibroblasts; Biocompatibility; TiO2 nanotubes; Electrochemical stability; Wettability; EIS;

Efficiency of the delivery of small charged molecules into cells in vitro by M.S. Venslauskas; S. Šatkauskas; R. Rodaitė-Riševičienė (130-135).
The effectiveness of the delivery of small charged molecules, including anticancer drugs into MH22 hepatoma cells in vitro was investigated. It was shown that for each kind of small molecules one can find a specific set of pulse strength–duration combinations that define electrotransfer of chosen compounds into the same amount of electroporated cells. Analysis of experimental data from the point of theory of hydrophilic aqueous pores and the estimation of the contribution of the electrostatic Born's energy to the change in free energy suggests that the main factors defining small molecules transfer through the membrane are: the charge and size of molecules, the permittivities of external medium, membrane material, and the electropores respectively as well as the size of electropores. The joint impact of all mentioned factors on transfer efficiency is essential.
Keywords: Electroporation; Drug electrodelivery; Molecule's electrotranfer;

Carbon nanotube-enhanced cell electropermeabilisation by Vittoria Raffa; Gianni Ciofani; Orazio Vittorio; Virginia Pensabene; Alfred Cuschieri (136-141).
The use of controlled electric fields to facilitate cell permeabilisation for enhanced cellular uptake of molecules is well established. The main limitation to the application of this technology in clinical practice is the requirements of high voltages which cause significant cell death in the target tissue. This paper presents a new modality for cell electro-permeabilisation based on the use of carbon nanotubes (CNTs) and external static electric fields. An explanation of the results based on the dielectric response of multiwall CNTs (MWCNTs) to these electric fields is proposed. The experimental data obtained indicate that this method of CNT-enhanced electro-permeabilisation provides an effective means of lowering the electric field voltage required for repairable cell electro-permeabilisation to below 50 V/cm and with an efficiency exceeding 80%.
Keywords: Carbon nanotubes; Static electric fields; Cell electropermeabilization;

Dependence of catalytic activity and long-term stability of enzyme hydrogel films on curing time by Joshua Lehr; Bryce E. Williamson; Frédéric Barrière; Alison J. Downard (142-146).
Enzyme hydrogels were prepared on carbon film electrodes using glucose oxidase and an epoxide crosslinking agent. The catalytic activity of the gels was found to depend strongly on curing time. The competing effects of increased mechanical stability and decreased enzyme activity as curing time increases resulted in the highest catalytic activity for films cured for 24 h at 25 °C. Weekly electrochemical measurements established that the long-term stabilities of all hydrogels cured for 24–72 h were similar, with close to half of the initial catalytic activity being retained after immersion for 3 months in agitated phosphate buffer solution at 25 °C.
Keywords: Hydrogel; Glucose oxidase; Poly(ethylene glycol) diglycidyl ether; Carbon; Hydroxymethylferrocene;

Enzymatic flow injection method for rapid determination of choline in urine with electrochemiluminescence detection by Jiye Jin; Masahiro Muroga; Fumiki Takahashi; Toshio Nakamura (147-151).
In order to determine trace choline in human urine, a flow injection analysis (FIA) system has been developed by coupling an enzyme reactor with an electrochemiluminescence (ECL) detector. The enzyme reactor is prepared by covalently immobilizing choline oxidase (ChOx) onto the aminopropyl-controlled pore glass beads, which are then carefully packed into a micro column. The enzyme reactor catalyzes the production of hydrogen peroxide that is in direct proportion to the concentration of choline. In this study, the enzymatically produced hydrogen peroxide was detected by an ECL detector positioned at the down stream of an enzyme reactor based on the luminol/H2O2 ECL system. Under the optimized condition, the enzymatic FIA/ECL provided high sensitivity for the determination of choline with the detection limit as low as 0.05 µM (absolute detection limit was at sub pmol level). The method was successfully applied in the determination of choline in the samples of human urine, and the analytical results were in good agreement with those obtained by using the microbore HPLC with an immobilized enzyme reactor-electrochemical detection system.
Keywords: Electrochemiluminescence; Flow injection; Enzyme reactor; Choline; Human urine;