Electrochemical detection of biomolecules incorporated in a supported lipid bilayer

MARÍA ANTONIETA DAZA MILLONE; GUILLERMO A. BENÍTEZ; ROBERTO C. SALVAREZZA; MARÍA ELENA VELA; TÂNIA BEATRIZ CRECZYNSKI-PASA; ANDRÉ AVELINO PASA

Sevilla

Congreso; 59th Annual Meeting of the International Society of Electrochemistry; 2008

Transport of biomolecules across biological membranes is a main issue in bionanoscience. Usually, physiological effects are due to the action of a few numbers of molecules. Therefore, high sensitivity techniques are necessary to study this process. As a basic model for transport across the membrane, the incorporation of methylene blue (MB) in a dimyristoylphosphatydilcholine (DMPC) bilayer supported on a gold substrate was studied. Gold substrates were flame annealed to form Au(111) terraces. These substrates were modified with dithiothreitol (DTT), an alkanedithiol with two hydroxyl groups in C2 and C3. Bonded by the two S, DTT makes a hydrophilic surface but it keeps gold conductive properties. This modification was made by incubation of gold substrates in a DTT 50µM ethanolic solution during 30min at 60ºC. Binding via two S was confirmed by XPS measurements and DTT monolayer formation from electrodesorption peak charges. Phospholipidic bilayers were formed by vesicle fusion method, i.e. by deposition of DMPC liposomes. DTT modified gold substrates were incubated in a liposome suspension during 90min at 30ºC. Coverage of the surface and self-healing (closure of holes) properties were studied by AFM measurements. The permeability of the membranes was evaluated using two kinds of molecules as probes: methylene blue (MB), a cell staining agent that can cross membranes and flavin adenine dinucleotide (FAD), an enzyme cofactor, that crosses the membrane only through specific protein transporters. DMPC-DTT-Au substrates were incubated in MB and FAD 0.1mM aqueous solutions during 30min at 37ºC. After rinsing with MilliQ water they were used as working electrodes in a three electrode glass cell (Pt as counterelectrode and SCE as reference). The figure shows cyclic voltammograms performed in phosphate buffer 0.1M at pH = 7.0 where it is possible to see the redox couple for MB but not for FAD. In summary, supported bilayers on DTT-Au modified surface have good physical properties (surface coverage and self-healing) and barrier properties when incorporated with MB, turning out into a basic whereas still biomimetic model for studying biomolecule transport.