The biocompatibility of biofuel cells operating inside the body.

In 1968 Wolfson et al. published the concept for producing energy inside the body using catalytic electrodes exposed to the body fluid as an electrolyte and utilising naturally occurring fuels such as glucose. Since then, the technology has advanced to enhance the levels of power using enzymes immobilised within three-dimensional bioelectrodes that are nanostructured. Current research in the field of enzymatic fuel cells is directed toward applying electrochemical and nanostructural expertise to increase the energy density, to increase the power density, to increase the operational stability, and to increase the voltage output. Nonetheless, biocompatibility remains the major challenge for increasing the life-time for implanted enzymatic biofuel cells. Here, we discuss the current issues for biocompatibility and suggest directions to enhance the design of biofuel cells so as to increase the life-time of implantation whilst maintaining sufficient performance to provide power for implanted medical devices.

Characterization of structure and activity of garlic peroxidase (POX(1B)).

Structural characterization and study of the activity of new POX(1B) protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure-function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX(1B) exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k(cat) and K(M) values were 411 and 400 mM(-1) s(-1) for 3,3',5,5'-tetramethylbenzidine and 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX(1B) was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX(1B) showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX(1B)) is suitable for the monitoring of different analytes and biocatalysis.

A new peroxidase from garlic (Allium sativum) bulb: its use in H2O2 biosensing.

Peroxidase POX(1) isoenzyme was purified from garlic (Allium sativum L.) bulb by ammonium sulfate precipitation, gel filtration and anion-exchange chromatography. Native-PAGE profile showed two isoforms, designated POX(1A) and POX(1B). POX(1B) seems to be more attractive for biosensor design since its K(m) (app) for H(2)O(2) is lower than that of POX(1A). In addition to its storage and operational stability, POX(1B) was found to be highly heat-stable, since almost 70% of its activity was conserved at 60 degrees C, whereas full activity was retained at 50 and 40 degrees C for 40 min. The optimal pH was approx. 5 and the optimal temperature was 30 degrees C. Next, gelatin was used as a matrix for enzyme immobilization on a gold electrode surface and electrochemical measurements were performed by using cyclic voltammetry. POX(1B)-based electrodes show great potential for application in H(2)O(2) monitoring of biological samples.

Chitosan improves stability of carbon nanotube biocathodes for glucose biofuel cells.

We demonstrate a novel combined chitosan-carbon-nanotube-enzyme biocathode with a greatly enhanced and stable long-term current density of -0.19 mA mL(-1). The fibrous microstructure of the electrode improves the performance of the biocathode by creating a protective microenvironment, preventing the loss of the electrocatalytic activity of the enzyme, and providing good oxygen diffusion.

Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria.

Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3) CFU mL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3) CFU mL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum.

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness.

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 µgm L(-1)-80 µgm L(-1) (6.5 µmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 µgm L(-1) to 80 µgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.

Direct monitoring of pollutants based on an electrochemical biosensor with novel peroxidase (POX1B).

A biosensor for the monitoring of phenolic compounds based on a new protein named POX(1B) purified from garlic which demonstrates similar biochemical properties to peroxidase is investigated. The enzyme was immobilized into chitosan microspheres with covalent link. The properties of the biosensor were analyzed with Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). FT-IR demonstrates the covalent attachment of POX(1B) into chitosan and SEM shows high dispersion of the POX(1B) into the chitosan microspheres. The redox potential of POX(1B) in chitosan is 147 mV vs. SCE, which is much higher than reported works using HRP, demonstrating excellent direct electrochemical behaviour of the POX(1B). The electrocatalytic activity of the obtained biosensor towards chlorophenols derivatives in a large range from 10 pM to 10 microM was demonstrated. The mediator free POX(1B)-based biosensor exhibited high sensitivity towards 2,6-dichlorophenol, 4-chlorophenol and pentachlorophenol. A detection limit of 1 pM in the case of 4-chlorophenol was demonstrated with kinetic constant K(m,app) of 0.42 microM with high rapidity of electrochemical response of the biosensor of 1 s.