Cristina Gutierrez-Sanchez

Gold Nanoparticles as Electronic Bridges for Laccase-Based Biocathodes

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme molecules onto an electroactive surface modified with functionalized gold nanoparticles (AuNPs). Here, we present such strategy for obtaining a DET-based laccase (Lc) cathode for O(2) electroreduction at low overpotentials. The stable nanostructured enzymatic electrode is based on the step-by-step covalent attachment of AuNPs and Lc molecules to porous graphite electrodes using the diazonium salt reduction strategy. Oriented immobilization of the enzyme molecules on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O(2) reduction can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc molecules that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate constant k(0) ≫ 400 s(-1).

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.

Oxygen biosensor based on bilirubin oxidase immobilized on a nanostructured gold electrode

Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase.

FTIR spectroelectrochemical characterization of the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough.

For the first time a complete characterization by infrared spectroscopy of a Ni–Fe–Se hydrogenase in its different redox states is reported. The Ni–Fe–Se hydrogenase was isolated from Desulfovibrio vulgaris Hildenborough. Two different electron paramagnetic resonance silent and air-stable redox states that are not in equilibrium were detected. Upon reduction of these states the catalytically active states Ni-R and Ni–C appear immediately. These states are in redox equilibrium and their formal redox potential has been measured. Putative structural differences between the redox states of the active site of the Ni–Fe–Se hydrogenase are discussed.

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O2. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O2. The inhibition of the Ni–Fe–Se hydrogenase activity by O2 could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O2 on the bridging site between Ni and Fe.

Third-generation oxygen amperometric biosensor based on Trametes hirsuta laccase covalently bound to graphite electrode

The response of low-density graphite electrodes hosting Trametes hirsuta laccase in a direct electron transfer regime is presented for real-time analysis of O2 concentrations. The use of contrasting immobilisation methods developed for biocathodes affords good reproducibility and reliability of the amperometric biosensor, which shows a limit of detection below 1 µM and a sensitivity slightly higher than 60 nA cm−2 M−1.

ATP Synthesis and Biosensing Coupled to the Electroenzymatic Activity of a Hydrogenase on an Electrode/Biomimetic Membrane Interface

Cells generate energy by coupling a proton gradient across a phospholipid bilayer membrane with the activity of a cross-membrane ATP synthase enzyme. [...]