Bioelectrochemistry (v.71, #1)

by Wlodzimierz Kutner (1).

A convenient method for the measurement of the catalytic activity of laccase is proposed based on the voltammetric determination of catalytic reaction substrates: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) and 1,4-benzenediol (hydroquinone). The measurement performed using microelectrodes working under spherical diffusion conditions is both accurate and simple, and allows to monitor parallely the consumption of substrate and formation of product of the catalytic reaction. The method proposed in this paper was compared with the two generally employed procedures based on oxygen measurement by Clark electrode and on spectrophotometry. The procedure described in the present paper was found to be simpler and more reproducible results were obtained than using Clark electrode. Compared to spectrophotometry a larger range of catalytic reaction substrates can be studied including colorless compounds.
Keywords: Laccase; Microelectrode; Michaelis–Menten model; Catalytic activity; Enzymatic kinetics; ABTS; Hydroquinone;

The monoolein-based liquid crystalline cubic phase was used as the matrix to incorporate redox enzymes — glucose (GOx), pyranose (PyOx) oxidases and laccase. Thin layer of the cubic phase embedding GOx or PyOx activated glucose oxidation in the presence and absence of appropriate mediators. The electrodes exhibited unchanged voltammetric response to glucose for not less than six days. The potentials and ratio of catalytic to diffusion currents could be modified by choosing appropriate electroactive probes as mediators. Ferrocenecarboxylic acid and Ru(NH3)6 2+ provided contact between the electrode and the enzyme. The sensitivity to glucose for glucose oxidase was 0.4 ± 0.05, 11 ± 3.1 μA/cm2/mM without mediator and with ferrocenecarboxylic acid respectively and 0.9 ± 0.06, 31 ± 5.6 μA/cm2/mM for pyranose oxidase without and with mediator. The system based on glucose oxidase and Ru(NH3)6 2+ as mediator was found useful due to the most negative potential of the process. The catalyses of oxygen reduction by two laccases: Cerrena unicolor and Trametes hirsuta embedded in the cubic phase together with 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS) as the mediator were found efficient and the reduction potential was positive enough to be considered in the application of lyotropic liquid crystals as a material for biofuel cells.
Keywords: Lipidic cubic phase; Lyotropic liquid crystals; Glucose oxidase; Pyranose oxidase; Laccase;

Immobilization of laccase on gold, silver and indium tin oxide by zirconium–phosphonate–carboxylate (ZPC) coordination chemistry by M. Mazur; P. Krysiński; A. Michota-Kamińska; J. Bukowska; J. Rogalski; G.J. Blanchard (15-22).
In this paper we present a simple method allowing for stable laccase immobilization on various conducting surfaces that retains the activity of the enzyme. The strategy for laccase immobilization presented in this paper relies on Zr4+ ion coordination chemistry that involves –COO terminal groups present on the protein. Using a host of techniques, including surface plasmon resonance (SPR), quartz crystal microbalance (QCM) gravimetry, atomic force microscopy (AFM), surface enhanced Raman scattering (SERS), resonance Raman scattering (RR) and electrochemical techniques, we show that laccase bound to a surface coordinatively through zirconium phosphonate/carboxylate (ZPC) functionalities forms a stable enzymatic layer with the enzyme retaining its activity to a significant extent.
Keywords: Zirconium phosphonate/carboxylate; Laccase immobilization;

Hybrid bioelectrocatalyst for hydrogen peroxide reduction: Immobilization of enzyme within organic–inorganic film of structured Prussian Blue and PEDOT by Andrzej Ernst; Oktawian Makowski; Barbara Kowalewska; Krzysztof Miecznikowski; Pawel J. Kulesza (23-28).
Fabrication of structured film (on glassy carbon substrate) composed of compact Prussian Blue (that has been prepared by alternate immersions and through assembling within ultra-thin layers of 4(pyrrole-1-yl)-benzoic acid, PPyBA) and poly(3,4-ethylendioxythiophene), PEDOT, is described. This functionalized film has been characterized by fast charge propagation, and it has served as a redox conducting template for permanent attachment of a model enzyme, horseradish peroxidase, HRP. The resulting organic–inorganic system acts as an effective hybrid bioelectrocatalyst for electroreduction of hydrogen peroxide, a model reactant for biosensors and biofuel cells. Among important issues are rigidity, permanence of enzyme attachment, morphology, hydrophilicity, and attractive mediating capabilities of the PEDOT-stabilized Prussian Blue based structured film.
Keywords: Composite film; Structured Prussian Blue film; 4(pyrrole-1-yl)-benzoic acid; Poly(3,4-ethylendioxythiophene); Horseradish peroxidase; Bioelectrocatalysis;

Series of liquid photopolymerizable compositions (LPhPC) based on oligouretanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemetacrylate (OCM-2), monomethacrylic ether of ethylene glycol and vinylpyrrolidone (VP) were tested. It was shown that the LPhPC, which contained VP (as basic hydrophylic matrix), OCM-2 (cross-linking agent) and OUM-2000T (to increase adsorption of polymer) was the most optimal. The blend contained 3 g/100 ml of enzyme. ISFET based biosensors for analysis of glucose and urea had the following characteristics: linear response in the range of concentrations 0.1–10 mmol/l, 0.05–20 mmol/l, angle of slope of concentration curve — 30 mV/pC, 38 mV/pC, and response time of approximately 10–15, 5–10 min, correspondingly. The value of K m for immobilized urease and β-glucose oxidase (GOD) achieved 0.85 and 3.1 mmol/l, respectively. It was established that under immobilization conditions at 20 °C the residual activity of GOD was about 35% from the initial level, the residual activity of horseradish peroxidase (HRP) and urease was 42% and 20%, respectively. In case of an immobilization of GOD at − 50 °C its residual activity reached almost 50% from the initial level. It was investigated how different sources of UV radiation and different substances (including specific and non-specific substrates) influenced stability of the enzymes in the LPhPC and in the prepared membrane at storage. Dynamics of changes of enzyme activity at the process of photo immobilization was characterized, and requirements for enzyme maximal storage were selected. The proposed LPhPC may be prepared in advance since enzymes do not lose their activity during 2 months. Therefore, two processes, i.e. manufacturing of a transducer and preparation of a biological membrane on its surface, can be combined in one. In order to achieve this, approaches of modern electronics, such as for example photolithography, can be used. The developed LPhPC is homogenous, non-active to biological substances, permeable for the analyzed sample, can be prepared using a simple immobilization procedure, and has a defined hydrophobic–hydrophilic balance and sufficient level of adhesion to transducer surfaces. These all cover the requirements to modern biosensors.
Keywords: Liquid photopolymerizable composition; Enzymes immobilization; ISFETs; Glucose; Urea;

Interaction of tin(II) and arsenic(III) with DNA at the nanostructure film modified electrodes by Adriana Ferancová; Miriam Adamovski; Peter Gründler; Jiří Zima; Jiří Barek; Jürgen Mattusch; Rainer Wennrich; Ján Labuda (33-37).
Biosensors based on DNA and DNA-carbon nanotubes film immobilized at the surface of a screen-printed carbon electrode were used for simple in vitro tests of chemical toxicity. The damage to DNA caused by tin(II) and arsenic(III) compounds as components of specific reaction media was evaluated by means of an electrochemical DNA marker, [Co(phen)3]3+, as the portion of original dsDNA which survives an incubation of the biosensor in the cleavage medium. The results were confirmed by the electrically heated electrode and by the measurement of the DNA guanine moiety signal.
Keywords: Tin; Arsenic; DNA damage; DNA biosensor; Voltammetry;

Electrical wiring of Pseudomonas putida and Pseudomonas fluorescens with osmium redox polymers by Suna Timur; Behzad Haghighi; Jan Tkac; Nurdan Pazarlıoğlu; Azmi Telefoncu; Lo Gorton (38-45).
Two different flexible osmium redox polymers; poly(1-vinylimidazole)12-[Os-(4,4′-dimethyl-2,2′-di'pyridyl)2Cl2]2+/+ (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N′-methylated-2,2′-biimidazole)3]2+/3+ (osmium redox polymer II) were investigated for their ability to efficiently “wire” Pseudomonas putida ATCC 126633 and Pseudomonas fluorescens (P. putida DSM 6521), which are well-known phenol degrading organisms, when entrapped onto cysteamine modified gold electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os2+/3+-functionalities are located. The bacterial cells were adapted to grow in the presence of phenol as the sole source of organic carbon. The performance of the redox polymers as mediators was investigated for making microbial sensors. The analytical characteristics of the microbial sensors were evaluated for determination of catechol, phenol and glucose as substrates in both batch analysis and flow analysis mode.
Keywords: Osmium redox polymers; Microbial biosensor;

Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure by Jan Hrbáč; Čeněk Gregor; Markéta Machová; Jana Králová; Tomáš Bystroň; Milan Číž; Antonín Lojek (46-53).
Electropolymerization regime of meso-tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin is optimized to yield films possessing both electrocatalytical and permselective properties towards nitric oxide oxidation. The sensor composed of electrochemically oxidized carbon fiber, covered solely with nickel porphyrin derivative layer electropolymerized using our method, is characterized by high selectivity towards nitrite (1:600), ascorbate (1:8000) and dopamine (> 1:80), determined by constant potential amperometry at 830 mV (vs. Ag/AgCl). Selectivity for ascorbate and dopamine as well as detection limit for NO (1.5 nM at S/N = 3) is 5-10 times better than parameters usually reported for Nafion® coated porphyrinic sensors. Nafion coating can further enhance selectivity properties as well as aids to the stability of the sensors’ responses.
Keywords: Nitric oxide sensor; Nickel porphyrin; Electropolymerization; Carbon fiber; Pretreatment;

The interaction of glassy carbon-supported thin wetting films of lecithin with some divalent cations is investigated by impedimetry and voltammetry. The influence of Ca2+, Mg2+, and Mn2+ on the film structure is explored in two different cases — the divalent cations are added to the electrolyte either before or after the formation of the film. When the film has been previously formed, the addition of divalent cations in millimolar concentrations leads to changes in the passive electrical parameters and the blocking properties of the films. On the one hand the dielectric properties of the film measured in 0.1 M KCl seem to improve after the interaction with divalent cations — the film capacitance decreases, the resistance and resistivity of the film increase. On the other hand the increase of the redox current in the presence of 1 mM Fe(CN)6 3−/4− in the electrolyte suggests the formation of some defects in the lipid structure of the film after the action of divalent cations. It is shown that the amount of these defects could be significantly decreased when the divalent cations are present in the electrolyte solution before the film formation. The effect of divalent cations on the film stability is tested by applying negative potential to the film. In 0.1 M KCl the films are not stable at potential of − 0.8 V (vs. Ag/AgCl) and are destroyed. The addition of divalent cations stabilizes the films and at certain millimolar concentrations the films remain intact after the action of the negative potential. The effect of Mn2+ is more pronounced, the Ca2+ and Mg2+ have smaller commensurate effect. It is proposed that the changes in the films’ properties could be related with more tight packing of the lipid molecules with the divalent cations inserted in the film and that some defects could be opened during the rearrangement of the lipids when the film has been previously formed.
Keywords: Solid supported thin lipid films; Divalent cations; Voltammetry; Impedimetry; Film stability;

Heavy metal sorption in the lichen cationactive layer by Kłos Andrzej; Rajfur Małgorzata; Wacławek Maria; Wacławek Witold (60-65).
Results of copper ion sorption in lichens owing to the ion exchange between the surroundings (aqueous solution) and the lichen cationactive layer have been presented. It indicates that the course of sorption of these ions, similarly as in the case of cations of other heavy metals, depends on the concentration and type of cations naturally found in lichen surroundings: H+, Na+, K+, Mg2+ and Ca2+. A determination method of heavy metal concentration in lichen surroundings has been proposed. It consists in exposure of transplanted lichens in the presence of salts that provide precisely determined, artificial salinity of precipitation with which the lichens are in contact. The studies were conducted on Hypogymnia physodes lichens.
Keywords: Lichens; Lichenoindication; Heavy metals;

Conducting polymers in modelling transient potential of biological membranes by Beata Paczosa-Bator; Teresa Blaz; Jan Migdalski; Andrzej Lewenstam (66-74).
The possibility of using conducting polymer (CP) films doped with biological ligands as artificial biological membranes to study potential formation mechanisms is presented. Calcium and magnesium ion-binding anionic sites — asparagine, glutamine, adenosinotriphosphate and heparin are incorporated into the poly(pyrrole) film during electrochemical polymerization. This approach allows the competitive calcium–magnesium ion-exchange to be inspected by open circuit measurements. After a close-to-Nernstian sensitivity of the CP membranes was induced by soaking in alkaline solutions of calcium or magnesium, dynamic experiments were performed by a change in the bulk concentration of magnesium or calcium ions. A characteristic transitory potential response, though distinctively different for the calcium and magnesium ions, was observed and explained using the diffusion layer model (DML).
Keywords: Poly(pyrrole); Ion-exchange kinetics; Ca–Mg competition; Potential transients;

A simple procedure of preparing low cost, planar and disposable reference electrodes for potentiometric applications is presented. This method is essentially the same as used for obtaining all-plastic ion-selective electrodes and thus promising for simple fabrication of complete cells. Commercially available aqueous dispersion of poly(3,4-ethylenedioxythiophene) doped by poly(4-styrenesulfonate) ions (PEDOT-PSS, Baytron P) is simply cast on a non-conducting plastic support (transparent foil for laser printers). This layer is covered by a non-selective poly(vinyl chloride) based membrane containing solid AgCl and KCl, added to obtain a stable potential. The conducting polymer layer plays a double role, of electrical contact and ion-to-electron transducer, enhancing the potential stability.The reference electrodes obtained exhibit independence of the kind and concentration of electrolyte applied as well as very low sensitivity to interferences: redox reactants and H+ ions; they are also characterized by both potential stability and low polarisability, sufficient for potentiometric applications.Cells of plastic electrodes (indicator and reference ones) are tested using an arrangement with Pb2+ or Ca2+ selective sensors. Potentiometric characteristic of such cells is satisfactory, well comparable with that using a classical electrode arrangement.
Keywords: Reference electrode; Poly(3,4-ethylenedioxythiophene); All-plastic ion-selective electrode; Lead ion sensor; Calcium ion sensor;

Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors by Bertrand Ngounou; Elchin H. Aliyev; Dmitrii A. Guschin; Yusif M. Sultanov; Ayaz A. Efendiev; Wolfgang Schuhmann (81-90).
The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis–Menten constant (K M app) representing the linear measuring range and the maximum current (I max app). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.
Keywords: Electrodeposition paints; Polymer library; Functionalization; Parallel synthesis; Immobilization; Biosensor optimization;

Enantioselective screen-printed amperometric biosensor for the determination of d-amino acids by Marzena Wcisło; Dario Compagnone; Marek Trojanowicz (91-98).
d-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of d-amino acids has been constructed by the immobilization of d-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for d-alanine in the concentration range from 5 to 200 μM. Excellent enantioselectivity towards d-amino acids was discovered. During the experiment, d-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.
Keywords: Chiral sensors; Enzymatic; Amperometry; Screen-printed biosensor; d-amino acids; d-alanine;

pH-tunable equilibria in azocrown ethers with histidine moieties by Elżbieta Jabłonowska; Barbara Pałys; Ewa Wagner-Wysiecka; Marzena Jamrógiewicz; Jan F. Biernat; Renata Bilewicz (99-106).
The crown ethers with electro- and photoactive azo moieties containing substituents with mobile protons such as in the –COOH groups of histidine, show unique effect of pH switched on/off presence of the azo form. The differences observed for the electrochemical behavior of azocrown ethers with N-acetylhistidine and imidazole moieties reveal the interference of a chemical reduction pathway in strongly acidified solutions. This chemical reduction process leads to the formation of a hydrazine derivative which can be detected by its further electroreduction on the electrode surface. The involvement of chemical reduction is seen clearly in the presence of mobile protons of the –COOH group and mercury as the electrode substrate. The behaviour of the N-acetylhistidine azomacrocyle is similar to that of compounds known to exist in quinone–hydrazone tautomeric equilibria.
Keywords: Histidine derivative; Azocrown ether; Azocompound; Tautomeric equilibria; Voltammetry; Macrocycle;