David Olea

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.

Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.

Dextran-lipase conjugates as tools for low molecular weight ligand immobilization in microarray development

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran-lipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500-40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran-BTL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09-136.56 ng L(-1)), lower limits of detection (0.007 ± 0.001 ng L(-1)), and lower IC50 values (4.4 ± 0.7 ng L(-1)). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.

Physicochemical characterization of Acidiphilium sp. biofilms.

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X-ray photoelectron spectroscopy (XPS), whereas surface-enhanced infrared absorption spectroscopy indicated the presence of redox-active proteins.