Electrochemical Behavior of Nitrate Reductase Immobilized in Self-Assembled Structures with Redox Polyviologen

Abstract

We report on a novel bioelectrode based on self-assembled multilayers of nitrate reductase (NR) intercalated with a cationic viologen-functionalized polyvinylpyridinium (PV) polymer specially synthesized for this purpose. Different samples of polymer proved to have varying substitution ratios, according to NMR experiments. The electrostatic self-assemblies were built up on a thiol-modified gold surface using a strategy previously described by Hodak et al. The electrochemical behavior of PV and the response of the electrodes toward nitrate were followed by cyclic voltammetry. The catalytic currents obtained were proportional to the number of NR-immobilized layers, as well as to the polymer substitution degree, proving that the enzyme remained active under immobilization conditions and that it can be regenerated by PV redox moieties. Experiments with quartz crystal microbalance electroacoustic impedance were carried out to follow quantitatively the multilayer film formation. The kinetics of the adsorption processes and the influence of the ionic strength are investigated. The evaluation of water and ion exchange during the reduction of PV is also achieved from electroacoustic and electrochemical experiments. Results indicate an important water exchange associated with the polymer redox behavior.

Charge Transport in Redox Polyelectrolyte Multilayer Films: The Dramatic Effects of Outmost Layer and Solution Ionic Strength

“…We found that k ET does not systematically depend on the nature of the film topmost layer or salt concentration and we note that the average k ET = (360 AE 130) s À1 is very close to the value k S = 250 s À1 for the same complex covalently tethered to a thiol layer with the same number of CH 2 groups as the MPS thiol. [55] On the other hand, for the complex Os(bpy) 3 2 + /3 + in solution, k 0 = (1.55 AE 0.03) 10 À2 cm s À1 , [64] which is much higher than the k 0 values in Table 1, but assuming D % 1-2 10 À6 cm 2 s À1 for the soluble species, one obtains k ET % 100-500 s…”

“…[1] The method, based on the alternate adsorption of positively and negatively charged macro-molecules, enables an unprecedented control over film thickness, composition, and electrochemical behavior. Following the first examples of LbL-modified electrodes in 1997, the toolbox of redox-active building blocks has grown to include redox proteins, [2][3][4][5] inorganic complexes, [6,7] nanocrystals, [8] and several types of redox polyelectrolytes. [2,[9][10][11][12][13][14] Electrochemically active polyelectrolyte multilayers [15] have received widespread interest due to their potential applications in sensing, [2,4,9,10] electrochromic devices, [7][8][9]11] and as platforms for fundamental studies of redox proteins.…”

Fabrication of Redox‐Mediator Supported Potentiometric Nitrate Biosensor with Nitrate Reductase

“…This reaction has been commonly used for amperometric detection of nitrate [6,15,16]. However, as also evident from Equation 1, there is a change in the H þ concentration as the nitrate is reduced to nitrite at the electrode surface.…”

“…So to overcome some of the drawbacks of using NADH as a cofactor for fabrication of potentiometric nitrate biosensor and to possibly improve sensitivity, selectivity and stability of the biosensor, the use of redox mediator may also be considered. Several redox mediators have been used previously for the fabrication of amperometric biosensors [6,15,16], but to our knowledge none has ever been used for fabrication of potentiometric nitrate biosensors. Another potential benefit in considering the replacement of NADH with a redox mediator is economic.…”

Triply Branched Viologen Stars: Synthesis and Polymerization by Peripheral Benzyl Coupling

“…Efforts have been made to fabricate modified electrodes based on viologen films [9] and by cation-anion interaction of polyviologens with a negatively charged electrode surface. [10] Electropolymerization is another possibility to fabricate such interfaces and, as electropolymerization proceeds only at electrode surfaces, it is a particularly suitable method for the modification of area-defined electrodes. Electrochemically induced electrode modification involves electrochemically assisted adsorption of a polyviologen [11] or cross-linking a polymer with viologen oligomers.…”