Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8-35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting 'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.

Effects of buffer composition and dilution on nanowire field-effect biosensors.

Nanowire-based field-effect transistors (FETs) can be used as ultra-sensitive and label-free biosensors for detecting protein-protein interactions. A way to increase the performance of such sensors is to dilute the sensing buffer drastically. However, we show here that this can have an important effect on the function of the proteins. Moreover, it is demonstrated that this dilution significantly affects the pH stability of the sensing buffer, which consequently impacts the charge of the protein and thus the response and signal-to-noise ratio in the sensing experiments. Three model systems are investigated experimentally to illustrate the impact on ligand-protein and protein-protein interactions. Simulations are performed to illustrate the effect on the performance of the sensors. Combining various parameters, the current study provides a means for evaluating and selecting the most appropriate buffer composition for bioFET measurements.

A step closer to membrane protein multiplexed nanoarrays using biotin-doped polypyrrole.

Whether for fundamental biological research or for diagnostic and drug discovery applications, protein micro- and nanoarrays are attractive technologies because of their low sample consumption, high-throughput, and multiplexing capabilities. However, the arraying platforms developed so far are still not able to handle membrane proteins, and specific methods to selectively immobilize these hydrophobic and fragile molecules are needed to understand their function and structural complexity. Here we integrate two technologies, electropolymerization and amphipols, to demonstrate the electrically addressable functionalization of micro- and nanosurfaces with membrane proteins. Gold surfaces are selectively modified by electrogeneration of a polymeric film in the presence of biotin, where avidin conjugates can then be selectively immobilized. The method is successfully applied to the preparation of protein-multiplexed arrays by sequential electropolymerization and biomolecular functionalization steps. The surface density of the proteins bound to the electrodes can be easily tuned by adjusting the amount of biotin deposited during electropolymerization. Amphipols are specially designed amphipathic polymers that provide a straightforward method to stabilize and add functionalities to membrane proteins. Exploiting the strong affinity of biotin for streptavidin, we anchor distinct membrane proteins onto different electrodes via a biotin-tagged amphipol. Antibody-recognition events demonstrate that the proteins are stably immobilized and that the electrodeposition of polypyrrole films bearing biotin units is compatible with the protein-binding activity. Since polypyrrole films show good conductivity properties, the platform described here is particularly well suited to prepare electronically transduced bionanosensors.