Enzyme etch
In their experiments, the engineers used an enzyme called DNase I as an "ink" in a process called dip-pen nanolithography – a technique for etching or writing at the nano-scale. The dip pen allowed them to inscribe precise stripes of DNase I ink on a gold plate, which they had previously coated with a thick forest of short DNA strands. The stripes of the enzyme were 100nm wide.
Once the researchers had created the stripes, they then activated the enzyme with a magnesium-containing solution. This changed the DNase I into a form that efficiently breaks down any DNA in its path. The stripes of activated enzyme carved out 400nm wide "troughs" in the DNA coating.
"We were surprised that the enzyme ‘ink’ worked so well, because it was simply deposited on the surface and could have washed away during the processing steps," says biomedical engineer Ashutosh Chilkoti of Duke’s Pratt School of Engineering, who leads the project. Chilkoti credits much of the experiment’s success to the laboratory skills of Jinho Hyun, who was a post-doctoral fellow in the group and is now an assistant professor at Seoul National University.
"We wanted to see if we could steal functionality from biology to make the complex structures we need," says Chilkoti. The outcome, he says, was everything he and his colleagues could have hoped for: "In an afternoon, we inexpensively created a nanostructure that would have taken weeks to develop using expensive, traditional methods of etching circuits into chips."
Now that the team has demonstrated that enzymes can "subtract" from the substrate to make precise troughs, the researchers envision many other possibilities. Instead of using enzymes that degrade DNA, for example, they could use other enzymes that link DNA strands together. That would allow them to make "additions" to the substrate, causing the DNA layer to grow thicker in certain places. Alternatively, they could use still other enzymes that make chemical changes in the DNA substrate itself, allowing them to build complex structures with what Chilkoti calls "different colored bricks".
The team also believes it could do away with the DNA entirely and use a different substrate. Chilkoti comments: "We used DNA because it is pretty robust, because you can buy synthetic DNA strands off the shelf, and because there are lots of enzymes that work on it. But there is nothing unique about it for this kind of application."
Team member Stephen Craig adds: "By harnessing the diverse power available in nature, it may be possible to selectively erase structures at one point, add structures at a second location, transform them from one state to another at a third location, and so on. The potential exists to create very small and very complex architectures."
"A lot more work is needed to optimise the process, but we feel this enzyme-inking technique has tremendous promise for wide applicability," says Chilkoti.
To date, dip-pen nanolithography has been primarily a bench top laboratory technique. Scaling up the technique to make it viable for manufacturing will require new instrumental technology such as dip-pen lithography machines with multiple, articulated tips that can move independently to deposit several different types of enzymes. Chilkoti envisions machines that can work on a sheet of chips using different enzymes, so that the chips can be snapped apart after the enzyme inking and processing. Such machines are already being commercially developed, so the day might not be too far off when enzyme-based nanomanufacturing might be possible on an industrial scale.
Enzymes are catalyst proteins used in many biological processes. These organic chemicals are also used in a wide range of industrial processes, from wastewater treatment and cheese making to dissolving blood clots after heart attacks. Many enzymes are commercially available and are well characterised.
The research was funded by the US National Science Foundation through a Nanotechnology Interdisciplinary Research Initiative (NIRT) grant.
