Atomic force microscopy lithography used to position neurons on gold

Biologists want to study cells in their natural environment if at all possible. When studying neurons this is even more important but can be quite a challenge. Since in vivo neural networks are too complex to easily observe biological processes, scientists often turn to in vitro methods to replicate and simplify networks of neurons and their environments.

However, cells as specialized as neurons require a special touch when working in vitro. Concerns include keeping the biomolecules required for neuron growth stable, producing neurons that are precisely arranged so that they can interact with each other, and keeping external conditions strictly regulated so that neuron behavior isn’t affected.

Researchers at the University of Wisconsin-Madison turned to atomic force microscopy (AFM) lithography to see if it can aid in creating neuronal assemblies. They used the technique to pattern poly-D-lysine (PDL) and laminin onto gold surfaces. These proteins can be used to control neuronal growth by confining the neurons and controlling their interconnectivity. The work was published in Biomaterials.

Specifically, they used an AFM technique called nanoshaving in which a spot on a PEG/Au substrate is selected by imaging the surface topography at low force, then the AFM tip (under relatively high force loads) shaves away a micron-sized area of PEG while the substrate is in contact with a pure solvent. The removed PEG molecules dilute into the solution. Then the solvent is replaced with a buffer solution containing either PDL or laminin protein, which bind to only the regions where the gold substrate is exposed.

The researchers found they could create protein features of ~50 nm and that isolated cortical neurons grown on protein squares ≥100 µm apart and surrounded by PEG did not form any neurites unless there were protein lines patterned between the growth squares.

Research paper: Positioning and guidance of neurons on gold surfaces by directed assembly of proteins using Atomic Force Microscopy