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Nanofabrication With Biomolecules
Nanofabrication With Biomolecules
with biomolecules
by Dominic C. Chow1,3, Matthew S. Johannes2,3, Woo-Kyung Lee2,3, Robert L. Clark2,3, Stefan Zauscher2,3, and Ashutosh Chilkoti1,3*
Bionanofabrication lies at the intersection of nanotechnology processes. Finally, we outline potential future directions, with
and biotechnology, and in many cases can trace its origins particular emphasis on fabrication processes and their
back to technologies that are at least a century old. Many control, integration, and characterization.
strategies employed in bionanofabrication, especially those
for nanolithography and nanopatterning of biomolecules Opportunities and challenges
(top-down nanofabrication), can trace their origins to The incorporation of ‘soft-wet’ biological components into
analogous strategies in conventional micro- or conventional nanofabrication platforms designed and built for
nanofabrication. For example, soft lithography, used to ‘hard-dry’ semiconductors, conductors, and dielectrics brings
deposit biomolecules over a large area in a parallel fashion10, both new opportunities and challenges since biomolecules
is an analog of photolithography used to create transistors on possess some unique properties:
a Si substrate, and arguably owes much to methods • A wide range of biomolecules, including nucleic acids,
developed in the printing industry. Similarly, dip-pen proteins, lipids, and oligosaccharides, react with other
nanolithography (DPN) directly places biomolecules on biological components by molecular recognition14,15,
surfaces at the nanoscale using micromachined cantilevers of which is important for bottom-up nanofabrication based
an atomic force microscope (AFM)11 and is the nanoscale on self-assembly;
analog of writing with quills. • Enzymes can catalyze the synthesis and removal of both
The early development of bionanofabrication has largely biological and synthetic molecules16,17;
focused on the creation of biologically active structures for • Biomolecular reactions (biotransformations) are highly
biosensing and diagnostics by fairly straightforward selective and site-specific17. There are many enzymes that
modifications and, in some cases, the extension of cleave DNA at particular sites (restriction enzymes) and
conventional nanofabrication techniques. More recently, link two pieces of DNA together (ligases)17. Proteases that
there is a growing interest in using biomolecules as active digest proteins at specific sites16,17 and enzymes that add
components in nanofabrication processes and the functional motifs to proteins18 are just a few examples of
development of new biomolecule-based materials systems. protein-modifying enzymes;
For example, enzymes can be deposited to synthesize and • Biomolecular reactions are often highly efficient under
excavate nanostructures of nucleic acids and proteins at the physiological conditions, so the yields of
nanoscale12,13. In addition, the use of biomolecules for biotransformations are substantially higher than those by
nanoscale assembly has become a promising and effective chemical syntheses19,20; and
solution to difficult positioning problems in the fabrication of • The use of biomolecules and biological processes is often
nanostructures (bottom-up nanofabrication). environmentally friendly, so treatment of the waste
The capability of bionanofabrication to manipulate products can be minimal and the by-products pose little
biomolecules with nanometer precision on surfaces in a health risk compared to the reagents used in
massively parallel fashion is potentially revolutionary semiconductor processing.
because it would enable the creation of novel devices with On the other hand, there are also significant constraints
performance metrics that go beyond those currently associated with the use of biomolecules in
envisioned. However, the direct application of bionanofabrication. These are:
nanofabrication techniques and processes to realize the • The ultrahigh-vacuum (UHV) conditions used in
potential of bionanofabrication remains a difficult, though, in conventional nanofabrication approaches are incompatible
our opinion, not insurmountable task. In this review, we with biomolecules, whose function and structural integrity
highlight the opportunities and challenges that researchers in most cases are destroyed in a high-vacuum
and scientists face in developing bionanofabrication environment;
processes. We provide specific examples of the uses of • Even in an aqueous environment, many proteins can easily
bionanofabrication in the areas of biosensing, nanomaterials, lose their native, active conformation after deposition
drug discovery, and synthetic biology. Next, we discuss because they can unfold and bind nonspecifically to a
current developments in bionanofabrication techniques and surface; and
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• Biological reactions, with some notable exceptions21,22, exploit the intrinsic functionality of biomolecules to create
must take place in an aqueous solution or buffer, which new nanostructures, processes, and nanomaterials systems.
limits their uses in many electronic applications. For example, bacterial S-layer proteins27,28 and DNA tiles
(Fig. 1)29 self-assemble into crystalline two-dimensional
Current applications and potential uses templates with highly controllable and predictable periodic
The unique challenges posed by the use of biomolecules in features for patterning metal nanostructures. The assemblies
nanofabrication are substantial, but surmountable. To have potential applications in DNA computing and
fabricate efficiently with biomolecules and make use of their nanoelectronics30-32.
unique properties, a radical shift away from the high-energy, Recently, the direct deposition of biomolecules has gained
high-vacuum processing paradigm of conventional micro- and considerable attention, because it allows the precise
nanofabrication is needed. Although the field of placement of biomolecules at the nanoscale. A variety of
bionanofabrication is still in its infancy, its impact and biomolecules such as collagen33, oligonucleotides34, biotin-
promise go far beyond biological applications where creative streptavidin nanostructures35, elastin-like polypeptides
uses of biomolecules remotely resemble those envisioned by (ELPs)36, and DNase I12 can be deposited to create
biologists who originally studied and isolated them. bionanostructures by DPN for biosensing and nanoscale
Genomics and proteomics surface modifications. In addition, the roles of novel
Early implementation of bionanofabrication targets genomics, biomolecules in miniaturized fluidic systems, and potentially
proteomics, and high-throughput biosensing and diagnostic nanofluidics, are exemplified by the recent use of thermally
applications. Bionanofabrication strategies are used to reduce or pH-responsive proteins as biological actuators in
the spot size of the deposited biomolecules, to increase the microfluidic systems37.
spot density of arrays, and to improve the throughput of Drug discovery and synthetic biology
deposition processes23-25. Through miniaturization of More recently, new and exciting opportunities for
diagnostic devices and equipment, the cost efficiency, speed, bionanofabrication have emerged in the fields of synthetic
and accuracy of micro- or nanoarray-based detection biology and recombinant protein engineering. The firm
technology can be improved substantially by reducing the integration of recombinant protein engineering techniques
amounts of reagents required, the reaction volumes, and such as phage display38, directed evolution39,40, and
detection errors26. combinatorial libraries41,42, as well as the discovery and
Nanostructures and nanomaterials isolation of novel proteins from extremophiles (microbes that
As bionanofabrication becomes increasingly mature and thrive in extreme environments)43,44, would further
sophisticated, more and more research efforts will turn to strengthen bionanofabrication approaches by providing
Fig. 1 DNA-based bionanofabrication by self-assembly of DNA molecules. (A) DNA strands, which have complementary sticky-end overhangs, self-assemble into a branched junction.
(B) These branched junctions can further self-assemble into DNA nanogrids owing to the orientation of the complementary sticky ends, as shown in the AFM images of DNA lattices.
(Reprinted with permission from102. © 2003 AAAS.)
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Fig. 2 Micropatterned DNA arrays used as templates for high-throughput gene synthesis
and protein expression. (A) Single-strand DNA immobilized on a microchip surface was
used as a template for generating double-strand DNA by polymerase chain reaction
(PCR). (B) Synthesis (left) and cleavage (right) of oligonucleotides from a microfluidic
microchip, visualized by fluorescent scanning microscopy. Details of microfluidic Fig. 3 Nanostructured biocompartment M13 bacteriophage for nanowire synthesis.
chambers and connecting channels are shown in the inset. (Reprinted with permission (A) Visualization of different processes in nanowire synthesis for the nucleation, ordering,
from45. © 2004 Nature Publishing Group.) and annealing of virus-particle assemblies. (B) Nucleated particles self-assemble
symmetrically on the virus for aggregation-based annealing. (C) The rigidity and packing
of the expressed peptides promote high-order self-assembly of M13 bacteriophage along
proteins with improved thermal stability, binding specificity, the preferred orientation in nucleated particles. (D) The bacteriophage contains
genetically modified capsid and ends that are encoded for in the enclosed phagemid DNA.
and solvent compatibility. (Reprinted with permission from48. © 2004 AAAS.)
This integrated approach, albeit at the microscale, is
exemplified by a recent report that micropatterned DNA polymerization of silica and Si at low-temperatures19,20. This
arrays could be used as templates for high-throughput gene discovery heralds the future development of Si deposition at
synthesis and protein expression (Fig. 2)45. In this specific both micro- and nanoscales with novel enzymes under
example, the use of patterned oligonucleotide sequences is ambient conditions, and blurs the distinction between
extended to drug discovery and lead validation, also providing biological and nonbiological components used in
a valuable tool for systems biology. This technology can bionanofabrication.
easily be applied and adapted to DNA nanoarrays,
significantly improving their throughput and processing Current developments in techniques
capability. and processes
Another emerging trend is the use of recombinant Taking soft lithography down to the nanoscale
nanostructured biocompartments such as vesicles, viruses, Soft lithography was first developed in the Whitesides
and phages, which allow predefined functional biological laboratory at Harvard in 199349. Initially, the process was
components to self-assemble into a sophisticated self- termed microcontact printing (µCP), in which
contained system. These biocompartments are ideal for polydimethylsiloxane (PDMS) stamps with micron features
bionanofabrication. They not only serve as nanoscale reaction were cast against negative masters generated by
chambers for mineralization and metallization46,47, but also photolithographic processes50. These stamps were coated in
provide a means to introduce docking sites to spatially direct alkanethiol inks and used to create self-assembled
the assembly of biological, metallic, and semiconducting monolayers (SAMs) on substrates through conformal contact
nanostructures (Fig. 3)48. in a massively parallel fashion. The technique has spawned
Finally, another major opportunity for bionanofabrication many other related techniques and variations10,51.
lies in harnessing the amazing biotransformation capabilities Most notably, in the context of this review, soft
of enzymes to potentially synthesize semiconducting lithography was first used to pattern proteins in a direct print
materials. For example, silicatein filaments and subunits have fashion in 199852; chicken immunoglobulins (IgGs) were
been isolated in a marine sponge and were shown to catalyze directly patterned onto glass and polystyrene surfaces at the
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nanoscale for protein recognition52, and on polymers using studies show how soft lithography can be used to fabricate
the biotin-streptavidin adapter system53,54. In addition to DNA arrays and point toward the simultaneous printing of
PDMS, other materials are being studied for their applicability multiple oligonucleotides.
to pattern biomolecules. By increasing the strength of the Scanning probe lithography for bionanofabrication
stamps used for printing, feature sizes of µCP can be reduced DPN is a versatile scanning probe-based technique for
to less than 100 nm (nanocontact printing, nCP) by avoiding fabricating both organic and biomolecular nanostructures. In
the complication of stamp collapse. For example, stamps general, DPN involves the coating of an atomic force
made out of polyolefin plastomers (POPs) exhibit better microscope (AFM) cantilever tip with the desired molecule to
mechanical stability than PDMS and are able to print be transferred. Once the tip is brought into contact with the
proteins, particularly fibrinogen, down to 100 nm feature target substrate, the molecules assemble on the surface
sizes55. The development of protein patterning was further because of their specific or nonspecific affinity. When the tip
extended to the printing of multiple proteins using is translated across the surface, the molecules are deposited
microchannels56 and single proteins (various antibodies) on a in the wake. Originally, this technique was used to directly
surface (line widths <100 nm) (Fig. 4)57. These studies deposit organic molecules on surfaces at the nanoscale,
highlight the potential impact and current limits of soft e.g. an alkanethiol onto Au61, an organosilane onto Si62, and
lithography for construction with proteins at the nanoscale. a positive onto a negative polyelectrolyte, or vice visa63. This
Soft lithography has also been used to directly pattern approach was made more generic in later work by
DNA. For example, Xiao and coworkers used µCP with precise nanopatterning a SAM of 16-mercaptohexadecanoic acid
alignment to fabricate and synthesize oligonucleotide arrays (MHA) on Au surfaces to serve as a template for
on surfaces in a step-wise, single-nucleotide fashion for DNA nanopatterning biomolecules. The advantage of this approach
hybridization studies58. Another study demonstrated the is that MHA can be reproducibly nanopatterned by DPN and
direct printing of DNA-surfactant molecules on the is easily functionalized with biomolecules by reaction with
microscale59. More recently, patterning of 20 bp amine groups, therefore the DPN process does not have to be
oligonucleotides and 500 bp and 1600 bp PCR fragments was optimized empirically for each combination of substrate and
accomplished with a feature size of about 800 nm60. These ink. For example, immunoglobulin G64, lysozyme64, biotin-
streptavidin35, ELP (Fig. 5)36, alkylamine-modified DNA65, and
cowpea mosaic virus66 could be nanopatterned with feature
sizes on the order of 100 nm by this approach.
Direct-write DPN, though more difficult, has also been
developed for the deposition of biomolecules onto surfaces.
For example, a hexanethiol-modified oligonucleotide ‘ink’ was
deposited on Au and oxidized Si substrates, with feature sizes
from 200 nm to microns34. Similarly, this strategy was
applied to DNA labeled with fluorescent molecules of
different colors for direct optical detection67. This direct-
write approach is also applicable to proteins, demonstrated
by the successful deposition of thiolated collagen and
collagen-like peptides onto Au substrates, generating 30 nm
wide line patterns for cell binding assays33. This approach has
been extended to catalyze biotransformations with nanoscale
spatial precision; in the first example of this approach,
Fig. 4 Nanocontact printing (nCP) of proteins. (A) Steps highlighting the nCP process. DNase I molecules were deposited locally by DPN to digest
Typically a polymeric stamp is cast against a master, coated with an inking solution, and
then brought into conformal contact with the target substrate, which transfers target an oligonucleotide SAM with nanoscale precision (Fig. 6)12.
molecules to the substrate. (B) AFM tapping mode image of rabbit antibodies patterned Conversely, this strategy should also be applicable to create
on a glass substrate using nCP. Patterned line widths are around 100 nm. (Reprinted with
permission from57. © 2003 American Chemistry Society.) DNA nanostructures enzymatically, for example, by terminal
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DNA
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36 December 2005
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