Selective biomimetic filtration and active transport of macromolecules. Chemical, nanofluidic and optical study – Golden Gates

Using a pluridisciplinary approach combining nanotechnology, nanochemistry and nanofluidics, we propose to make and study smart, selective, and active filters of proteins, nucleic acids and water-soluble polymers by mimicking the nuclear pore complex i.e. by grafting copolymers with blocks of varied hydrophilicity at the surface of polycarbonate or Silicon Nitride nanopores drilled in ultrathin membranes by Focused Ions Beam. The grafted chains form a semi-dilute polymer brush filling the pore. This random network acts as a steric barrier for large macromolecules passing through the pore unless suitable attractive interactions are established between the brush and the macromolecules. The pattern of hydrophilicity of the brush controls the compatibility with the macromolecules or nanoparticles to be selected and ensures the selectivity of the pores. We will synthetize different systems and compare their properties:
1) Native nucleoporins (FG (Phenylalanine-Glycine) Nups) as found in biological nuclear pores,
2) Synthetic double hydrophilic copolymers (derived from various hydrophilic and hydrophobic oxazoline monomers) grafted onto or from the pore surface by reactive ends,
3) Polymers and copolymers selectively grafted inside nanopores via diazonium anchors using SECM (scanning electrochemical microscopy). The initial characterization of the grafted layers will be conducted by classical techniques, TEM, SEM, XPS, SECM,AFM, and flow measurements.

The filtration, transport and gating properties of these hairy nanopores will be assessed at the single molecule-single pore level by optical, hydrodynamical, electrical and electrochemical techniques in comparison with recent theories. The optical detection is novel. It relies on the deposition of a thin gold layer on the pores and on the illumination by visible light in the zero-mode waveguide regime, where the wavelength of the light is larger than the pore diameter. An evanescent light wave is then localized at the entrance of the pores. Fluorescently labelled macromolecules entering or exiting a pore are excited by the evanescent wave and emit fluorescence light. They are thus visualized in real time. Our optical and electrical setup will be equipped with a very sensitive pressure control and we will be able to perform flow-driven filtration experiments at the single molecule level. We will study the transport kinetics (at the scale of the millisecond with the optical detection and the scale of the microsecond with the electrical detection) of macromolecules and colloids of increasing hydrophoby and their interactions with the pore surface and the grafted copolymer layer. One aim is to rationalize the best conditions for realizing the stealth and fast transport of a given class of macromolecules and colloidal particles. We will probe these stealth particles as transporters of other macromolecular cargoes. Concentration gradients of transporters will be made to generate directional transport. Active transport will also be generated using electrical and electrochemical driving forces.

Microscopic measurements will be correlated with macroscopic permeation and filtration measurements. Realistic estimates of the pores selectivity and of the flow rate show that microfabricated membranes with arrays of nanopores could be useful and cost effective for the separation and production of high added value macromolecules such as recombinant proteins, justifying our participation to the present call.