“Biological recognition is a property that promises to be very useful in nanotechnology,” Levi Gheber told nanotechweb.org. “At such small scales, one has to rely on ‘smart’ materials, which have some knowledge of where they should go, in order to assemble a larger structure. The inorganic chemistry world is pretty poor in such specific recognition interactions, while the biological world is full of them.”
It’s not just DNA that can carry out recognition interactions - antibodies/antigens, receptors and their targets, and enzymes and their substrates all do so as well. “In fact, the spectrum of these interactions is much richer than those of DNA,” said Gheber. “We decided to make use of proteases that recognize the specific cleavage sites on the appropriate proteins and employ them as a smart ‘etchant’.”
The scientists used the proteolytic enzyme trypsin. Proteolytic enzymes hydrolyze peptide bonds, cleaving proteins at specific sites along the amino acid sequence: trypsin cleaves on the carboxyl side of lysine and arginine residues. Gheber and colleagues treated a layer of bovine serum albumin (BSA), a 607 amino acid protein, with the enzyme. BSA contains 60 lysines and 26 arginines, so cleavage by trypsin is expected to cause a major collapse of the BSA’s three-dimensional structure.
To control patterning by the enzyme, Gheber and colleagues applied a solution of trypsin from a nanopipette attached to the head of a scannning probe microscope. Holding the nanopipette in contact with the protein layer produced round wells as the enzyme etched away the BSA. Longer contact times created deeper and wider wells. For example, a 200 nm pipette produced wells with widths of 0.6 microns, 1.2 microns and 2.3 microns and depths of 0.9, 1.9 and 2.4 microns when held in place for 20 s, 60 s and 120 s, respectively. To create channels, the team moved the sample while holding the pipette in contact with the surface. A 50 nm aperture pipette produced a channel 340 nm wide and about 660 nm deep when the sample moved at about 3 microns/s.
Control experiments with water-filled pipettes actually produced mounds and ridges rather than wells and trenches, as a result of swelling of the BSA film. This indicates that, under normal conditions, the rate of etching by the trypsin is faster than the rate of swelling due to the presence of water. In fact, the researchers found that using a solution of 10 parts soybean trypsin inhibitor to one part trypsin also produced mounds and ridges. So the team could use the concentration of inhibitor as a tool to modulate the effect of the trypsin.
“We believe that [the technique] will be useful in the areas of nano-biochips, nano-fluidics and lab-on-a-chip,” said Gheber. “The fact that the structures are being patterned in a biological surface means that they can readily have biological activity. This is basically different from all other approaches.” According to Gheber, scientists will typically choose materials that are convenient for patterning purposes and then attach biological molecules to the pattern once it is formed. “Using our approach, it is possible to skip the step of attachment to the substrate or at least to simplify it very much,” he said.
The researchers reported their work in Nano Letters.