The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.

For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of À 0.8 V [vs. Ag/AgCl (1 M KCl)] under ambient air conditions thanks to an additional enzymatic O 2 -scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16 nA/μM TMAO in a useful measuring range of 2-110 μM with a detection limit of LOD = 2.96 nM (S/N = 3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 � 2 s, the biosensor is stable over prolonged daily measurements (n = 20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.It can also catalyze other N-oxide compounds, like 4-methyl [a] B.

An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of −0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO.

The deposition of in-situ formed gold nanoparticles at the liquid/liquid (L/L) interface is studied by means of thin-organic-film-modified electrodes (TFE). The degree of ordering and aggregation of gold nanoparticles can be tuned by adding a lipophilic and hydrophilic thiol in the organic and aqueous phase, respectively. The ordered thiol-anchored gold nanoparticles exhibit pronounced catalytic effect toward electron-transfer reactions across the L/L interface.

A coupled electron-ion transfer reaction at thin organic-film-modified electrodes (TFE) is studied in the presence of glucose oxidase (GOx) under voltammetric conditions. TFE consists of a graphite electrode modified with a nitrobenzene solution of decamethylferrocene (DMFC) as a redox mediator and tetrabuthylammonium perchlorate as an organic-supporting electrolyte, in contact with aqueous buffer solutions containing percholarte ions and GOx. The redox turnover of DMFC coupled with perchlorate transfer across water|nitrobenzene interface composes the coupled electronion transfer reaction. Glucose oxidase strongly adsorbs at the liquid|liquid interface affecting the coupled electron-ion transfer reaction by reducing the surface area of the liquid interface, prompting coadsorption of the transferring ion and lowering down slightly the rate of the ion transfer reaction. Although the enzyme exists as a polyvalent anion over the pH interval from 5.6 to 7, it does not participate directly in the ionic current across the liquid interface and percholrate remains the main transferring ion. Raman spectroscopic data, together with the voltammetric data collected by three-phase droplet electrodes, indicate that the adsorption of the enzyme does not depend either on the redox mediator (DMFC) or the organicsupporting electrolyte, while being driven by intrinsic interactions of the enzyme with the organic solvent. The overall electrochemical mechanism is mathematically modeled by considering linear adsorption isotherm of the transferring ion, semi-infinite mass transfer regime, and phenomenological second-order kinetic model.