PATTERN TRANSFER FABRICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application No. 10201608596S, filed on 13 October 2016, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention generally relates to nano technology. In particular, the present invention relates to nanostructures and manufacture of nanostructures.

BACKGROUND OF THE INVENTION

[0003] Biopolymers are increasingly attractive as green alternatives for constructing biocompatible and biodegradable electronic/photonic devices. These biopolymers have direct applicability for biomedical applications, such as implantable devices. One approach is to use materials that integrate both the structural and functional optical components into a single element. This application places certain requirements on the substrate material, such as biocompatibility, transparency, toughness, flexibility, and others.

[0004] A major challenge in designing alternative green devices and providing alternatives biocompatible and biodegradable electronic/photonic devices are the fact that to date very few biopolymers have been shown to possess the above properties. Even when the biopolymers having the above properties have been identified, the method of manufacturing the alternative devices above (e.g. such as a protein film and a protein-metal hybrid film) is not straightforward. In view of the above, there is a need to provide an alternative method of manufacturing a protein film comprising a patterned nanostructure and a protein-metal hybrid film comprising a patterned nanostructure. There is also a need to provide an alternative protein film and an alternative protein-metal hybrid film.

SUMMARY OF THE INVENTION

[0005] In one aspect, there is provided a method of manufacturing a protein film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) applying a protein solution to a template shaped to correspond with the patterned nanostructure, wherein the template comprises structures with a width of 20 nm and below; (b) allowing the proteins in the protein solution to self-assemble to form a protein film; and (c) removing the protein film from the template. In another aspect, there is provided a method of manufacturing a protein-metal hybrid film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) applying a protein solution to a template shaped to correspond with the patterned nanostructure, wherein the template comprises structures with a width of 20 nm and below; (b) allowing the proteins in the protein solution to self-assemble to form a protein film; (c) removing the protein from the template; and (d) applying a metal layer to a surface of the protein film, wherein the surface comprises the patterned nanostructure. In one embodiment, the protein is a suckerin protein. In another embodiment, the suckerin protein is selected from the group consisting of a suckerin monomer, a suckerin trimer, a suckerin pentamer, and a suckerin octamer. In yet another embodiment, the template comprises structures with a width of more than Onm to < 20nm. In yet another embodiment, the structures are protrusions. In yet another embodiment, the structures are of regular shape or wherein the structures are of regular shape selected from the group consisting of triangle, square, rectangle, and hexagon. In yet another embodiment, the structures in the template are spaced apart from each other at a distance from about lOnm to about 400nm. In yet another embodiment, the protein solution comprises from about 0.5 wt% to about 5 wt% of protein. In yet another embodiment, the template is a template for an optical device. In yet another embodiment, the template is made of silicon. In yet another embodiment, (b) is carried out in vacuum. In yet another embodiment, (b) comprises allowing the proteins in the protein solution to self-assemble for more than 0 hour to up to 24 hours. In yet another embodiment, the protein film is of about ΙΟΟμπι to about 500μπι thick. In yet another embodiment, removing the protein film from the template in (c) comprises lifting the protein film with mechanical tools or allowing the protein film to attach to an adhesive material which is brought in contact with the protein film and removed after attachment of the protein film to the adhesive material. In yet another embodiment, the protein-metal hybrid film is a protein-gold hybrid film. In yet another embodiment, (d) comprises applying a gold layer to the surface of the protein film. In yet another embodiment, applying a gold layer comprises evaporating gold films to the surface of the protein film. In yet another embodiment, the gold films are of about lnm to about 300nm thick.

[0006] In yet another aspect, the present invention provides a protein film comprising a patterned nano structure, wherein the protein is a self-assembling protein, and the patterned nano structure has a width of 20nm and below. In yet another embodiment, the protein film is manufactured according to the method as described herein.

[0007] In yet another aspect, the present invention provides a protein-metal hybrid film comprising a patterned nanostructure, wherein the protein is a self-assembling protein, and the patterned nanostructure has a width of 20nm and below. In yet another embodiment, the protein-metal hybrid film is manufactured according to the method as described herein. In yet another embodiment, the nanostructure is biocompatible and/or biodegradable. In yet another embodiment, the protein film as described herein or the protein-metal hybrid film as described herein further comprising a polymer or an active agent.

[0008] In yet another aspect, the present invention provides an optical device comprising a protein-metal hybrid film as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0010] Figure 1 shows data related to genetic engineering of Suckerins. Figure 1A shows a set of amino acid sequences depicting the molecular architecture of two suckerins from Dosidicus gigas (DG-19) and Sepioteuthis lessoniana (SL-lb). The amino acid residues of beta-sheet forming regions are bolded. The amino acid residues of amorphous glycine rich regions are italicized. Proline residues that flank the beta-sheet forming regions are underlined. Figure IB shows a graphical illustration depicting schematic of multimeric suckerins generated by genetic engineering. Figure 1C shows a Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) result depicting the molecular weight analysis of multimeric suckerins produced by bacterial production. Thus, Figure 1 illustrates examples of the generated suckerin multimers.

[0011] Figure 2 shows a schematic illustration depicting one example of the fabrication process of hybrid protein-gold nanostructures. Figure 2A shows a graphical illustration depicting a silicon template with sub-20-nm-wide protrusions. The silicon templates were fabricated by using inductively-coupled plasma (ICP) drying etching based on the mask of hydrogen silsesquioxane (HSQ) resist defined by electron beam lithography (EBL). Figure 2B shows a graphical illustration depicting suckerin protein film as casted onto the sub-20- nm silicon template. Figure 2C shows a graphical illustration depicting the tape placed onto the suckerin protein resist and followed by the template stripping process. Figure 2D shows a graphical illustration depicting the patterned suckerin protein nanostructures with sub-20-nm gaps. Figure 2E shows a graphical illustration depicting the evaporation of gold film onto the suckerin protein nanostructures. Thus, Figures 2A to 2D illustrate an exemplary method of manufacturing a protein film comprising a patterned nanostructure and Figures 2A to 2E illustrate an exemplary method of manufacturing a protein-gold hybrid film comprising a patterned nanostructure.

[0012] Figure 3 shows a set of Scanning Electron Micrograph (SEM) images depicting suckerin protein resist having sub-20-nm dimensions of different shapes. Figure 3A shows a Scanning Electron Micrograph (SEM) image depicting a silicon template generated by electron beam lithography. Figures 3B, 3C, and 3D show a set of Scanning Electron Micrograph (SEM) images depicting free-standing suckerin protein film casted onto silicon templates having square (Figure 3B), triangular (Figure 3C) and hexagonal (Figure 3D) features. Thus, Figure 3 illustrates that the patterned protein nanostructures can have various shapes whilst maintaining gap size of less than 20 nm.

[0013] Figure 4 shows data depicting optical characterization results of the hybrid protein-gold nanostructures. Figure 4A shows an optical microscope image depicting hybrid protein-gold nanostructures having varying pitch and varying dosage. The color and the color intensity of each square vary. As the dose time increases and the pitch size (Λ) decreases, the color and the color intensity becomes darker. The reflectance optical spectrum of each square

(i.e. each feature) within the dotted box (i.e. the rectangle drawn with dashed ( ) line) is measured and the result is depicted on Figure 4B. Figure 4B shows a set of spectra depicting the reflectance optical spectrum of features (i.e. the squares in Figure 4A) with varying pitch size (from 60nm to 360nm; the difference of the pitch size between one reflectance optical spectrum to the next is 20nm) outlined by dotted box in Figure 4A. The dashed line labeled as "Dip." on Figure 4B marked the position of the observable dips in the optical reflectance spectrum. The dip position refers to the wavelength of having minimum reflectance value. The dashed line also indicates that increase of pitch size correspond to a red shift (i.e. increase of wavelength or λ) in the reflectance optical spectrum. As the pitch size is increased, the color of the feature becomes lighter (as shown in Figure 4A). Figure 4C shows a Scanning Electron Micrograph (SEM) image of Feature 9 (i.e. a feature having a dose time of 2.5μ8 and a pitch size of 220 nm, which correspond to the ninth square from the bottom right corner of Figure 4A, inside of the dotted box) depicting the evidence of the presence of sub-20-nm gap size in the features even after the gold evaporation process. Thus, Figure 4 illustrate that the hybrid gold-protein nanostructures with strong localized plasmon resonance can be obtained by evaporating 45-nm-thick gold films onto the demolded protein nanostructures with sub-20-nm dimensions.

[0014] Figure 5 shows a schematic illustration depicting one example of a representative manufacturing process of hybrid protein-metal nanostructures. Figure 5A shows a graphical illustration depicting a template with sub-20-nm-wide protrusions. Figure 5B shows a graphical illustration depicting protein film as casted onto the sub-20-nm template. Figure 5C shows a graphical illustration depicting the adhesive material placed onto the protein resist and followed by the template stripping process. Figure 5D shows a graphical illustration depicting the patterned protein nanostructures with sub-20-nm gaps. Figure 5E shows a graphical illustration depicting the application of metal film onto the protein nanostructures. Thus, Figures 5A to 5D illustrate an exemplary method of manufacturing a protein film comprising a patterned nanostructure and Figures 5A to 5E illustrate an exemplary method of manufacturing a protein-metal hybrid film comprising a patterned nanostructure.

[0015] Figure 6 shows a schematic illustration depicting one example of cross section of a portion of a template and a portion of a corresponding protein film that is manufactured using said template. The size of the gap (or trenches) of the protein film corresponds to the size of the structures on template. Figure 6A shows a portion a template and Figure 6B shows a portion of a protein film. The schematic illustration is not drawn to scale.

[0016] Figure 7 shows a set of spectra depicting the result of molecular weight determination of monomeric, trimeric, pentameric, and octameric forms of the suckerin protein using Matrix Assisted Laser Desorption/Ionization - Time Of Flight (MALDI-TOF) analysis. Figure 7 A shows a MALDI-TOF spectrum corresponding to the monomeric form of suckerin that has molecular weight of about 14.9KDa. Figure 7B shows a MALDI-TOF spectrum corresponding to the trimeric form of suckerin that has molecular weight of about 37.7KDa. Figure 7C shows a MALDI-TOF spectrum corresponding to the pentameric form of suckerin that has molecular weight of about 60.4KDa. Figure 7D shows a MALDI-TOF spectrum corresponding to the octameric form of suckerin that has molecular weight of about 93.8KDa. Thus, Figure 7 illustrates that even though the repetitive nature of the proteins causes them to migrate differently during SDS-PAGE (as shown in Figure 1C), MALDI- TOF verifies the predicted molecular weight of the proteins.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0017] Biopolymers (such as proteins) are becoming increasingly attractive as green alternatives for constructing biocompatible and biodegradable electronic and photonic devices, which have direct applicability to biomedical applications, such as implantable devices. In order to provide such devices, one approach is to use materials that integrate both the structural and functional optical components into a single element. This design places certain requirements on the substrate materials, such as biocompatibility, transparency, toughness, flexibility, and the like. To date, very few biopolymers have been shown to possess the above properties thereby limiting the ability to design functional green devices. Even when a biopolymer having the above properties is discovered, the method of using said biopolymer to manufacture green devices is not obvious.

[0018] In view of the above problems, there is a need to provide an alternative method of manufacturing a protein film comprising a patterned nanostructure and a protein-metal hybrid film comprising a patterned nanostructure. There is also a need to provide an alternative protein film and an alternative protein-metal hybrid film.

[0019] The inventors of the present disclosure have found an alternative method of manufacturing a protein film comprising a patterned nanostructure and a protein-metal hybrid film comprising a patterned nanostructure. In other words, the present disclosure describes the direct patterning of protein (such as genetically engineered suckerin) into nanostructures with dimension of sub-20-nm, and the realization of compact optical color filters by protein- metal (such as protein-gold) hybrid nanostructures. There are several variants of the method of manufacturing the protein-film or the protein- metal hybrid film as described herein. In the first variant, as shown for example in Figures 5A and 5B, a protein solution is applied to a template and the protein in the protein solution is allowed to self-assemble or to aggregate to form a protein film. The second variant of the method can be thought of as an inverse of the first variant. In the second variant, the protein film is obtained and it is then imprinted (or stamped or impressed) with a template. The step of removing the protein film from the template, as shown for example in Figure 5C, is identical for both the first variant and the second variant of the method of manufacturing. By following the first variant or the second variant of the method of manufacturing, a protein film comprising a patterned nanostructure, as shown for example in Figure 5D, is obtained.

[0020] Further to the above, in order to obtain a protein-metal hybrid film comprising a patterned nanostructure, as shown for example in Figure 5E, an additional step of applying a metal layer to a surface of the protein film (i.e. to the surface comprising patterned nanostructure) has to be performed on the protein-film manufactured according to the first variant or the second variant of the manufacturing method described above. Therefore, the third variant of the manufacturing method comprises providing a protein-film manufactured according to the first variant followed by the step of applying a metal layer on the patterned surface of the protein-film. The fourth variant of the manufacturing method comprises providing a protein-film manufactured according to the second variant followed by the step of applying a metal layer on the patterned surface of the protein-film. The variants of the manufacturing methods are further described in the aspects listed on the following four paragraphs.

[0021] Thus, in one aspect, the present disclosure provides a method of manufacturing a protein film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) applying a protein solution to a template shaped to correspond with the patterned nanostructure, wherein the template comprises structures with a width of 20 nm and below; (b) allowing the proteins in the protein solution to self-assemble to form a protein film; and (c) removing the protein film from the template.

[0022] In another aspect, the present disclosure provides a method of manufacturing a protein film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) obtaining a protein film comprising the self-assembling protein; (b) imprinting the protein film from (a) with a template shaped to correspond with the patterned nanostructure to form a patterned nanostructure on the protein film, wherein the template comprises structures with a width of 20 nm and below; and (c) removing the protein film from the template.

[0023] In yet another aspect, the present disclosure provides method of manufacturing a protein-metal hybrid film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) applying a protein solution to a template shaped to correspond with the patterned nanostructure, wherein the template comprises structures with a width of 20 nm and below; (b) allowing the proteins in the protein solution to self-assemble to form a protein film; (c) removing the protein from the template; and (d) applying a metal layer to a surface of the protein film, wherein the surface comprises the patterned nanostructure.

[0024] In yet another aspect, the present disclosure provides a method of manufacturing a protein-metal hybrid film comprising a patterned nanostructure, wherein the protein that is forming the protein film is a self-assembling protein, the method comprising: (a) obtaining a protein film comprising the self-assembling protein; (b) imprinting the protein film from (a) with a template shaped to correspond with the patterned nanostructure to form a patterned nanostructure on the protein film, wherein the template comprises structures with a width of 20 nm and below; and (c) removing the protein film from the template; and (d) applying a metal layer to a surface of the protein film, wherein the surface comprises the patterned nanostructure.

[0025] As used herein, the term "applying a protein solution" refers to any suitable method for putting or spreading a protein solution on the patterned surface of a template. In one example, "applying a protein solution" is performed by dripping the protein solution using an instrument such as a pipette and allowing the protein solution to dry on the template. As described above, the proteins that are applicable for manufacturing biocompatible and biodegradable electronic and photonic devices (such as a protein film and/or a protein metal hybrid film described herein), have to possess certain characteristics. Thus, in one example, the self-assembling protein is capable of forming non-covalent nano-confined beta sheets. As used herein, the term "self-assembling protein" refers to a protein that can aggregate or self- aggregates in a protein solution. In other words, during the self-assembling or self- aggregating process, the protein itself will form the nanostructures on the surface of the protein-film by conforming to the template. Without wishing to be bound by theory, the intrinsic property of a protein to form non-covalent nano-confined beta sheets is useful for the formation of free-standing films that can be molded in to sub-20nm features.

[0026] Any type of protein that is capable of forming non-covalent nano-confined beta sheets is suitable to be used in the method of manufacturing as described herein. Recently, silk has become one of the most extensively studied and developed biopolymer for optical applications because of its good mechanical and optical properties, biodegradability and biocompatibility. Other biopolymers, such as keratins, have been shown to be amenable to soft-lithography techniques for forming micro and nano -patterned optical elements. Thus, in one example, the self-assembling protein includes, but is not limited to, a silk protein, a suckerin protein, and the like. When the silk protein is used in the method of manufacturing described herein, the silk protein can originate from any source. In one example, the silk protein originates from silkworm or spider or the like.

[0027] In one example, the protein used in the method of manufacturing described herein is a suckerin protein. Suckerins are one family of proteins recently identified in the teeth as found in the suckers that line the arm and tentacles of squids (or the sucker teeth of squids). These teeth play an important grappling role in squid predation. Suckerins self-assemble into robust supramolecular networks via hydrogen bonding and thus can be used to produce materials that have mechanical properties capable of matching with synthetic polymers, despite being composed solely of proteins held together by weak non-covalent interactions. Moreover, these proteins can be produced recombinantly and easily processed to form functional components such as hydrogels and films with a range of mechanical properties through controlled cross-linking. Sequence analysis and structure analysis have shown that suckerins are molecularly organized in a hierarchical manner, comprising repeating units of silk- like beta sheet forming motifs and amorphous glycine -rich domains. The inventors have surprisingly found that modifying the numbers of repeating units (i.e. the number of monomers) that a suckerin protein has can affect the toughness and the flexibility of the protein film or the protein-metal hybrid film obtained from the suckerin protein. Thus, in one example, the suckerin protein includes, but is not limited to, a suckerin monomer, a suckerin dimer, a suckerin trimer, a suckerin tetramer, a suckerin pentamer, a suckerin hexamer, a suckerin heptamer, a suckerin octamer, a suckerin nonamer, and the like. As shown for example in Figure 1C of the present disclosure, a suckerin monomer, a suckerin trimer, a suckerin pentamer, and a suckerin octamer have been synthesized. In some example, the suckerin proteins described herein are exemplified in Figure 1C.

[0028] The suckerin proteins used for the method of manufacturing described herein have been obtained via genetic modification. In one example, the suckerin dimer, or the suckerin trimer, or the suckerin tetramer, or the suckerin pentamer, or the suckerin hexamer, or the suckerin heptamer, or the suckerin octamer, or the suckerin nonamer, or the like is obtained by genetic modification of the wild type suckerin monomer. The wild type suckerin can originate from any organism that produces suckerin proteins, such as squids. It is commonly known in the art that there is common species of squid that give rise to almost all known squid species. This common species of squid is known to diverge into various species more than 350 million years ago. Thus, even though some species of squid are distantly related, they are still capable of producing suckerins. Thus, in one example, the wild type suckerin monomer is from an organism (such as a squid) that includes, but is not limited to, Dosidicus gigas (DG-19), Sepioteuthis lessoniana (SL-lb), Sepia esculenta, Loligo vulgaris, Loligo pealei, Todarodes pacificus, Euprymna scolopes, and the like.

[0029] The inventors have surprisingly found that in order to obtain localized lateral gap plasmon resonance or to achieve a strong localized plasmon resonance, a protein-metal hybrid film manufactured according to the method described herein needs to have sub-20nm gap size. As used herein, the term "localized plasmon resonance" refers to the excitation of the collective oscillation of electrons on metal. The localized plasmon resonance can be measured using any method known in the art. In one example, the localized plasmon resonance is measured by using the micro -spectrometer to measure the reflectance spectra. The localized plasmon resonances lie on the wavelength position, which is corresponding to the dip wavelength in the reflectance spectrum. The optical reflectance spectra of the samples can also be measured using any method known in the art. In one example, the optical reflectance spectra of the samples were measured by using a CRAIC UV-VIS-NIR micro- spectrophotometer model QDI 2010 (equipped with a 36x objective lens with NA of 0.5). The size of the gaps on the surface of a protein-film or a protein-metal hybrid film depends of the width of the structures on the template used in the manufacturing process described herein. Thus, in one example, the template comprises structures with a width of about 20nm, or about 19nm, or about 18nm, or about 17nm, or about 16nm, or about 15nm, or about 14nm, or about 13nm, or about 12nm, or about l lnm, or about lOnm, or about 9nm, or about 8nm, or about 7nm, or about 6nm, or about 5nm, or about 4nm, or about 3nm, or about 2nm, or about lnm, or below about 20nm, or below about 19nm, or below about 18nm, or below about 17nm, or below about 16nm, or below about 15nm, or below about 14nm, or below about 13nm, or below about 12nm, or below about l lnm, or below about lOnm, or below about 9nm, or below about 8nm, or below about 7nm, or below about 6nm, or below about 5nm, or below about 4nm, or below about 3nm, or below about 2nm, or below about lnm. As would be understood by a person skilled in the art, the width of the structures on the template used in the manufacturing process is not Onm or is more than Onm. In one example, the template comprises structures with a width from 1 lnm to 20nm.

[0030] In one example, as shown for example on Figure 5A and Figure 6A, the structures comprised on a template used in the manufacturing method described herein are protrusions. As used herein, the term "protrusions on a template" refers to structures that are extending from the surface of a template. Due to their sizes, the structures that are extending from the surface of a template (or the protrusions on a template) are considered as nanostructures. In one example, the height of the protrusions on a template is from about 20nm to about 40nm, or from about 40nm to about 60nm, or from about 60nm to about 80nm, or from about 80nm to about lOOnm, or from about lOOnm to about 120nm, or from about 120nm to about 140nm, or from about 140nm to about 160nm, or from about 160nm to about 180nm, or from about 180nm to about 200nm, or from about 20nm to about 200nm, or from about 40nm to about 180nm, or from about 60nm to about 140nm, or from about 80nm to about 120nm. In one example, the height of the protrusions is about 20nm, or about 40nm, or about 60nm, or about 80nm, or about lOOnm, or about 120nm, or about 140nm, or about 160nm, or about 180nm, or about 200nm. In one example, the height of the protrusions is about lOOnm. The protrusions on the template allows for the formation of trenches or gaps on the surface of a protein-film or a protein-metal hybrid film. Without wishing to be bound by theory, sub-20- nm trenches (or gaps) in the protein-metal hybrid film are able to support the localized plasmon resonance so as to confine the optical fields into the sub-20-nm trenches thereby the protein-metal hybrid film is able to function as a color filter. On the other hand, if the template comprises sub-20-nm depressions instead of protrusion, the protein-film or the protein-metal hybrid film manufactured using said template will comprise protrusions having width of less than 20 nm and trenches or gaps having width of more than 20 nm. A fabricated protein-metal hybrid film comprising gaps having width of more than 20 nm may not be able to support the localized plasmon resonance.

[0031] In one example, the structures comprised on a template used in the manufacturing method described herein are of regular shape or irregular shape. Non-limiting example of the irregular shapes include but are not limited to As used herein, the term "irregular shapes" may also refer to abstract shapes or shapes that are not considered as regular polygons. Non-limiting example of the regular shape includes, but is not limited to, triangle, square, rectangle, pentagon, hexagon, octagon, any other regular polygons, and the like. In some examples, the structures on the template used in the method of manufacturing described herein can be faithfully replicated on the protein film.

[0032] In addition to determining the width of the structures (i.e. the protrusions) on the template used in the method of manufacturing described herein, the width of the spaces between the structures (i.e. how far apart is one structure from the next) have to be determined. Thus, in one example, the structures in the template are spaced apart at from about lOnm to about 20nm, from about 20nm to about 30nm, from about 30nm to about 40nm, from about 40nm to about 50nm, from about 50nm to about 60nm, from about 60nm to about 70nm, from about 70nm to about 80nm, from about 80nm to about 90nm, from about 90nm to about lOOnm, from about lOOnm to about 120nm, from about 120nm to about 140nm, from about 140nm to about 160nm, from about 160nm to about 180nm, from about 180nm to about 200nm, from about 200nm to about 220nm, from about 220nm to about 240nm, from about 240nm to about 260nm, from about 260nm to about 280nm, from about 280nm to about 300nm, from about 300nm to about 320nm, from about 320nm to about 340nm, from about 340nm to about 360nm, from about 360nm to about 380nm, from about 380nm to about 400nm. In one example, the structures in the template are spaced apart at about lOnm, or about 20nm, or about 30nm, or about 40nm, or about 50nm, or about 60nm, or about 70nm, or about 80nm, or about 90nm, or about lOOnm, or about 120nm, or about 140nm, or about 160nm, or about 180nm, or about 200nm, or about 220nm, or about 240nm, or about 260nm, or about 280nm, or about 300nm, or about 320nm, or about 340nm, or about 360nm, or about 380nm, or about 400nm. As the template provided herein can be prepared according to any method known in the art, a person skilled in the art appreciate that the method to adjust the width of the spaces between the structures varies according to the method used to prepare the template.

[0033] As shown for example on Figure 6B, the width of the protrusions on the protein- film or the protein-metal hybrid film will be determined by the width of the spaces between the structures on the template. The width of the protrusion on the protein-film can also be referred as "pitch size". As used herein, the term "pitch size" also refers to the distance between the two nearest periodically repeated structures on the template (or protrusion on the template).. Due to their sizes, the protrusions on the protein-film are considered as nanostructures. Changes in the pitch size affect the color of the protein-metal hybrid film. As shown for example on Figure 4B, increasing the pitch size causes a red shift in the reflectance optical spectrum. As used herein, the term "optical reflectance spectrum" refers to the reflectance values at each wavelength. As used herein, the term "relative reflectance" refers to the measured reflectance from one sample, with respect to the reflectance spectrum as measured from the flat region. The optical reflectance spectrum of the samples can be measured using any method known in the art. In one example, the optical reflectance spectra of the samples were measured by using a CRAIC UV-VIS-NIR micro -spectrophotometer model QDI 2010 (equipped with a 36x objective lens with NA of 0.5). The pitch size of a protein-metal hybrid film can be adjusted by adjusting the width of the spaces between the structures on the template used in the method of manufacturing described herein.

[0034] In order to manufacture a protein film or a protein-metal hybrid film using the manufacturing method described herein, it is useful to be able to dissolve the protein in solution and to be able to obtain a homogeneous protein solution. The inventors have found that the protein solution having certain wt% is useful because the protein readily dissolves and does not form aggregates in solution. Thus, in one example, the protein solution comprises from about 0.5 wt% to about 1 wt%, or from about 1 wt% to about 1.5 wt%, or from about 1.5 wt% to about 2 wt%, or from about 2 wt% to about 2.5 wt%, or from about 2.5 wt% to about 3 wt%, or from about 3 wt% to about 3.5 wt%, or from about 3.5 wt% to about 4 wt%, or from about 4 wt% to about 4.5 wt%, or from about 4.5 wt% to about 5 wt% of protein, or from about 5 wt% to about 10 wt% of protein, or from about 10 wt% to about 15 wt% of protein, or from about 15 wt% to about 20 wt% of protein. In one example, the protein solution comprises about 0.5 wt%, or about 1 wt%, or about 1.5 wt%, or about 2 wt%, or about 2.5 wt%, or about 3 wt%, or about 3.5 wt%, or about 4 wt%, or about 4.5 wt%, or about 5 wt% of protein, or about 10 wt% of protein, or about 15 wt% of protein, or about 20 wt% of protein. In one example, the protein solution comprises about 0.5 wt% of protein. In one example, the solvent for the peptide solution is hexafluoro-2-propanol or Hexafluoroisopropanol (HFIP). In one example, the protein solution comprises a suckerin protein. The inventors have surprisingly found that suckerin protein is useful for the method of manufacturing as described herein because processing of suckerin protein (e.g. dissolving suckerin protein to form protein solution) can be performed using mildly acidic solvent (such as acetic acid).

[0035] Non-limiting example of the use of the protein film or the protein-metal hybrid film manufactured according to the method described herein is for optical applications. Thus, in one example, the template used in the method of manufacturing described herein is a template for an optical device. In one example, the optical device includes, but is not limited to, a lens, a microlens array, an optical grating, a pattern generator, a beam reshaper, a color sensor, a compact spectrometer, and the like.

[0036] The template used in the manufacturing method described herein can be made of any material that is suitable for making a template. In one example, the template used in the manufacturing method as described herein is made of a material that includes, but is not limited to, silicon, glass, metal, metal oxide, silicon dioxide, silicon nitride, indium tin oxide, ceramic, sapphire, combinations thereof, and the like. In one example, the template used in the manufacturing method as described herein is made of silicon.

[0037] The silicon template used in the method of manufacturing described herein can be made by following any suitable protocol known in the art. A non-limiting example of the protocol for formation of the silicon template is provided in Experimental Section, subtitled "Fabrication Process of Silicon Template". In one example, the silicon template is formed by dry etching based on a mask defined by electron beam lithography. In one example, the dry etching used for the formation of the silicon template is inductively-coupled plasma (ICP) dry etching. In one example, the mask used in the dry etching process includes, but is not limited to, a mask of hydrogen silsesquioxane (HSQ), a mask of polymethyl methacrylate (PMMA) and a mask of ZEP (or ZEP resist).

[0038] In the method of manufacturing described herein, the step of "allowing the proteins in the protein solution to self-assemble to form a protein film" or the step of "imprinting the protein film with a template shaped to correspond with the patterned nanostructure to form a patterned nanostructure on the protein film, wherein the template comprises structures with a width of 20 nm and below" is referred as "(b)". Thus, in one example, (b) is carried out in vacuum. In one example, wherein when (b) comprises the step of "allowing the proteins in the protein solution to self-assemble to form a protein film", the length of time to perform (b) is for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 12 hours, up to 14 hours, up to 16 hours, up to 18 hours, up to 20 hours, up to 22 hours, or up to 24 hours. As would be understood by a person skilled in the art, the length of time to perform (b) is more than 0 hour. In one example, wherein when (b) comprises the step of "allowing the proteins in the protein solution to self-assemble to form a protein film", the length of time to perform (b) is for about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 7 hours, or about 8 hours, or about 9 hours, or about 10 hours, or about 12 hours, or about 14 hours, or about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours. In one example, wherein when (b) comprises the step of "allowing the proteins in the protein solution to self-assemble to form a protein film", the length of time to perform (b) is for about 6 hours.

[0039] The method of manufacturing described herein allows for formation of a protein film or a protein-metal hybrid film having variety of thickness. As shown for example on Figure 6B, the thickness of the protein film or the protein-metal hybrid film is the distance measured from surface of film that does not have protrusions to the top of the protrusions on the film. Thus, in one example, the protein film is of about ΙΟμπι to about 20μπι thick, about 20μπι to about 30μπι thick, about 30μπι to about 40μπι thick, about 40μπι to about 50μπι thick, about 50μπι to about 60μπι thick, about 60μπι to about 70μπι thick, about 70μπι to about 80μπι thick, about 80μπι to about 90μπι thick, about 90μπι to about ΙΟΟμπι thick, about ΙΟΟμπι to about Ι ΙΟμπι thick, or about Ι ΙΟμπι to about 120μπι thick, or about 120μπι to about 130μπι thick, or about 130μπι to about 140μπι thick, or about 140μπι to about 150μπι thick, or about 150μπι to about 160μπι thick, or about 160μπι to about 170μπι thick, or about 170μπι to about 180μπι thick, or about 180μπι to about 190μπι thick, or about 190μπι to about 200μπι thick, or about 200μπι to about 210μπι thick, or about 210μπι to about 220μπι thick, or about 220μπι to about 230μπι thick, or about 230μπι to about 240μπι thick, or about 240μπι to about 250μπι thick, or about 250μπι to about 260μπι thick, or about 260μπι to about 270μπι thick, or about 270μπι to about 280μπι thick, or about 280μπι to about 290μπι thick, or about 290μιη to about 300μιη thick, or about 300μιη to about 320μιη thick, or about 320μιη to about 340μηι thick, or about 340μιη to about 360μιη thick, or about 360μιη to about 380μηι thick, or about 380μιη to about 400μιη thick, or about 400μηι to about 420μιη thick, or about 420μιη to about 440μηι thick, or about 440μιη to about 460μιη thick, or about 460μιη to about 480μηι thick, or about 480μιη to about 500μιη thick. In one example, the protein film is of ΙΟμιη thick, or about 20μιη thick, or about 30μιη thick, or about 40μιη thick, or about 50μιη thick, or about 60μιη thick, or about 70μιη thick, or about 80μm thick, or about 90μιη thick, about ΙΟΟμιη thick, or about Ι ΙΟμιη thick, or about 120μm thick, or about 130μιη thick, or about 140μιη thick, or about 150μιη thick, or about 160μιη thick, or about 170μιη thick, or about 180μm thick, or about 190μιη thick, or about 200μιη thick, or about 210μιη thick, or about 220μιη thick, or about 230μm thick, or about 240μιη thick, or about 250μιη thick, or about 260μιη thick, or about 270μιη thick, or about 280μm thick, or about 290μιη thick, or about 300μιη thick, or about 320μιη thick, or about 340μιη thick, or about 360μm thick, or about 380μιη thick, or about 400μιη thick, or about 420μιη thick, or about 440μιη thick, or about 460μιη thick, or about 480μm thick, or about 500μιη thick. In one example, the protein film is of about 300μπι thick.

[0040] As shown for example on Figure 5C, the method of manufacturing described herein comprises removal of a protein film from the template. In one example, after a protein film is removed from the template, the template is reusable or can be re-used. Without wishing to be bound by theory, the multimeric design of the protein and the increase molecular weight improves the toughness of the film manufactured according to the method described herein. Therefore, when combined with a gentle lift-off process developed for demolding (i.e. removal of a protein film from the template), the structural integrity of the film is enhanced and thereby minimizing deformation in the final film. Thus, in one example, removing the protein film from the template in (c) comprises lifting the protein film with mechanical tools, or allowing the protein film to attach to an adhesive material which is brought in contact with the protein film and removed after attachment of the protein film to the adhesive material, or the like.

[0041] In one example, the mechanical tools used for removing the protein film from the template are forceps. The inventors have found that forceps are useful for removing thicker film. Thus, in one example, the thickness of the protein film that can be removed using forceps is about 200μπι to about 210μπι thick, or about 210μπι to about 220μπι thick, or about 220μιη to about 230μηι thick, or about 230μηι to about 240μηι thick, or about 240μηι to about 250μιη thick, or about 250μηι to about 260μηι thick, or about 260μηι to about 270μιη thick, or about 270μηι to about 280μηι thick, or about 280μηι to about 290μηι thick, or about 290μηι to about 300μηι thick, or about 300μηι to about 320μηι thick, or about 320μιη to about 340μηι thick, or about 340μηι to about 360μηι thick, or about 360μηι to about 380μιη thick, or about 380μηι to about 400μηι thick, or about 400μηι to about 420μηι thick, or about 420μηι to about 440μηι thick, or about 440μηι to about 460μηι thick, or about 460μιη to about 480μηι thick, or about 480μηι to about 500μηι thick. In one example, the thickness of the protein film that can be removed using forceps is about 200μιη thick, or about 210μιη thick, or about 220μιη thick, or about 230μιη thick, or about 240μιη thick, or about 250μιη thick, or about 260μιη thick, or about 270μιη thick, or about 280μιη thick, or about 290μιη thick, or about 300μιη thick, or about 320μιη thick, or about 340μιη thick, or about 360μιη thick, or about 380μιη thick, or about 400μιη thick, or about 420μιη thick, or about 440μιη thick, or about 460μιη thick, or about 480μm thick, or about 500μιη thick. In one example, the thickness of the protein film that can be removed using forceps is about 300μιη thick.

[0042] As described above and as shown for example on Figure 5C, besides removing the protein film with mechanical tool, the protein film can also be removed from the template by allowing the protein film to attach to an adhesive material which is brought in contact with the protein film and removed after attachment of the protein film to the adhesive material. A person skilled in the art is aware that the adhesive material used in the method of manufacturing described herein is an adhesive material that will allow for the protein-film to maintain its integrity when the adhesive material is removed. In other words, removal of the adhesive material from the protein-film will not tear, break, deform, or destroy the protein- film. Thus, in one example, the adhesive material is an adhesive tape. Any type of adhesive tape that can attach to the protein film can be used in the manufacturing method described herein. Thus, in one example, the adhesive tape is a dissolvable adhesive tape. When an adhesive tape is used to remove the protein film from the template, in one example, the manufacturing method described herein further comprising removing the adhesive tape. Any suitable method can be employed for removing the adhesive tape from the protein film. In one example, removing the adhesive tape from the protein film comprises removing by mechanical force, or removing by dissolving the adhesive tape, or the like. [0043] As described above and as shown for example in Figure 5E, the manufacturing method described herein also comprises the step of applying a metal layer on the surface comprising patterned nanostructure of a protein-film thereby obtaining a protein-metal hybrid film. In one example, the protein-metal hybrid film described herein comprises a hybrid film of protein and a metal that includes, but is not limited to, gold, silver, aluminum, titanium, chromium, platinum, copper, tin, indium, cadmium, lead, tungsten, iron, nickel, selenium, silicon, strontium, palladium, vanadium, zinc, zirconium, alloys and oxides thereof, any combination thereof, and the like. As shown for example in Figure 2E, in one example, the protein-metal hybrid film is a protein-gold hybrid film.

[0044] In the method of manufacturing described herein, the step of "applying a metal layer to a surface of the protein film, wherein the surface comprises the patterned nanostructure" is referred as "(d)". Thus, in one example, (d) comprises applying a gold layer to the surface of the protein film. The application of the gold layer to the surface of the protein film can be performed using any application method known in the art. In one example, applying a gold layer comprises evaporating gold films to the surface of the protein film. The evaporation of gold films on the surface of the protein film can be performed using any suitable instrument. In one example, the instrument used to evaporate gold film on the surface of protein film is Electron beam evaporator (Denton). The gold films applied of the surface of the protein film can have a variety of thickness. In one example, the gold films applied on the surface of the protein film are of about lnm to about 5nm thick, or about 5nm to about lOnm thick, or about lOnm to about 15nm thick, or about 15nm to about 20nm thick, or about 20nm to about 25nm thick, or about 25nm to about 30nm thick, or about 30nm to about 35nm thick, or about 35nm to about 40nm thick, or about 40nm to about 45nm thick, or about 45nm to about 50nm thick, or about 50nm to about 55nm thick, or about 55nm to about 60nm thick, or about 60nm to about 65nm thick, or about 65nm to about 70nm thick, or about 70nm to about 75nm thick, or about 75nm to about 80nm thick, or about 80nm to about 85nm thick, or about 85nm to about 90nm thick, or about 90nm to about 95nm thick, or about 95nm to about lOOnm thick, or about lOOnm to about HOnm thick, or about HOnm to about 120nm thick, or about 120nm to about 130nm thick, or about 130nm to about HOnm thick, or about HOnm to about 150nm thick, or about 150nm to about 160nm thick, or about 160nm to about 170nm thick, or about 170nm to about 180nm thick, or about 180nm to about 190nm thick, or about 190nm to about 200nm thick, or about 200nm to about 210nm thick, or about 210nm to about 220nm thick, or about 220nm to about 230nm thick, or about 230nm to about 240nm thick, or about 240nm to about 250nm thick, or about 250nm to about 260nm thick, or about 260nm to about 270nm thick, or about 270nm to about 280nm thick, or about 280nm to about 290nm thick, or about 290nm to about 300nm thick. In one example, the gold films applied on the surface of the protein film are of about lnm thick, or about 5nm thick, or about lOnm thick, or about 15nm thick, or about 20nm thick, or about 25nm thick, or about 30nm thick, or about 35nm thick, or about 40nm thick, or about 45nm thick, or about 50nm thick, or about 55nm thick, or about 60nm thick, or about 65nm thick, or about 70nm thick, or about 75nm thick, or about 80nm thick, or about 85nm thick, or about 90nm thick, or about 95nm thick, or about lOOnm thick, or about l lOnm thick, or about 120nm thick, or about 130nm thick, or about 140nm thick, or about 150nm thick, or about 160nm thick, or about 170nm thick, or about 180nm thick, or about 190nm thick, or about 200nm thick, or about 210nm thick, or about 220nm thick, or about 230nm thick, or about 240nm thick, or about 250nm thick, or about 260nm thick, or about 270nm thick, or about 280nm thick, or about 290nm thick, or about 300nm thick. In one example, the gold films applied on the surface of the protein film are of about 45nm thick.

[0045] In addition to finding an alternative method of manufacturing a protein film comprising a patterned nanostructure and a protein-metal hybrid film comprising a patterned nanostructure, the inventors of the present disclosure have found an alternative protein film comprising a patterned nanostructure and an alternative protein-metal hybrid film comprising a patterned nanostructure. The inventors have surprisingly found that the patterned nanostructures on the protein film or on the protein-metal hybrid film are free-standing, mechanically robust, and flexible.

[0046] Thus, in yet another aspect, the present disclosure provides a protein film comprising a patterned nanostructure, wherein the protein is a self- assembling protein, and the patterned nanostructure has a width of 20nm and below. In one example, said protein film is manufactured according to the method of manufacturing a protein film as described herein.

[0047] In yet another aspect, the present disclosure provides a protein-metal hybrid film comprising a patterned nanostructure, wherein the protein is a self-assembling protein, and the patterned nanostructure has a width of 20nm and below. In one example, the protein- metal hybrid film is manufactured according to the method of manufacturing a protein-metal hybrid as described herein. [0048] In order for the protein film or the protein-metal hybrid film to be applicable for biomedical application, it has to be biocompatible and biodegradable. As used herein, the term "biocompatible" refers to materials that are compatible with living tissue and/or a living system by not being toxic, injurious, or physiologically reactive, and/or not causing immunological rejection. The biocompatibility of a material can be determined using any method known in the art. In one example, the patterned nanostructure on the protein film as described herein or the patterned nanostructure on the protein-metal hybrid film as described herein is biocompatible. In one example, the protein film as described herein or the protein- metal hybrid film as described herein is biocompatible.

[0049] As used herein, the term "biodegradable" refers to materials that are capable of being decomposed by bacteria or other living organisms and thereby avoiding pollution. The biodegradability of a material can be determined using any method known in the art. In one example, the patterned nanostructure on the protein film as described herein or the patterned nanostructure on the protein-metal hybrid film as described herein is biodegradable. In one example, the protein film as described herein or the protein-metal hybrid film as described herein is biodegradable.

[0050] The protein film described herein or the protein-metal hybrid film described herein can also comprise additional substance. The present disclosure paves the way for further processing of a protein-film or a protein-metal hybrid film through incorporating with other functional elements, such as enzymes and chemical probes for biocompatible and biodegradable sensors, as well as a flexible nanoplasmonics platform. Furthermore, because the manufacturing process described herein does not require the use of an external heat source to form the protein film (such as a heated plate), co-deposition of heat- sensitive materials such as enzymes is possible. Thus, in one example, the protein film described herein or the protein-metal hybrid film described herein further comprising an additional polymer. In one example, the protein film described herein or the protein-metal hybrid film described herein further comprising an active agent. Any active agent that is compatible with the protein film described herein or the protein-metal hybrid film described herein can be added. Thus, in one example, the active agent added to the protein film described herein or the protein-metal hybrid film described herein include, but is not limited to, therapeutic agents, cells, proteins, peptides, nucleic acid analogues, nucleotides, oligonucleotides, nucleic acids, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, hormones, hormone atagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics, antimicrobial compounds, anti-inflammation agents, antifungals, antivirals, toxins, prodrugs, chemotherapeutic agents, small molecules, dyes, amino acids, vitamins, antioxidants, combinations thereof, and the like.

[0051] As described above, the protein film or the protein hybrid film is useful for optical applications. In one example, protein-film or hybrid protein-metal film comprising suckerins is considered as a suitable material for building optical devices. Metallic nanostructures can be used to produce printed color images with resolutions beyond the diffraction limit of light. In one example, the present disclosure demonstrates the patterning of suckerin protein into nanostructures with dimension of sub-20-nm, and the realization of compact optical color filters by protein-gold hybrid nanostructures. Through adjusting the pitch size and gap size of the hybrid protein-gold nanostructures, these films could be used as protein-based optical color filters, which potentially could be extended further for protein-based high-resolution color printing beyond the diffraction limit. Thus, in yet another aspect, the present disclosure provides the use of the method described herein, or the protein film described herein, or the protein-metal hybrid film described herein in the manufacture of an optical device. In yet another aspect, the present disclosure provides a method of manufacturing an optical device comprising attaching a protein film or a protein-metal hybrid film as defined herein to an optical device. In yet another aspect, the present disclosure provides an optical device comprising a protein-metal hybrid film as defined herein. In one example, the optical device comprising a protein-metal hybrid film as defined herein is an optical filter in reflection mode. As used herein the term "optical filter in reflection mode" refers to an optical component, which could change or alter the reflection spectrum.

[0052] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a protein film" includes a plurality of protein films, including mixtures and combinations thereof.

[0053] As used herein, the terms "increase" and "decrease" refer to changes of a certain measurable values such as changes in pitch size. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale. The term "change", as used herein, also refers to the difference between a measurable value with another measurable value. However, this term is without valuation of the difference seen. [0054] As used herein, the term "about" in the context of concentration of a substance, thickness or width of an object (such as a protein film or a protein-metal hybrid film), length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.

[0055] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0056] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0057] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0058] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[0059] Material and Methods

[0060] Suckerin Multimer Generation.

[0061] The suckerins multimer were derived from a Sepioteuthis lessoniana suckerin (suckerin- 1) that harbors both beta-sheet forming and glycine rich domains. A codon- optimized gene (SEQ ID NO: 1, expressing protein as defined on SEQ ID NO: 2) for bacterial expression was ordered from IDTDNA and concatemers were generated using a one-pot reaction orthogonal end protection method. Three different sets of primers (SEQ ID NOs: 3 to 8) were used to generate PCR amplicons, as corresponding to a left chain-stopping unit, middle unit and a right chain-stopping unit. The left unit was digested with Bsal, the middle unit was digested with Bsal and BsmBI and the right unit was digested with BsmBI. Digested amplicons were pooled at ratios of 1:2: 1, 1:4: 1 and 1:8: 1 and ligated. The ligated fragments were analyzed on a 1% agarose gel and high molecular weight fragments were gel isolated, digested with Nhel and Xhol and cloned into the pET28a expression vector. Clones harbored an N-terminal HIS -tag. Insert fragments were confirmed by test digestion and sequencing.

[0062] Protein Expression.

[0063] Multimeric clones (SEQ ID NOs: 9, 11, 13, or 15) were transformed into T7 Express strain of E.coli cells (New England BioLabs) and streaked onto selective LB agar plates (50 μ§/ιηΕ kanamycin) followed by an overnight incubation at 37°C. Single colonies were then inoculated into selective LB media (50μ^ηή _^ kanamycin) and incubated overnight under constant shaking at 37°C. Glycerol cell stocks were made by adding 20% (v/v) glycerol for storage at -80°C. Pre-cultures were made from glycerol stocks and incubated overnight. Phosphate Buffered Super Broth (35g/L tryptone, 20g/L yeast extract, 2.5g/L sodium chloride, 1.12g/L potassium phosphate monobasic, 6.27g/L potassium phosphate dibasic) was inoculated with the pre-culture at a ratio of 1: 100 and cultured in baffled flasks under similar conditions to an optical density (OD) of 0.4-0.6 prior to protein induction with 0.5mM IPTG. Cell cultures were kept on the shaker at 37°C for another 4 hours and spun down at 4,200 rpm for 30 minutes thereafter. Cells were re-suspended twice in 50mL of 20mM tris buffer (pH 8.0) and spun down for 10 minutes at 10,000 rpm prior to storage at -20°C.

[0064] Protein Purification by Tangential Flow Filtration.

[0065] Harvested cells were resuspended in 35mL of lysis buffer (50mM tris buffer (pH 7.4), 200mM sodium chloride, ImM PMSF) and were homogenized under high shear by passing it 6 times through a microfluidizer. The collected lysate was then spun down at 19,000 rpm for 1 hour and the supernatant decanted. The remaining inclusion body pellet was then resuspended in 50mL of Wash 1 (5mM EDTA, 2M urea, 2% (v/v) Triton-X, lOmM DTT, lOOmM tris buffer (pH 7.4)) and transferred to the primary reservoir of the Tangential Flow Filtration (TFF) device. An additional 200mL of Wash 1 was added to the secondary reservoir and the TFF was allowed to run until approximately 12.5mL of sample is left in the primary reservoir and with the secondary reservoir empty. The retentate was then collected and spun down at 5,000 rpm for 15 minutes. The inclusion bodies were washed twice with Wash 2 (5mM EDTA, 5mM DTT, lOOmM tris buffer (pH 7.4)) by resuspending it in 40mL of Wash 2 and spinning it down at 5,000 rpm for 15 minutes. Subsequently, the proteins (SEQ ID NOs: 10, 12, 14, or 16) were eluted with 12.5mL of 5% acetic acid and clarified by spinning at 19,000 rpm for 30 minutes. Thereafter, the soluble fraction was dialyzed against 1L of deionized water for 16 hours, followed by a fresh buffer exchange and dialyzed for another 8 hours. The dialyzed protein solution was then lyophilized for 48 hours.

[0066] Protein Purification and Processing.

[0067] Multimeric clones were transformed into T7 Express strain of E.coli cells (New England BioLabs) and cultured in high density growth medium. Suckerin proteins were expressed as inclusion bodies, homogenized with high shear and purified by tangential flow filtration. The high throughput of tangential flow filtration enables purification of the proteins in large batches with excess volumes of wash buffers and minimal handling efforts. Due to a high isoelectric point of greater than pH 9, suckerin proteins are soluble in 5% acetic acid and could be processed in aqueous conditions. High fidelity replication of sub-20-nm gaps (Figure 3) onto suckerin films was thus obtained by solution casting of multi-meric suckerins under vacuum conditions, demonstrating the potential for suckerin proteins as nanopatterned substrates. When stiffer films are desired, suckerin monomer and suckerin trimer were used. Pentamer was largely used for its high yield and robustness. [0068] Fabrication Process of Silicon Template.

[0069] Hydrogen silsesquioxane (HSQ, 2% wt. in methyl isobutyl ketone, Dow Corning XR-1541) e-beam resist was spin coated onto the silicon substrate at the speed of 5k revolutions-per-minute (rpm), resulting in a thickness of -30 nm. Electron beam lithography (EBL) was done using an Elionix ELS-7000 system with an acceleration voltage of 100 keV, and beam current of 500 pA and an exposure dose of 12 mC cm ^" . After the e-beam exposure, the samples were developed by the NaOH/NaCl salt solution (1% wt./4% wt. in de-ionized water) for 60 seconds and immersed in de-ionized water for 60 seconds to stop the development. Next, the sample was immediately rinsed by acetone and isopropanol alcohol (IPA), followed by a continuous flow of nitrogen air gun for 1 minute to dry the sample. The next process is silicon etching by using an inductively-coupled-plasma (ICP, Unaxis shuttle lock system SLR-7701-BR), where the detailed etching conditions were DC power of 150 watts, coil power of 300 watts, Cl ₂ /HBr with the flow rates of 18 seem and 22 seem, process pressure of 10 mtorr, temperature at 6 °C, and an etching time of 46 seconds.

[0070] Experimental Result

[0071] Genetic engineering of Suckerins

[0072] Suckerins are composed of a family of modular proteins with a distinct architecture that facilitates the formation of beta- sheet-reinforced polymer networks. Thirty eight (38) different suckerins from squid and cuttlefish that are part of a multigene family were recently identified. A distinct feature of suckerin is that they are composed of a series of repetitive beta-sheet forming modules (Ml) and amorphous Gly-rich modules (M2) with precise placement of proline residues, which constrain beta-sheet nanocrystal size. Each of the 38 different suckerin proteins has distinct molecular weights and molecular architectures. These observations suggest that polymer networks may be tuned through genetic engineering the arrangement of the modules and the molecular weight of the proteins. Previously, it was shown that one suckerin derived from the Humboldt squid Dosidicus gigas suckerin- 19 (39KDa) could be produced recombinantly. In this work, a different suckerin from bigfin reef squid Sepioteuthis lessoniana (SL. suckerin -lb) was used. This suckerin is about a third the size (11.13KDa), but it has a similar modular architecture with alternating Ml and M2 modules, like suckerin- 19 with shorter amorphous Gly-rich domains (Figure 1A). This suckerin was selected as a basis to engineer suckerins of different molecular weights to tune its mechanical properties. [0073] Initially, it was found that protein films casted from the monomeric SL.suckerin-lb to be highly stiff and brittle making it hard to handle. The toughness of the protein was modulated through genetic engineering of the recombinant protein. A codon-optimized version of the monomer was synthesized and a series of concatenated genetic constructs were generated using a one -pot reaction orthogonal end protection method (Figure IB). The clones that corresponded to trimeric (37.7KDa), pentameric (60.4KDa), and octameric (93.9KDa) forms of the protein were isolated. Each clone was well-expressed as inclusion bodies and were easily purified to high purity (>95%) (Figure 1C). Because of the repetitive nature of the proteins, the protein migrated differently during SDS-PAGE. The molecular weight was accurately determined by MALDI-TOF analysis (Figure 7). Interestingly, the octamer did not ionize which may be due to propensity to aggregate or self-assemble during the drying process. It was also noticed that the octamer would run as high molecular weight smear in SDS-PAGE when performed at higher voltages which was reduced when the voltage was reduced. Moreover, proteins films with higher molecular weights are more pliable and easier to handle. This characteristic is likely to be due to the higher amount of inter-chain entanglement.

[0074] Sub-20-nm Patterning of Protein Nanostructure and Hybrid Protein-Gold Optical Characteristics.

[0075] A simple casting procedure to produce suckerin films from nano-patterned silicon substrates with sub-20-nm features was developed. The silicon templates were patterned by the dry etching process of using inductively-coupled plasma (ICP), and the etching mask was HSQ pattern as defined by electron beam lithography (EBL). After that, the silicon substrate was treated by trichloro (lH,lH,2H,2H-perfluorooctyl) silane. Lyophilized pentamer was dissolved to a concentration of 5 mg/mL in 0.22 μιη filtered 5% acetic acid and vacuum casted for 6 hours onto the silicon template as shown in Figure 2B. The samples were then demolded with adhesive tape and secured onto glass slides as shown in Figure 2C, where the features of the sub-20-nm protrusion features of the silicon template is inversely transferred onto the suckerin protein with sub-20-nm gaps. Thereafter, gold film was evaporated onto the patterned films.

[0076] Figure 3 presents the scanning electron micrograph (SEM) characterization results of the patterned protein nanostructures with various shapes. These SEM images of protein nanostructures were taken after evaporating 2-nm-thick Chromium (i.e. Cr) so as to avoid the charging effect during SEM imaging. It shows that the typical gap size of the patterned protein nanostructures is less than 20 nm. These characterization results demonstrate that suckerin protein could be used as a simple high resolution patterning resist with the advantage of bio-compatibility, as compared to the existing approach by using gold and optical adhesive (OA) glue.

[0077] By evaporating 45-nm-thick gold films onto the demolded protein nanostructures with sub-20-nm dimensions, the hybrid gold-protein nanostructures with strong localized plasmon resonance was obtained. By increasing the pitch size, a red shift in the reflectance optical spectrum was obtained, as corresponding to the observable color change in features. Figure 4A shows the corresponding observable color of the features with varying pitch and varying dosage, wherein Figure 4B presents the measured optical reflectance spectrum. The term "dosage" or "dose time" as used herein refers to the time that electron beam lithography exposes the HSQ resist. The length of the "dose time" can be controlled using the software interface of the electron beam lithography instrument. Moreover, SEM of Feature 9 (Figure 4C) serves as evidence of the sub-20-nm gap size that is present in the features even after the gold evaporation process.

[0078] In the present disclosure, the use of nanopatterned gold-suckerin hybrid substrates with a strong localized plasmon resonance was demonstrated. An array of plasmonic colors was prepared by controlling the pitch size and lateral gap size of these structures, where an array of plasmonic colors appears after evaporating 45-nm-thick gold onto the patterned protein nanostructure, where the localized plasmon resonance is ranging from 500-700 nm (Figure 4B). Considering the wide spread environmental damage from the use of organic solvents for polymer processing, suckerin proteins stands out as a viable option as a highly processible "green" polymer. The high tyrosine content of suckerins have also been reported to reduce gold(III) chloride in-situ to form gold nanoparticles opening the possibility of a facile method of developing plasmonic surfaces for display and sensing applications.

[0079] The top-down patterning of suckerin protein with the resolution of sub-20-nm based on the silicon template with nanostructures was demonstrated. By evaporating 45-nm- thick gold films onto the patterned suckerin protein nanostructures, the hybrid protein-gold nanostructures demonstrate the functionality of ultra-compact optical color filters, which also pave a way towards nanoplasmonics platform with flexible substrates.