Biological nanopores control the specific transport and sequestration of diverse biomolecules between different biological compartments. Mimicry of such pores could open up highly selective and efficient molecular separation. While current techniques such as nanopore analytics focus on using small pores (&lt;10 nm) for controlling and sensing single molecules, the characterisation and control of the molecular separation and mass transport of biomacromolecules (e.g. proteins) using larger nanopores are less developed.<br/>Towards developing such nanopores for biomacromolecular transport, we demonstrate how the diffusion of fluorescent labelled proteins through nanopore channels may be tracked by exploiting nanoscale confined illumination in a nanoporous planar optical waveguide. We fabricate nanoporous anodic aluminium oxide (AAO) waveguides with pore diameters matching those of the nuclear pore complex found on the membrane of the cell nucleus (30-50 nm). The pores were functionalised with poly(ethylene glycol) (PEG) to decrease the nonspecific binding of proteins. The diffusion of proteins of different sizes and net charges as well as free dyes was characterised. As control, the pores were constricted (closed) by addition of a crosslinked organic matrix (poly(tannic acid)) in order to measure the diffusion of the molecules only on the surface of the AAO film.<br/>As expected, the surface diffusion on top of the closed pores, simulating the absence of nanopores, showed similar kinetics for all molecules despite their differences in size. On the other hand, the diffusion of the free dye through the open pores is faster than for the proteins. In addition, proteins with the same molecular weight but different net charges showed different kinetics because of electrostatic interactions that can restrict pore entry. Our technique brings a new concept of measuring molecular diffusion through nanopores which can be invaluable for the development of biomimetic macromolecular transport.

Characterizing molecular diffusion through nanopores using nanoporous anodic alumina waveguides

254th National Meeting (2017)

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