The development of new methods for preparing three-dimensional structures of the nano- or the micrometer scale is of great interest in various technological fields such as biotechnology, microelectronics, microfluidics and biomaterials.
In microelectronics, research have focused on the development of new approaches for miniaturizing classical 2D integrated circuits into smaller 3D-structures in which interconnected silicon wafers (3D-IC (3D-integrated circuits)) or chips (3D-packaging) are stacked. The so-called “3D-technology” enables to provide greater functionality and higher component density while decreasing the form factor. One of the biggest challenges of 3D-technologies remains the creation of vertical electrical connections between stacked chips or stacked silicon wafers using nano- or microstructures such as nanowires. Nowadays, microelectronic devices are mainly available by conventional top-down processes such as photolithography. But, for the foreseeable future, such technologies are believed to remain inappropriate for the large-scale production of microelectronic devices.
Nanotechnology research thus put a great emphasis on the “Bottom-Up” strategies. These strategies are based on the self-assembly of molecular building blocks for creating three dimensional nano- or microstructures to be integrated in larger and functional microdevices. Appropriate molecular building blocks encompass inorganic compounds able to form crystals as well as biological molecules such as DNA, peptides and proteins. In that respect, proteins are very attractive building blocks because of their physical sizes, their highly specific interactions, their high degree of organization and their ability to be chemically functionalized or coated.
Several publications describe nanowires obtained from protein fibres.
Scheibel et al. (PNAS, 2003, 1000, 4527-4532) describe the use of self-assembling amyloid fibres from Saccharomyces cerevisiae prion determinant to construct nanowire elements. The said nanowire elements were subsequently metallized with silver or gold so as to produce conductive metal wires of 100 nm wide.
Patolsky et al. (Nature Materials, 2004, 4, 692-695) describe gold wires (1-4 μm long and 80-200 nm high) exhibiting high electrical conductivity which are obtained by polymerization of G-actin labelled with Au (gold) nanoparticles followed by the catalytic enlargement by metallization process. In the same way, Yao et al. (Analytical and Bioanalytical Chemistry, 395, 1563-1566) describe semiconductor nanowires obtained by conjugation of quantum dots to actin filaments.
However, few studies relate to methods for actually preparing three-dimensional protein structures more complex than nanowires.
Nakamura et al. (MEMS proceedings, 2007, Japan) describe that, when G-actin and fascin solution was confined and polymerized in Polydimethylsiloxane (PDMS) micro chambers, the shape of the actin bundles may follow the geometry of the chambers. It clearly appears that such a method is quite limited and only enables to obtain very few flat actin structures.
Brough et al. (Soft Mater, 2007, 3, 541-546) has proposed a method for preparing three-dimensional actin structures with nano-resolution patterns in the XY direction and on the micron pattern on Z direction while controlling the precise location of said structures on substrates such as silicon wafers. This method is based on the use of streptavidin nanopatterns for immobilizing biotinylated gelsolin-F-actin complex and subsequently promoting the polymerization of actin filaments. Gelsolin is indeed a capping agent which binds the barbed of actin filament. Consequently, the polymerization of actin filaments only occurred from pointed ends and is thus very slow. The resulting polymerized actin filaments are unbranched and display a length of at most 3-4 μm. Even if the actin filaments grew in out-of-plane direction from the nanopatterns, no well-orientated and well-defined three-dimensional actin structure could be actually observed.
Huang et al. (Langmuir 2006, 22, 8635-8638) describes a method similar to that of Brough et al. This method is based on the immobilization of pre-formed actin filaments on patterns. The immobilized actin filaments are then aligned along a desired direction using an electric field.
US 2005/0106629 reports a method for preparing a three-dimensional actin column based on the use of a first surface (nucleating plate) patterned with nucleating agent and a second surface (binding plate) patterned with capture agent. As explained in Example 3 of this document, the two patterned surfaces are brought into contact and aligned within a small fluid chamber under an inverted microscope. The fluid chamber is filled with a polymerization mixture to initiate column growth from nucleating plate. For enabling the growth of the actin column, the binding plate is moved away from the nucleating plate at a rate approximately equal to the rate of the column growth but not faster. In other words, the column grows as a bundle of parallel actin filaments, each actin filament being in contact by its first end to a nucleating agent on the nucleating plate and being connected by its second end to a capture agent on the binding plate. Even if said method virtually enables to control the location of the three-dimensional structures of actin, it clearly appears that such a method is very difficult to implement in mass production process since the distance of the two surfaces must be increased with a very precise rate, during the polymerization process, in order to promote the unidirectional growth of actin filaments. Moreover, since US 2005/0106629 does not provide any experimental result, the experimental feasibility of this method remains to be demonstrated.
There is thus a need for novel methods enabling the preparation of three-dimensional structures of actin on the nano- or micrometer scale while controlling precisely the shape, the orientation and the spatial location of said structures.