Bioelectrochemical application of some PQQ-dependent enzymes.

This paper focuses on the use of PQQ-dependent enzymes (PQQ enzymes) in amperometrical biosensors and gives emphasis on their innovative designs and applications. The study covers some aspects in the evolution of biosensors based on PQQ enzymes. Main attention is focused on the electrochemical properties of PQQ enzymes as very promising materials for the formation of electrochemical biosensors. Immobilization approaches and redox mediators recently used in PQQ enzymes based biosensors are reviewed. The acceptance of polypyrrole as a very promising immobilization matrix for some PQQ enzymes is discussed.     

Horse heart cytochrome c entrapped into the hydrated liquid-crystalline phases of phytantriol: X-ray diffraction and Raman spectroscopic characterization

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413 nm excitation, and non-resonance Raman technique with 785 nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q(230) and Q(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q(230) phase at 1 wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568 cm(-1) (γ(21)) and ν(48) mode at 633 cm(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5 wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature.     

Cyclometalated N,N-dimethylbenzylamine ruthenium(II) complexes [Ru(C6HRRR-o-CH2NMe2)(bpy)(RCN)2]PF6 for bioapplications: synthesis, characterization, crystal structures, redox properties, and reactivity toward PQQ-dependent glucose dehydrogenase

Cyclometalated derivatives of ring-substituted N,N-dimethylbenzylamines with controlled redox potentials as potent mediators of bioelectrochemical electron transport are reported. The cycloruthenation of R¹R²R³C₆H₂CH₂NMe₂ (R¹, R², R³ = H, Me, ^tBuO, MeO, NMe₂, F, CF₃, CN, NO₂) by [(η⁶-C₆H₆)RuCl(μ-Cl)]₂ in the presence of NaOH/KPF₆ in acetonitrile or pivalonitrile affords cyclometalated complexes [(η⁶-C₆H₆)Ru(C₆HR¹R²R³-o-CH₂NMe₂)(RCN)]PF₆ [R = Me (1) and R = CMe₃ (2)] in good yields. Reactions of complexes 1 and 2 with 2,2′-bipyridine (bpy) in acetonitrile or pivalonitrile result in dissociation of η⁶-bound benzene and the formation of [Ru(C₆HR¹R²R³-o-CH₂NMe₂)(bpy)(RCN)₂]PF₆ [R = Me (3) and R = CMe₃ (4)]. All new compounds have been fully characterized by mass spectrometry, ¹H/¹³C NMR, and IR spectroscopy. An X-ray crystal structural investigation of complex 1 (R¹/R²/R³ = H/H/H) and two complexes of type 3 (R¹/R²/R³ = MeO/H/H, MeO/MeO/H) has been performed. Acetonitrile ligands of 3 are mutually cis and the σ-bound carbon is trans to one of the bpy nitrogens. Measured by the cyclic voltammetry in MeOH as solvent, the redox potentials of complexes 3 for the Ru^II/III feature cover the range 320-720 mV (versus Ag/AgCl) and correlate linearly with the Hammett constants. Complexes 3 mediate efficiently the electron transport between the active site of PQQ-dependent glucose dehydrogenase (PQQ = pyrroloquinoline quinone) and a glassy carbon electrode. Determined by cyclic voltammetry the second order rate constant for the oxidation of the reduced (by d-glucose) enzyme active site by Ru^III derivative of 3 (R¹/R²/R³ = H) (generated electrochemically) is as high as 4.8 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.     

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H₂O₂ takes place at more negative potentials (0.1-0.4 V versus Ag/AgCl). Moreover, H₂O₂ oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.     

Probing Reactivity of PQQ-Dependent Carbohydrate Dehydrogenases Using Artificial Electron Acceptor

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k _ox = 660 M⁻¹ s⁻¹, while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.     

Fabrication and phase variation in annealed Cu3Se2 nanowire arrays

We have found for the first time that Cu₃Se₂ nanowired products encased inside the alumina pores by electrodeposition demonstrate the promising nonlinear optical properties in UV-vis-NIR spectra region. Furthermore, the annealing of these products results in the formation of Cu_2−xSe with x depending on the annealing temperature. Optical nonlinearities of as-grown and thermally treated copper selenide nanowire arrays were evidenced using the standard open-aperture Z-scan and laser pump-probe techniques.     

AFM study of complement system assembly initiated by antigen-antibody complex

The shape and size of complement system C1 components assembled on a SiO₂ surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO₂ substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO₂ substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO₂. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.