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Posted: Mar 19, 2013

Novel electron beam lithography technique works with plant viruses

(Nanowerk Spotlight) Plant viruses have recently become the focus of intense research in the field of nanotechnology due to their promising applications as biotemplates for bottom-up nanofabrication. The Tobacco Mosaic Virus (TMV) plays a special role: It was the first virus that was isolated and characterized, its structure is very simple, and it is chemically and physically unusually resilient. TMV is a classical model of 1D self-assembled protein particles. The particle length is 300 nm and its coat proteins assemble to well-defined 18 nm thick tubes with 4 nm wide channels. TMV and other plant viruses have shown the capability to be filled and/or coated with metallic and magnetic materials to form wires or clusters (see for instance: "Nanotechnology uses plant viruses for materials development").
"Perhaps the biggest advantage of plant viruses in nanotechnology is that we know exactly which chemical groups are located in which position," says José María Alonso, a former postdoctoral researcher at the Self-Assembly Group, led by Alexander Bittner, at CIC nanoGune in Donostia-San Sebastián, Spain (Alonso is now a researcher at the Laboratory of Organic Chemistry of the Wageningen University). "We use the channel and outer surface for modifications with the aim of making new nanoscale devices such as conductive wires, magnetic tubes, and small containers for liquids."
In order to build nanoscale devices based on plant viruses it is necessary to incorporate the virus with standard micro- and nanofabrication techniques – something that still remains a considerable challenge.
In new work, reported in the February 22, 2013 online issue of Nanotechnology ("Integration of plant viruses in electron beam lithography nanostructures"), researchers at the CIC nanoGUNE (Donostia-San Sebastián, Spain) and CEMES-CNRS (Toulouse, France) have now shown that TMV particles are compatible with electron beam lithography (EBL) processes and can be integrated in nanostructures made of positive and also of negative EBL tone resists.
SEM micrograph of a TMV particle underneath a PMMA barrier
SEM micrograph of a 900 nm long line of three TMV particles (white) underneath a PMMA barrier (blue) on a silicon wafer (red). (Image: Dr. Alonso)
"Two major challenges had to be taken into account when integrating TMV with EBL processes," Alonso explains to Nanowerk. "First, although TMV tolerates many more solvents than other viruses, conventional EBL developers cause denaturation. This led to the investigation of alternative methods to remove the polymer masking layer based on less reactive solutions. The second challenge is the thermal sensitivity of the TMV. As a biological supramolecular assembly, TMV loses its structural integrity, with an apparent collapse to irregular particles, above 90°C. Again, this makes TMV much more tolerant than most other viruses, but is still not sufficient for conventional processing in EBL."
The novelty in this new fabrication technique is that the researchers used a lithography scheme that relies on extremely low processing temperatures of 50°C – the temperature of the polymer resist used to cover TMV – and on development of tone resists with organic solvents that were chosen for their biocompatibility. As a result, viral particles maintain their biochemical functionality after all fabrication steps, which was verified through selective immunocoating of the TMV.
As a proof of concept to demonstrate the post-lithography biochemical functionality of TMV, the scientists performed selective immunocoating of the viral particles with primary and with secondary gold-labelled antibodies, and used immobilized TMV as a direct immunosensor.
Alsonso explains that this concept can be broadened by coating TMV with other peptides or proteins, and especially with various epitopes – in fact plant viruses are excellent vehicles for vaccine production, when they are coated with relevant epitopes.
"We believe that our fabrication methods should work also for other types of sensitive materials that are incompatible with standard EBL processing, e.g. DNA, RNA, protein fibers/tubes, or soft polymers," he says. "Moreover, taking into account the dimensions of TMV, our structures are ideal templates to study nanofluidic events. Such ‘virus nanofluidics’ is in fact very much basic science, operating close to the ultimate (molecular) scale, i.e below 5 nm.” "
This work is an example of the first steps that nanotechnology researchers are taking to integrate nanobiostructures into typical solid-state device nanofabrication techniques.
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