WO2017045080A1 - Functional protein structures and methods of making and using same - Google Patents

Abstract

Functional three-dimensional constructs incorporating biological materials produced by additive manufacturing techniques are provided. The biological material can be a protein that is incorporated into the three-dimensional construct via a light-inducible functional group and 0 retains its native function. In one embodiment, the biological material is bacteriorhodopsin and the construct incorporates a silver inorganic catalyst to produce hydrogen upon exposure to light.

Description

FUNCTIONAL PROTEIN STRUCTURES AND METHODS

OF MAKING AND USING SAME

Reference to Related Applications

[0001] This application claims priority to, and the benefit of, U.S. provisional patent application No. 62/219585 filed 16 September 2015, the entirety of which is hereby incorporated by reference herein.

Technical Field

[0002] Some embodiments of the present invention relate to three-dimensional printing or additive manufacturing of functional constructs incorporating biological materials. Some embodiments of the present invention relate to three-dimensional printing or additive manufacturing of protein constructs. Some embodiments of the present invention relate to three- dimensional printing or additive manufacturing of functional protein constructs. Some embodiments of the present invention relate to methods of making or using functional protein constructs produced by three-dimensional printing or additive manufacturing.

Background

[0003] Three-dimensional printing or, more broadly, additive manufacturing is a technique of considerable interest. This technique can be used to rapidly fabricate three-dimensional objects. This technique has been used to print biomaterials, for example to produce tissues for implantation into living organisms, or to repair damaged tissue and reinsert the repaired tissue into living organisms.

[0004] The use of additive manufacturing to produce constructs incorporating biomaterials for carrying out a desired function, for example photosynthesis to produce energy, has not been described to the knowledge of the inventors. There remains a need for constructs produced from biological materials, for example proteins, DNA, RNA or the like, that can carry out specific functions, as well as methods for making and using such constructs to solve specific problems.

[0005] For example, a current problem of concern is that the world's power supply is based predominantly on the use of non-renewable energy resources such as fossil fuels including oil, coal and natural gas. The rapid consumption of fossil fuels has caused unacceptable environmental problems such as greenhouse effects, which could lead to disastrous climatic consequences if left unchecked. New renewable and clean energy resources are required in order to solve such global energy and environmental issues.

[0006] Biological materials such as proteins may have a role to play in providing such new and renewable clean energy resources. For example, the light harvesting proton pump

bacteriorhodopsin, from Archaea, e.g. Halobacteria, has been considered for use in the construction of nanophotocatalysts using a platinum/titanium oxide nanocatalyst (see e.g.

Balasubramanian et al, Nano Letters 2013, 13, 3365-3371, which is incorporated by reference herein for all purposes). Bacteriorhodopsin is used as a proton pump to provide sunlight-driven proton transfer across a membrane to produce an electrochemical gradient, which is converted to chemical energy in the form of ATP. Balasubramanian et al. used the protons so generated with a Pt/Ti0₂ nanocatalyst to produce hydrogen.

[0007] The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

Summary

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

[0009] One aspect of the invention provides a method of making a functional construct incorporating a biological molecule, comprising obtaining the biological molecule and immobilizing the biological molecule in a hydrogel matrix by cross-linking suitable monomers to form the hydrogel matrix using a 3D printer. The 3D printer may use either light or heat to cross-link the suitable monomers. In some embodiments, a spatial position of at least two biological molecules in the hydrogel matrix is controlled by using different (i.e. first and second) wavelengths of light to selectively localize each one of the at least two biological molecules in the hydrogel matrix, by providing each of the two biological molecules with different (i.e. first and second) light-inducible functional groups that can be used to couple the hydrogel matrix by application of the different wavelengths of light (i.e. the first light-inducible functional group is coupled to the hydrogel via a reaction initiated by the first wavelength of light, and the second light-inducible functional group is coupled to the hydrogel via a reaction initiated by the second wavelength of light. In some embodiments, a spatial position of at least a third biological molecule in the hydrogel is controlled by providing the third biological molecule with a third light-inducible functional group that is coupled to the hydrogel via a reaction initiated by a third wavelength of light. The different wavelengths of light can be blue, green or red wavelengths of light in some embodiments. In alternative embodiments, a spatial position of a fourth biological molecule in the hydrogel is controlled to be in the same hydrogel as one of the first, second or third biological molecules by providing the fourth biological molecule with one of the first, second or third light-inducible functional groups, respectively.

[0010] In one aspect, the biological molecules are proteins, and the proteins are engineered to contain a functional group to allow selective crosslinking of the protein to the hydrogel matrix. In some embodiments, the functional group comprises a light-inducible functional group. The light-inducible functional group can be an allyl, vinyl, lactone, thiol, epoxide, amino, azide, alkyne, alkene, or aldehyde functional group in some embodiments.

[0011] In one aspect, the biological molecules are membrane proteins, and the membrane proteins are combined with a membrane protein stabilizing agent prior to being incorporated into the functional construct. The membrane protein stabilizing agent is provided with the light- inducible functional group, for example the membrane protein stabilizing agent can be a functionalized amphipol or a functionalized beta-sheet peptide, and the functional group can be selectively reactive with suitable monomers that are polymerized to provide a hydrogel, for example by exposure to light of a suitable wavelength, optionally in combination with a photoinitiator.

[0012] In some embodiments, two or more different wavelengths of light are used to selectively localize two or more different biological molecules at predetermined spatial locations within a functional construct. In one such embodiment, the biological molecules are first and second proteins, and the proteins are combined with suitable polymerizable monomers that can form a hydrogel. The first protein is configured to cross-link with the suitable monomers at a first wavelength of light via a first light-inducible functional group. The second protein is configured to cross-link with the suitable monomers, or with second suitable monomers, at a second wavelength of light via a second light-inducible functional group that can be reacted at a different wavelength of light than the first light-inducible functional group. A 3D printer is used to control the spatial application of light having the first and second wavelengths, so that the light is used to both polymerize the suitable monomers and selectively couple either the first protein or the second protein to the hydrogel formed by the polymerized monomers at predetermined locations within the functional construct.

[0013] In one aspect, the functional construct comprises a hydrogen electrode.

Bacteriorhodopsin is stabilized with a functionalized beta-sheet peptide having an allyl functional group, and the allyl functional group is crosslinked to methacrylate monomers by polymerization using light having a wavelength of approximately 405 nm in the presence of a photoinitiator. Silver nanoparticles are incorporated in the hydrogel matrix of the functional construct to catalyze the production of hydrogen from the protons produced by the

bacteriorhodopsin upon exposure to light. In some embodiments, glassy carbon is incorporated in the hydrogel matrix of the functional construct to act as an electrode.

[0014] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.

Brief Description of the Drawings

[0015] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0016] Figure 1 shows an example embodiment of a method for producing a functional construct incorporating a membrane protein.

[0017] Figure 2 shows schematically the immobilization of a membrane protein according to one example embodiment.

[0018] Figure 3 shows schematically the immobilization of a membrane protein according to a second example embodiment. [0019] Figure 4 shows schematically the immobilization of a membrane protein according to a third example embodiment.

[0020] Figure 5 shows schematically an example embodiment of a functionalized beta-sheet stabilized membrane protein immobilized in a hydrogel produced by crosslinking of suitable monomers.

[0021] Figure 6 shows the mass spectrum of the cross-linkable peptide octenyl BP1 having the structure (20) used in one experimental example.

[0022] Figures 7A, 7B, 7C and 7D show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of an example embodiment of a glassy

carbon/bacteriorhodopsin/silver electrode (glassy carbon/ bR/Ag NP Electrode). Figure 7A shows a TEM image of silver nanoparticles. Figure 7B shows an SEM image of glassy carbon at 10 μηι magnification, with the inset showing the cross-sectional SEM image at 3 μηι magnification. Figure 7C shows a cross-sectional SEM image of the final electrode construct at 40 μηι magnification. Figure 7D shows a cross-sectional SEM image of the final electrode construct at 2 μηι magnification.

[0023] Figure 8 shows a scanning electron microscopy (SEM) image of an example embodiment of a 3D printed glassy carbon/bacteriorhodopsin/silver electrode construct (glassy carbon/ bR/Ag NP electrode) at 2 mm magnification.

[0024] Figures 9A and 9B show CV and IV (current voltage) curves of an example embodiment of a 3D printed glassy carbon/bacteriorhodopsin/silver electrode construct (glassy carbon/ bR/Ag NP electrode).

[0025] Figure 10 shows gas chromatography (GC) results showing the evolution of H₂ from an exemplary embodiment of a 3D printed glassy carbon/bacteriorhodopsin/silver electrode construct.

Description

[0026] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense. [0027] By modifying techniques developed for three-dimensional (3D) printing and additive manufacturing, the inventors have developed methods for printing constructs incorporating biological materials for carrying out desired functions. In some embodiments, the biological material is a protein. In some embodiments, the biological material is printed together with a different type of material, for example an inorganic catalyst, to produce a composite construct. The inventors refer to this technique of producing a functional construct incorporating a biological material using additive manufacturing concepts as "4-dimensional printing" or 4D printing.

[0028] As used herein the term, photopolymerizable or "light-inducible functional group" means a functional group that can be caused to participate in a coupling reaction by the addition of appropriate reactants and light having an appropriate wavelength and power. The light-inducible functional group itself may not be activated by the application of light, but is available to participate in a reaction with a molecule that is generated by the application of light, for example, appropriate reactive species generated by the application of light having an appropriate wavelength and power to a photoinitiator.

[0029] In some embodiments, the biological material is a membrane protein. In some embodiments, the biological material is a membrane protein that is stabilized and localized on a functional construct in any suitable manner, for example using beta-sheet peptides as described by Tao et al, Nature Meth. 10(8):759 (2013), which is incorporated by reference herein for all purposes, functionalized beta-sheet peptides as described in PCT publication No. WO

2016/029308, which is incorporated by reference herein for all purposes, or functionalized amphipols, for example as described in Delia Pia et al, J. Membrane Biol. (2014) 247:815-826, which is incorporated by reference herein for all purposes. In some embodiments, the membrane protein is anchored by reacting a functional group on the functionalized beta-sheet peptide or functionalized amphipol on a suitable matrix supporting or containing the construct.

[0030] In some embodiments, the functionalized beta-sheet peptides have the general sequence acetyl-(octyl)Gly-Ser-Leu-Ser-Leu-Asp-(octyl)Gly-Asp-NH₂ (SEQ ID NO: l) having the structure (1) below and comprise any suitable functional group. One or more of the amino acids can be an N-methyl amino acid. The functionalized beta-sheet peptide may, for example, be any suitable beta-sheet peptide of any suitable length, e.g. between eight and fifteen amino acids including any value therebetween, e.g. nine, ten, eleven, twelve, thirteen or fourteen amino acids, or longer, having alternating hydrophobic and hydrophilic residues.

(1)

[0031] In alternative example embodiments, the functionalized beta-sheet peptide could have any suitable sequence having alternating polar and apolar residues and a hydrophobic moiety at each end. The N-methyl amino acid of structure (1), which is provided on the second leucine in the illustrated embodiment, could be placed in different locations, combined in any desired number, or omitted in altemative embodiments. In some embodiments, the amino acid used at a particular location could be substituted by a different naturally-occurring amino acid or a modified amino acid that has similar properties. For example, in some embodiments, the functionalized beta-sheet peptide could have a sequence comprising from N-terminal to C- terminal any one of the amino acids listed in column 1 of Table 1 below at the first position, any one of the amino acids listed in column 2 of Table 1 below at the second position, and so on, to provide a beta-sheet peptide having alternating polar and apolar residues and a hydrophobic moiety at or near each end of the peptide. In some embodiments, additional amino acids could be present at either the C-terminal or N-terminal end of the beta-sheet peptide.

Table 1. Potential amino acid sequences for construction of functionalized beta-sheet peptides.

[0032] The functionalized beta-sheet peptides can have any suitable functional group at any suitable location, which can be an azide, an alkyne, an alkene, a vinyl, an azidophenyl, or a thiol in some embodiments. In some embodiments, the functional group is any suitable light- inducible functional group, for example, a lactone (e.g. propiolactone), epoxide, vinyl or allyl functional group. In some embodiments, the light-inducible functional group is a group capable of participating in a light-induced click reaction, for example as described in Tasdelen and Yagci, Angew. Chem. Int. Ed. 2013, 52, 5930-5938, which is incorporated by reference herein in its entirety. Such light-inducible functional groups include azides (for reaction with alkynes under UV or visible light or cyclopropenone under UV light), alkynes (e.g. for participation in copper(I)-catalyzed azide-alkyne cycloaddition, for reaction with cyclooctynes or thiols under UV or visible light), thiols (e.g. for participation in thiol-alkene/alkyne reactions under UV or visible light), or aldehydes (e.g. for participation in light-induced oxime reactions with an alkoxy amine or reaction with alkenes under UV light).

[0033] The functionalized beta-sheet peptides can be provided with a bifunctional crosslinker, which can contain a photo-reactive functional group. In some embodiments, the functional group may be attached to a C-terminal end of the beta-sheet peptide, or to an N-terminal end of the beta-sheet peptide. In other embodiments the functional group may be attached to a moiety located between the ends of the peptide, or e.g. at position 2, position 4 or position 6 of the exemplary potential amino acid sequences listed in Table 1. In some embodiments multiple functional groups may be attached to the functionalized beta-sheet peptide.

[0034] In some embodiments, a modified or non-naturally occurring amino acid is added at either or both of the N-terminus or the C-terminus of the peptide. In such embodiments, the functionalized beta-sheet peptide has a sequence that is longer than eight amino acids, for example nine amino acids or ten amino acids, or more. For example, in one example embodiment, a propargyl glycine (commercially available) is added to the C-terminus of a beta- sheet peptide having the sequence of SEQ ID NO: 1 via conventional peptide synthesis methods to make a 9 amino-acid sequence having the structure (2) with the peptide sequence acetyl- (octyl)Gly-Ser-Leu-Ser-Leu-Asp-(octyl)Gly-Asp-(propargyl)Gly-NH₂ (SEQ ID NO:2) having an alkyne light-inducible functional group provided by the propargyl glycine. The N-methyl amino acid of structure (2) is provided on the second leucine within the sequence, but could be provided on any desired amino acid residue or omitted in alternative embodiments. In alternative embodiments, more than one N-methyl amino acid could be present.

Similarly, glycine derivatives and other modified amino acids such as allylglycine,

cyclopropylglycine, azido-pentanoic amino acid, and the like could be incorporated using the same strategy.

[0035] In some embodiments, a modified or non-naturally occurring amino acid is inserted within the peptide sequence. In some such embodiments, the modified or non-naturally occurring amino acid is propargyl glycine, allylglycine, cyclopropylglycine, azido-pentanoic amino acid, octenyl glycine, or the like. Other amino acids besides glycine could be modified and inserted into the functionalized beta-sheet peptide in altemative embodiments. In some such embodiments, one or more additional amino acids may be added to either or both of the N- terminus or the C-terminus of the peptide. In some such embodiments, the one or more additional amino acids is added to maintain the alternating polar residue— non-polar residue sequence of the peptide. In such embodiments, the functionalized beta-sheet peptide has a sequence that is longer than eight amino acids, for example ten amino acids or more, due to the insertion of at least two additional amino acids into the peptide sequence.

[0036] In another example embodiment, the beta-sheet peptide comprises an azide end- functionalized beta-sheet peptide wherein the azide group comprises a light-inducible functional group. In one such example embodiment, the beta-sheet peptide comprises an azide end- functionalized beta-sheet peptide having the structure (3). Structure (3) has the general peptide sequence acetyl-azidohomoalanine-(octyl)Gly-Ser-Leu-Ser-Leu-Asp-(octyl)Gly-Asp-NH₂ (SEQ ID NO:3). Structure (3) comprises nine amino acids. The N-terminal amino acid of structure (3) is an exemplary modified amino acid, azidohomoalanine, which can be synthesized according to reported procedures. In structure (3), the N-methyl group is provided on the second leucine. In alternative embodiments, additional N-methyl groups could be provided within the peptide, or the N-methyl group(s) could be provided on amino acids other than the second leucine, or the N- methyl groups could be omitted altogether. Structure (3) can be photopolymerized with alkyne monomers via the addition of a copper catalyst, or using photo-induced click chemistry. In alternative embodiments, the azide light-inducible functional group in structure (3) can be replaced with any desired light-inducible functional group.

(3)

[0037] In another example embodiment, the functionalized beta-sheet peptide comprises an alkyne end-functionalized beta-sheet peptide having an alkyne light-inducible functional group, which can be photopolymerized with thiol groups. In one such example embodiment, the functionalized beta-sheet peptide comprises an alkyne end-functionalized beta-sheet peptide having the structure (4), wherein the alkyne end functionality is provided at the N-terminus of the peptide. Structure (4) has the general peptide sequence acetyl-(propargyl)Gly-(octyl)Gly- Ser-Leu-Ser-Leu-Asp-(octyl)Gly-Asp-NH₂ (SEQ ID NO:4). Structure (4) comprises nine amino acids. In structure (4), the N-methyl group is provided on the second leucine. In alternative embodiments, additional N-methyl groups could be provided within the peptide, or the N-methyl group(s) could be provided on amino acids other than the second leucine, or the N-methyl groups could be omitted altogether. In alternative embodiments, the alkyne functional group could be replaced with any suitable light-inducible functional group.

(4)

[0038] In another example embodiment, the functionalized beta-sheet peptide comprises an alkene mid-functionalized beta-sheet peptide having an alkene light-inducible functional group. In one such example embodiment, the functionalized beta-sheet peptide comprises an alkene mid-functionalized beta-sheet peptide having the structure (5). Structure (5) has the general peptide sequence acetyl-(octyl)Gly-Ser-Leu-Ser-amino hexenoic acid-Leu-Asp-(octyl)Gly-Asp- Gly-Leu-NH₂ (SEQ ID NO:5). Structure (5) comprises 11 amino acids. In structure (5), the N- methyl group is provided on the second leucine. In alternative embodiments, additional N- methyl groups could be provided within the peptide, or the N-methyl group(s) could be provided on amino acids other than the second leucine, or the N-methyl groups could be omitted altogether. In alternative embodiments, the alkene functional group could be provided on any other residue of the beta-sheet peptide, and could be replaced with any suitable light-inducible functional gro

(5)

[0039] In alternative embodiments, an alkyne mid-functionalized beta-sheet peptide having an alkyne light-inducible functional group is provided by substituting the vinyl-modified amino acid used to provide the alkene mid-functionalized beta-sheet peptide with a corresponding propargyl-modified amino acid. Thus, any suitable functional group can be positioned at any desired location within the functionalized beta-sheet peptide by incorporating the

correspondingly modified amino acid at the desired location within the peptide sequence.

[0040] In some embodiments, the functional group is provided at the N-terminus of the functionalized beta-sheet peptide. In one example embodiment, the functional group is provided on the acetyl group of the peptide, for example a peptide having the sequence of SEQ ID NO: 1, such that the peptide has the general structure (6):

(6)

wherein Ri can be any suitable light-inducible functional group, including for example an allyl, vinyl, lactone, thiol, epoxide, amino, azide, alkyne, alkene, or aldehyde functional group, or the like.

[0041] In some embodiments, the functional group is provided at the C-terminus of the peptide. In one example embodiment, the functional group is provided at the C-terminus of a peptide having SEQ ID NO: 1, such that the peptide has the general structure (7):

(7)

wherein R₂ can be any suitable light-inducible functional group, including for example an allyl, vinyl, lactone, thiol, epoxide, amino, azide, alkyne, alkene, or aldehyde functional group, or the like.

[0042] In some embodiments, the protein is crosslinked directly to a matrix that forms part of the functional construct (i.e. without linkage indirectly through another molecule such as a beta- sheet peptide or amphipol). For example, where the protein is not a membrane protein, the protein may be stable in aqueous solution, and need not be stabilized by using, for example, beta- sheet peptides, amphipols, or the like. Such proteins can be crosslinked directly to the matrix of the functional construct in any suitable manner, including by photo-activated coupling of an appropriate light-inducible functional group provided on the protein with suitable monomers that are polymerized to form the matrix of the functional construct.

[0043] In some embodiments, a hydrogel is used to provide the matrix that supports or forms the three-dimensional construct. Examples of materials that can be used to make suitable hydrogels that can be used for this purpose include acrylic polymers, epoxy and amine functional polymers, thiols and lactones. In some embodiments, the polymer used is a synthetic polymer. In some embodiments, the polymer used is a natural polymer.

[0044] In some embodiments, a plurality of different cross-linking strategies are employed in order to obtain the desired spatial positioning of various components of the construct. In some embodiments, the curing wavelength of different components used in the construct (i.e. the wavelength of light required to cross-link that component to the matrix of the construct) is different. For example, in some embodiments, curing is carried out using blue light having a wavelength of approximately 405 nm, green light having a wavelength of approximately 525 nm, and/or red light having a wavelength of approximately 635 nm. By providing appropriate light- inducible functional groups on each one of the components to be incorporated into the construct, the spatial position of such components within the construct can be controlled, because the spatial position at which the light inducible functional groups will react to form a covalent linkage with the functional construct is limited to regions exposed to light of the appropriate wavelength and power. It will be obvious to one skilled in the art that the wavelength of light used need not be exactly the specified wavelength, and that a variety of different wavelengths could be used. In some embodiments, curing is carried out using blue light having a wavelength in the range of 365 nm to 445 nm, including any value therebetween, e.g. 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430 or 440 nm. In some embodiments, curing is carried out using green light having a wavelength in the range of 485 nm to 565 nm, including any value therebetween, e.g. 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555 or 560 nm. In some embodiments, curing is carried out using red light having a wavelength in the range of 595 nm to 675 nm, including any value therebetween, e.g. 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665 or 670 nm. In some embodiments, curing is carried out using ultraviolet light, e.g. having a wavelength in the range of 240-400 nm or any value therebetween, e.g. 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380 or 390 nm. In some embodiments, curing is carried out using any suitable wavelength of light to polymerize the cross-linkable monomers and light-inducible functional groups used to provide a layer of a functional construct.

[0045] In some embodiments, a plurality of different layers of biological materials are formed to provide a functional construct. In some embodiments, each one of the plurality of different layers of biological materials contains a different biological material. Some or all of the plurality of different layers of biological materials can contain two or more different biological materials. For example, in one example embodiment, a functional construct is made with three different proteins. Each one of the proteins is immobilized using a different crosslinking agent having a unique wavelength of light required to crosslink that crosslinking agent and thereby immobilize the protein. In some such embodiments, a crosslinking agent that can be crosslinked using blue light is used to immobilize the first protein; a crosslinking agent that can be crosslinked using green light is used to immobilize the second protein; and a crosslinking agent that can be crosslinked using red light is used to immobilize the third protein. In this way, each of the first, second and third protein layers can be immobilized using a unique wavelength of light, so that each portion of the construct can be cured independently. In some embodiments, the order in which the different colours of light are used is varied. In this way, a custom scaffold can be built to support and immobilize any desired protein, including a membrane protein stabilized by an appropriate membrane protein stabilization agent having an appropriate light-inducible functional group. Scaffolds built in a similar manner may have other uses beyond the stabilization of functional proteins intended to carry out specific tasks.

[0046] In one example embodiment, a functional construct is made with four different proteins, i.e. first, second, third and fourth proteins. The functional construct has the first and second proteins in a first hydrogel layer of the functional construct; the third protein in a second hydrogel layer of the functional construct; and the fourth protein in a third hydrogel layer of the functional construct. Each one of the proteins is provided with a light-inducible functional group, which can be directly on the protein itself or on a stabilizing agent associated with the protein (for example, a functionalized beta-sheet peptide). The first and second proteins are provided with the same light-inducible functional group, and are reacted with first suitable monomers under conditions appropriate to both cross-link the first suitable monomers to form a first hydrogel layer and to react the light-inducible functional group with the hydrogel layer and/or the first suitable monomers, to immobilize the first and second proteins in the first hydrogel layer. The first hydrogel layer is formed using light having a first wavelength, for example, red light. The third protein is provided with a different light-inducible functional group and is reacted with second suitable monomers (which may differ from the suitable monomers used to provide the first hydrogel layer) under conditions appropriate to both cross-link the second suitable monomers and immobilize the third protein in the second hydrogel layer. The second hydrogel layer is formed using light having a second wavelength, for example green light. The fourth protein is provided with a different light-inducible functional group and is reacted with third suitable monomers (which may differ from the suitable monomers used to form the first and second hydrogel layer) under conditions appropriate to both cross-link the third suitable monomers and immobilize the fourth protein in the third hydrogel layer. The third hydrogel layer is formed using light having a third wavelength, for example, blue light. The fact that the first, second and third light-inducible functional groups all react under different conditions (i.e. different wavelengths of applied light) can help to ensure that each one of the proteins is crosslinked only in the appropriate hydrogel layer and at the appropriate position within the functional construct. The foregoing method can be varied appropriately to incorporate any desired number of molecules and layers into the functional construct.

[0047] In some example embodiments, other elements such as catalysts (whether organic or inorganic) or other non-biological molecules are also incorporated into the functional construct. In some embodiments, the catalysts or other non-biological molecules can be physically entrained within the crosslinked polymeric matrix of the functional construct. In some embodiments, the catalysts or other non-biological molecules can be functionalized to be covalently linkable to the suitable monomers that are polymerized to form a hydrogel matrix, or to the hydrogel matrix itself. For example, in the example embodiment characterized by the inventors, silver nanoparticles were physically entrained within the hydrogel matrix of the functional construct to act as an inorganic catalyst to catalyze the formation of hydrogen gas (H₂) from protons (H⁺). Glassy carbon was functionalized with a vinyl functional group to allow it to be crosslinked to the hydrogel matrix of the functional construct, and was used as an electrode to evaluate the development of hydrogen gas by the functional construct. In some embodiments, carbon could be incorporated into the functional construct as an electrode for use in a specific application, e.g. for use as an electrode in a hydrogen fuel cell.

[0048] In one example embodiment, the biological material is a protein. An example embodiment of a method 30 for producing a functional construct using a membrane protein is illustrated with reference to Figure 1. At step 32, the protein to be used in making the construct is obtained in any suitable manner. For example, molecular biology techniques can be used to provide a nucleotide construct (e.g. a plasmid) from which the protein can be expressed in a suitable organism (e.g. a suitable strain of Escherichia coli). The protein can be expressed, obtained and purified using appropriate techniques to provide a purified protein suitable for incorporation into a functional construct.

[0049] At step 34, in the case of a membrane protein, the membrane protein is stabilized in any suitable manner. For example, the membrane protein can be stabilized using membrane protein stabilizing agents such as functionalized beta-sheet peptides, as described in PCT publication No. WO 2016/029308, or using functionalized amphipols. In some embodiments in which the membrane protein stabilizing agents are functionalized beta-sheet peptides, the membrane protein is wrapped with a plurality of the functionalized beta-sheet peptides to provide a membrane-protein-functionalized beta-sheet peptide complex at step 34.

[0050] In some embodiments, the membrane protein stabilizing agent that is used to stabilize the membrane protein includes a functional group that allows for cross-linking of the stabilizing agent, for example a light-inducible functional group. In alternative embodiments, the membrane protein or other groups on the membrane protein stabilizing agent could subsequently be functionalized (i.e. after formation of the membrane protein-stabilizing agent complex) to allow for cross-linking of the membrane protein and/or the stabilizing agent, e.g. via a light- inducible functional group.

[0051] In some embodiments where the protein to be incorporated into the construct is not a membrane protein or where such membrane protein can be stabilized without the use of stabilizing agents such as beta-sheet peptides or amphipols, step 34 is omitted. In some such embodiments, the protein is engineered to contain a functional group that allows crosslinking of the protein to the matrix of the construct, for example an amino group (-NH₂), a thiol group (- SH), an allyl group (-C=C), a lactone group, or an epoxide group (as shown below) on the protein can be used to crosslink the protein to the matrix of the construct. Any suitable functional group that can react with the suitable monomers that are polymerized to form the matrix, or with the matrix of the functional construct, could be used to crosslink the protein to the matrix of the construct.

Allyl group Lactone group Thiol group Epoxide group

Amino group

[0052] At step 36, depending on the protein being incorporated into the construct, functional monomers or oligomers of the protein are synthesized and purified. For example, in some embodiments, a desired protein is recombinantly expressed in a suitable host and purified using molecular biology techniques, as are known to those skilled in the art.

[0053] In embodiments in which more than one protein is to be incorporated into the functional construct, steps 32 to 36 are repeated as necessary for each protein that is to be incorporated into the functional construct.

[0054] At step 38, the various components that are to be incorporated into the functional construct are combined. In some embodiments, the components are combined in ratios intended to optimize the functionality of the final construct. Optimal ratios of components to be incorporated into a construct can be determined empirically by one skilled in the art.

[0055] In some embodiments, photo-initiators are added at step 38. Examples of photo-initiators that can be used include 4'-phenoxyaceophenone, 3 -hydroxy benzophenone, 2,2' dimethoxy-2- phenylacetophenone, diphenyl(2,4,6- trimethylbenzoyl) phosphine oxide, anisoin, Irgacure™, and the like, although any suitable photoinitiator can be used. In some embodiments, photo- initiators are not added during the process of preparing the construct.

[0056] At step 40, light curing of the construct is carried out using a 3D printer. In some embodiments, the 3D printer is used to supply light to crosslink components of a mixture together (including in some embodiments both the monomers used for polymerization to produce the hydrogel and the stabilized protein component) and stabilize the orientation/spatial position of the various components of the construct in the resultant crosslinked hydrogel matrix. In some embodiments, the 3D printer is used to produce a finished product, i.e. functional construct, having a specific desired shape and size (for example, a component sized to be inserted into a particular apparatus) from such a mixture. In some embodiments, for example as may be used in microfiuidic applications, the 3D printer is used to control the spatial deposition of various components used to make the overall construct.

[0057] In some embodiments, the wavelength of light applied by the 3D printer is spatially controlled so that only one of the biological materials is immobilized at a given location within the functional construct. For example, if a functional construct is to include three different proteins, the first protein can be stabilized by a first protein stabilizing agent comprising a first light-inducible functional group that can be polymerized by blue light. The second protein can be stabilized by a second protein stabilizing agent comprising a second, different, light-inducible functional group that can be polymerized by green light. And the third protein can be stabilized by a third protein stabilizing agent comprising a third, different, light-inducible functional group that can be polymerized by red light. In some embodiments, the proteins are membrane proteins and the protein stabilizing agents are membrane protein stabilizing agents, e.g. functionalized amphipols or functionalized beta-sheet peptides.

[0058] Any suitable polymerization process can be used to prepare the functional construct by cross-linking to form a hydrogel matrix. In one example embodiment, the polymerization process is a ring-opening polymerization initiated by an acid-generating photo initiator. In one such example embodiment, the acid-generating photo initiator is Irgacure™ PAG 290

(tetrakis(2,3,4,5,6-pentafluorophenyl)borate; tris[4-(4-acetylphenyl)sulfanyl phenyl] sulfonium) having the structure (8):

(8)

and the monomer used for polymerization is a lactone, for example, propiolactone having the structure (9):

(9)

When exposed to blue light having a wavelength of approximately 405 nm, the acid-generating photo initiator generates acid, and the acid so formed initiates the polymerization by ring- opening of the lactone monomer, e.g. the propiolactone monomer, as shown in Scheme 1, to produce a polymer having the general structure (10) that provides the hydrogel matrix of the construct.

Scheme 1. Acid-catalyzed ring-opening to polymerize propiolactone monomer.

(9) (10)

[0059] In some such embodiments where the membrane protein is stabilized by a functionalized beta-sheet peptide, the functionalized beta-sheet peptide is designed to include a lactone functional group, e.g. a propiolactone group, attached to a side chain of one or more of the amino acids comprising the functionalized beta-sheet peptide, as the light-inducible functional group, for example a functionalized beta-sheet peptide having the general structure (11) below having the sequence acetyl-(octyl)Gly-Ser-Leu-Ser-Xaa-Leu-Asp-Ser-(octyl)Gly-NH2 (SEQ ID NO:6), in which Xaa is a custom amino acid having a propiolactone functional group. Thus, the application of blue light having a wavelength of approximately 405 nm will result in cross- linking of the functionalized beta-sheet peptide to the polypropiolactone (or polylactone) polymer formed from the polymerized propiolactone monomer, to yield a cross-linked bio- functional layer. The lactone functional group has an activation energy of approximately 80 kJ/mol to initiate polymerization.

(11)

[0060] Figure 2 shows schematically an example embodiment of the polymeric cross-linked functional layer produced using this ring-opening polymerization process. A membrane protein, illustrated schematically as 50, is stabilized by a plurality of beta-sheet peptides, illustrated schematically as 52, to provide a membrane protein-functionalized beta-sheet peptide (MP-FBP) complex, shown schematically as 54. The application of blue light having a wavelength of approximately 405 nm (shown schematically as 55) in the presence of an acid-generating photo initiator, for example having the structure (8), causes acid catalyzed ring-opening polymerization to polymerize the propiolactone monomer having the structure (9) and the functionalized beta- sheet peptides (via the light-inducible functional group, i.e. lactone group) to yield a polylactone polymeric complex 56 comprising the polymer (10) covalently j oined to the MP-FBP complex 54

[0061] In another example embodiment, the polymerization process is an epoxy amine crosslinking of oligomers using green light having a wavelength of approximately 525 nm. In this example embodiment, epoxy functionalized oligomers having the general structure (12) are crosslinked with amine functionalized oligomers having the general structure (13) to yield a polylactone polymer having the general structure (14) using green light as a catalyst according to Scheme 2. In this example embodiment, a functionalized beta-sheet peptide having an epoxide functional group as the light-inducible functional group, for example having the general structure (15) having SEQ ID NO:6 in which Xaa is a custom amino acid with an epoxide functional group could be used. The structures (12), (13) and (14) are not intended to be limiting but are drawn only to show polymers with amine and epoxy functional groups. In some such embodiments, the functionalized beta-sheet peptides are provided with epoxide functional groups as the light-inducible functional group in order to allow the functionalized beta-sheet peptides to be crosslinked together with the resulting polymer, e.g. via free amino groups of the amine monomers.

[0062] The mechanism by which this crosslinking proceeds is a nucleophilic ring-opening of the epoxy functional group by the free amine groups of the amine-functionalized oligomers (epoxy-amine reaction), as shown in Scheme 3. Similarly, free amino groups on the beta-sheet peptides, whether on the side chain of amino acids forming the beta-sheet peptides or at a free end of the beta-sheet peptides can react with the epoxide functional groups to cross-link the beta- sheet peptides (and therefore the stabilized membrane protein) to the hydrogel produced by the cross-linking reaction. In some embodiments, no photoinitiator is used to initiate this reaction (i.e. the energy from light irradiation is sufficient to initiate the cross-linking reaction).

Scheme 3. Nucleophilic ring-opening of epoxy functional group by free amine group.

[0063] Figure 3 shows schematically an example embodiment of the polymeric cross-linked functional layer produced using this polymerization process. A membrane protein, illustrated schematically as 150, is stabilized by a plurality of beta-sheet peptides, illustrated schematically as 152, to provide a membrane protein-functionalized beta-sheet peptide (MP-FBP) complex, shown schematically as 154. The application of green light having a wavelength of

approximately 525 nm (shown schematically as 155) in the presence of an epoxy functionahzed oligomer, for example having the general structure (12), causes ring-opening polymerization to polymerize the amine functionahzed oligomers having the general structure (13) and the functionahzed beta-sheet peptides to yield a polymeric complex 156 comprising the resulting polymer (14) covalently joined to the MP-FBP complex 154.

[0064] In another example embodiment, the polymerization process is a free radical

polymerization of acrylic monomers as shown in Scheme 4 using a photoinitiator and red light. In one example embodiment, the monomers are hydroxy ethyl acrylate (16) and tripropylene glycol diacrylate (17), but other suitable monomers could be used. In one example embodiment, the photoinitiator is 2,4,5,7-tetraiodo-3-hydroxy-6-fluorone (18), although any suitable photoinitiator could be used. The polymerization produces a polymeric hydrogel having the general structure (19). In one example embodiment, the light has a wavelength of approximately 635 nm. The mechanism of this exemplary crosslinking reaction is shown in Scheme 5. The allyl polymerization reaction has an activation energy of approximately 65 kJ/mol.

Scheme 4. Crosslinking of hydroxy ethyl acrylate and tripropylene glycol diacrylate monomers.

(17) (16) (18) (19) Scheme 5. Mechanism of free radical polymerization of acrylic monomers.

[0065] In this example embodiment, the functionahzed beta-sheet peptides used to stabilize a membrane protein would have a vinyl or allyl functional group as the light-inducible functional group. For example, in some embodiments, functionalized beta-sheet peptides having the structure (20) having SEQ ID NO: 6 where Xaa is a custom amino acid with an allyl functional group could be used.

(20)

[0066] Figure 4 shows schematically an example embodiment of the polymeric cross-linked functional layer produced using this polymerization process. A membrane protein, illustrated schematically as 250, is stabilized by a plurality of beta-sheet peptides, illustrated schematically as 252, to provide a membrane protein-functionalized beta-sheet peptide (MP-FBP) complex, shown schematically as 254. The application of red light having a wavelength of approximately 635 nm (shown schematically as 255) causes free radical polymerization of the acrylic monomers and the allyl side chains of the functionalized beta-sheet peptides to yield a polymeric complex 256 comprising the resulting polymer covalently j oined to the MP-FBP complex 254 via the light inducible allyl functional group.

[0067] Figure 5 shows schematically an example embodiment of a functionalized beta-sheet stabilized membrane protein immobilized in a hydrogel produced by crosslinking of suitable monomers. The construct of Figure 5 could be obtained using any of the three different methods of crosslinking described above, or any other suitable method of crosslinking to form a hydrogel linked to or associated with the membrane protein-stabilizing units, for example, the

functionalized beta-sheet peptides. Generally, the membrane protein 50 is immobilized within the cross-linked hydrogel matrix as part of a membrane-protein-functionalized beta-sheet peptide complex 54 to yield the polymeric complex 56.

[0068] In some embodiments, a 3D printer is used to produce a functional construct by using one, two or all three of the exemplary methods of immobilizing a protein in a hydrogel layer described above, or any suitable combination of methods of immobilizing the protein in the hydrogel layer while providing control over the location at which the protein is immobilized. In one example embodiment, photopolymerization in a 3D printer is based on stereolithographic techniques, for example projection micro stereolithography, in which a digital micromirror device incorporated into the optical path of the laser. This allows a layer of polymerized hydrogel/immobilized proteins to be built out of solution containing the monomers and the stabilized proteins. Additionally, the localized generation of radicals from photoinitiators can be adjusted not only by modifying the wavelength of light used, but also by adjusting parameters such as laser power, focal spot size, and depth of focus to allow control over the conversion of liquid monomer/protein solution into a solid via photo-polymerization. In some embodiments, this technique can provide a lateral resolution of up to 1 micrometre. In some embodiments, oxygen concentration can also be controlled to restrict polymerization to occur only around the area of focus of the laser beam of the 3D printer. In some embodiments, the difference in activation energy between the coupling reactions used to couple a first light-inducible functional group associated with a first protein and the coupling reactions used to couple a second light- inducible functional group associated with a second protein can be exploited to help regulate the initiation of polymerization as a functional construct is being produced. Thus, the spatial position of the protein within the functional construct can be controlled with precision.

[0069] In some embodiments, microfluidics techniques are used and the 3D printed construct is configured so that a product is produced at a first location on a microfluidic chip or other structure using a first biological material printed on the microfluidic chip using 3D printing, and that product is then passed to a second location on the microfluidic chip or other structure as a reactant for use by a second biological material printed on the microfluidic chip or other structure printed using 3D printing. In some such embodiments, a metabolic cascade incorporating a plurality of such biological materials could be set up on a microfluidic chip or other structure that mimics a naturally occurring metabolic pathway, for example a metabolic pathway found in humans. Such metabolic cascades have potential utility, for example, in testing potential drug candidates for adverse interactions in an in vitro setting. For example, where a drug candidate supplied to the metabolic cascade interferes with one or more steps in the cascade, it may be concluded that the drug candidate may cause side effects when used in vivo. In some cases, the drug candidate might be modified and the modified drug candidate passed through the same in vitro metabolic cascade, to examine how specific modifications to the drug candidate might be used to minimize potentially negative interactions.

[0070] In one example embodiment, photosynthesis is used as a model system for efficient solar energy production. A bio-inspired artificial photosynthesis system was created, and has the potential for use in low carbon energy production. In one example application, the inventors have used advanced three-dimensional (3D) printing to create bio-inspired hybrid nanocomposite electrodes for use in a water splitting application to produce hydrogen. A hybrid nano-bio hydrogel electrode was created using glassy carbon, bacteriorhodopsin (a proton-pumping protein), water-soluble polymers and metal nanoparticles (silver). The electrode was successfully prepared by 3D printing and exploited for direct photo electrochemical (PEC) water splitting. The 3D printed electrode displayed excellent catalytic performance, good stability, and enhanced hydrogen generation activity under light irradiation.

[0071] The experimental results described herein demonstrate that a functional bioink incorporating a stabilized membrane protein can be printed using 3D printing techniques, while still retaining the functionality of the membrane protein. The functional bioink can be printed to have any desired structure (for example, the shape of a specific logo as shown in Figure 8) while still retaining protein function, to produce functional constructs for any desired application. From the experimental results described below, it can be soundly predicted that two or more biological molecules, including two or more proteins, could be incorporated into a functional construct with each 3D printed biological molecule retaining its function, and that the biological molecules, including proteins, as well as other components of the functional construct such as inorganic catalysts or other non-biological molecules, can be spatially separated and deposited in specific locations within a functional construct as may be necessary to ensure the proper functioning of the functional construct.

[0072] With 3D printing techniques, it is possible to manufacture nano/microscale complex structures that are not feasible to produce by other manufacturing techniques. Based on the results described herein, it may be possible to design and create novel hybrid electrodes with improved activity and durability for use in areas such as solar cells, miniature batteries, and supercapacitors.

[0073] The same general principles as are demonstrated herein, i.e. immobilization of a protein within a hydrogel layer, optionally in the presence of one or more co-catalysts, including organic or inorganic cocatalysts, can be applied to other proteins to produce other 4D printed constructs for carrying out specific functions. For example, ATPases could be immobilized within a hydrogel layer and used to synthesize ATP, or glucosidases could be immobilized within a hydrogel layer and be used to break down specific sugars.

[0074] Certain embodiments of the present invention are further described with reference to the following examples, which are intended to be illustrative and not restrictive in nature.

Examples

Example 1.0 - Synthesis of Composite 3D Printed Electrode

[0075] Polymer hydrogels were prepared based on custom synthesized resins using methacrylic acids and lactones. The polymer hydrogels were utilized to crosslink an allyl beta-sheet peptide wrapped membrane proteins (stabilized bacteriorhodopsin, a membrane protein that functions as a proton pump). The polymer hydrogels also allow the incorporation of a carbon electrode and silver catalyst for efficient conversion of H⁺ to H₂ and characterizations. In this example embodiment, the substrate for the electrode was micron-sized particles of glassy carbon prefunctionalized with an allyl functional group to allow crosslinking of the polymer hydrogel to the substrate. The silver catalyst was mechanically incorporated into the construct as silver nanoparticles, which become physically entrained within the polymer hydrogel after crosslinking.

[0076] Generally, bacteriorhodopsin, a membrane protein that acts as a proton pump, was obtained, wrapped with a functionalized beta-sheet peptide containing a vinyl functional group, and crosslinked with a polymer containing a vinyl functional group using blue light. Silver nanoparticles were added to catalyze the formation of hydrogen, and glassy carbon was used as an electrode. The overall construct was shown to split water to form hydrogen (H₂) when exposed to light. Such a construct has potential use in applications such as fuel cells.

[0077] In more detail, recombinantly expressed bacteriorhodopsin (bR) in beta-D- decylmaltoside (DDM) was used for wrapping with the functionalized beta-sheet peptide. 2.69 μg of peptide (octenyl modified beta-peptide BP1 (1) having the structure (20) with an allyl light-inducible functional group) was mixed with the purified protein (3.5 μί) in 980 of 100 mM Tris (pH 7.5) buffer for 1 hour at 4 °C. Bacteriorhodopsin can be purchased commercially, for example, from Sigma- Aldrich. Then detergents were removed by dialysis against detergent- free buffer (6 kDa cutoff membrane), for two days at 4 °C. The removal of DDM was monitored by thin-layer chromatography. Complete detergent exchange of DDM to the functionalized beta- sheet peptide in bacteriorhodopsin samples was achieved in ~2 days. Stability of

bacteriorhodopsin after peptide wrapping was verified by circular dichroism (CD) spectroscopy.

[0078] Figure 6 shows the mass spectrum of the cross-linkable peptide octenyl BP1 having the structure (20), confirming that this is the desired functionalized beta-sheet peptide construct. Scheme 6 shows the crosslinking reaction used to prepare the exemplary functional construct.

Scheme 6. Crosslinking reaction used to prepare exemplary electrode construct.

[0079] Hydrogels were prepared using light-induced cross-linking of monomers in a 3D printer using light having a wavelength of 405 nm (i.e. blue light) to initiate crosslinking. Thermal 3D printers that use heat to initiate crosslinking could be used only where the level of heat produced in the crosslinking process will not harm the biomaterial to be incorporated into the construct (e.g. heat can denature proteins). Freshly prepared hydroxy ethyl methacrylate monomer (60 wt%), di(ethylene glycol) dimethacrylate cross-linker (3.1 wt%), high-viscosity grade hydroxy ethyl cellulose (16 wt%), photo-initiator (0.9 wt% Irgacure™ 1700, which is a mixture of 25 % bis(2,6-dimethoxybenzoyl)-2,4,4-trimethyl pentylphosphineoxide and 75 % 2-hydroxy- 2-methyl-l-phenyl-propan-l-one), bacteriorhodopsin wrapped with cross-linkable functionalized beta-sheet peptides having structure (20) (0.9 wt%), and glassy carbon powder (2-12 μηι size, 20 wt%) were mixed without any air bubbles and placed on an Autodesk 3D printer. A 22.5 mW/cm², continuous wave, diode laser emitting light at a wavelength of 405 nm was fitted in Autodesk 3D printer. The electrode shape is fabricated by Max® Design 2012 software version 14.0 (Autodesk, Inc., USA) and saved in STL format. The design was imported to the 3D printer's software, MakerWare Version 2.4.0.17 (MakerBot Industries, LLC, USA). During 3D printing, gelation was almost instantaneous upon irradiation. After the printing the electrode was washed thoroughly with water to remove any unreacted monomer and finally kept in 1 mM PBS buffer.

[0080] Without being bound by theory, it is believed that the construct described above functions as follows. Bacteriorhodopsin (bR), is a proton-pumping protein in the purple membrane of Halobacteriumsalinarum. This protein was selected for study due to its exceptional stability toward thermal, chemical, and photochemical degradation and its unique photoelectric and photochromic properties. Bacteriorhodopsin acts as a proton pump and electron transporter. In this particular construct, it is believed that bacteriorhodopsin acts as an active catalyst component when exposed to light for electrochemical hydrogen generation by the existence of synergistic effects between bacteriorhodopsin and silver nanoparticles. It is believed that bacteriorhodopsin provides its biological function of pumping protons to the H₂-evolving silver cocatalyst following light absorption, prompting the adsorption and formation of Ag-H on the silver nanoparticle surfaces, which accelerates the hydrogen generation process. For example, silver nanoparticles have previously been shown to be capable of generating hydrogen (see e.g. Yang et al., ACSApp. Mater. Interfaces 2013, 5, 8231-8240, which is incorporated by reference herein for all purposes). [0081] In alternative embodiments, carbon nanotubes could be used in place of glassy carbon. However, the inventors have found in preliminary testing that better results are achieved using glassy carbon under the tested conditions than using carbon nanotubes.

[0082] Preliminary results obtained from these studies are shown in the figures.

[0083] Figures 7 A, 7B, 7C and 7D show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. Figure 7A shows a TEM image of silver nanoparticles having a diameter of approximately 16 nm. Figure 7B shows an SEM image of glassy carbon showing the porous nature of this substrate at 10 μηι, with the inset showing the porous nature of this substrate at 3 μηι magnification. Figure 7C shows a cross-sectional SEM image of a portion of the final electrode construct with crosslinked hydrogel matrix, showing the uniformity of the structure obtained (i.e. there is an absence of visible defects in the cross-sectional structure) at 40 μηι magnification. Figure 7D shows a cross-sectional SEM image of the final electrode construct containing bacteriorhodopsin silver nanoparticles, and glassy carbon immobilized in the crosslinked hydrogel matrix at 2 μηι magnification. The generally even structure of the construct indicates an absence of aggregation of the various components of the construct, indicating that a generally even distribution of components within the construct was obtained by the 3D printing technique used.

[0084] Figure 8 shows a scanning electron microscopy (SEM image) of an example embodiment of a 3D printed glassy carbon/bacteriorhodopsin/silver electrode construct (glassy carbon/ bR/Ag NP electrode). In this example, the electrode is 3D printed to have the specific shape of a logo, iⁿgenuity. In alternative embodiments, the electrode can be 3D printed to have any desired shape, e.g. circular, tubular or other shape, as may be necessary for the electrode to be inserted into any given apparatus.

[0085] Figures 9A and 9B show CV and IV (current voltage) curves of the electrode

performance of the example embodiment described above. Figure 9A shows the results of cyclic voltametry (CV) experiments of the Ag/BR/glassy carbon hybrid electrode tested in a standard three-electrode system on a CHI 660E electrochemical workstation at room temperature. Pt and Ag/AgCl (KC1 saturated) were used as the counter electrode and reference electrode, respectively. A 1.0 M PBS aqueous solution of pH 7 was used as the electrolyte. The red colored peaks (labeled bR/Ag/C) represent the reduction and oxidation potential of Ag, as clearly demonstrated at a scan rate of 25 mV/s. For comparison, an electrode without Ag nanoparticles (black line, labelled Control) scanned as a control experiment.

[0086] Figure 9B shows polarization curves of the Ag/bR/Glassy Carbon hybrid electrode before and after irradiation of light (100 mW/cm², visible light) at pH 7 in 1.0 M PBS with a scan rate of 2 mV s^"1 at room temperature. The current density was normalized to the electrochemically active surface area (ECSA) of the electrode, which was obtained by CV experiment.

Photocurrent generated before irradiation is 605 μΑ cm ², while the current after irradiation is 815 μΑ c f ² at - 300 mV vs RHE (reversible hydrogen electrode). This result further indicates synergism between the silver nanoparticles and the bacteriorhodopsin, which enhances the hydrogen generation properties of the tested exemplary bio-hybrid electrode.

[0087] Figure 10 shows gas chromatography results for gas evolved from an exemplary

Ag/bR/glassy carbon hybrid electrode construct irradiated with light (100 mW/cm², visible light) at pH 7 in 1.0 M PBS at room temperature. The first large peak corresponds to H₂ generated by the electrode construct, while the second, smaller peak, corresponds to 0₂ generated by the electrode construct.

[0088] In commercial embodiments, a plurality of Ag/bR/glassy carbon hybrid electrodes could be used together to provide a sufficient current or power output, or to generate a sufficient amount of H₂, for a given application.

Example 2.0 - Synthesis of Custom Functionalized Beta-Sheet Peptides

[0089] A functionalized beta-sheet peptide having the structure (20) containing an octenyl light- inducible functional group was synthesized using commercially available octenyl alanine with Boc tert-butyloxycarbonyl) protecting group having the structure (21):

(21)

[0090] The resulting structure (20) can be used to make the corresponding functionalized beta- sheet peptide (15) bearing an epoxide light-inducible functional group via epoxidation using meta-chloro peroxy benzoic acid (mCPBA). Structure (15) can further be converted to the functionalized beta-sheet peptide (11) having a propiolactone light-inducible functional group by reaction with carbon monoxide at 60 atm in the presence of 2 mol % of cobalt carbonate catalyst.

[0091] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are not to be limited by the preferred embodiments described herein, but are to be interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.

Claims

WHAT IS CLAIMED IS:

1. A functional construct incorporating a biological material and produced by three- dimensional printing, wherein the biological material comprises a protein, DNA or RNA.

2. A method of making a functional construct incorporating a biological material, wherein the biological material optionally comprises a protein, comprising:

obtaining the biological material; and

immobilizing the biological material in a hydrogel matrix that forms part of the functional construct by cross-linking suitable monomers to form the hydrogel matrix and by cross-linking the biological material to the hydrogel matrix using a 3D printer.

3. A method as defined in claim 2, comprising controlling a spatial position of at least two proteins in the hydrogel matrix by using different wavelengths of light to selectively localize each one of the at least two proteins in the hydrogel matrix, wherein the different wavelengths of light are optionally selected from the group comprising blue light, green light and red light.

4. A method as defined any one of claims 2 or 3, wherein a photointiator is used to initiate cross-linking of the suitable monomers, wherein the photoinitiator optionally comprises 4'-phenoxyaceophenone, 3-hydroxy benzophenone, 2,2' dimethoxy-2- phenylacetophenone, diphenyl(2,4,6- trimethylbenzoyl) phosphine oxide, anisoin, 2,4,5,7-tetraiodo-3-hydroxy-6-fluorone, tetrakis(2,3,4,5,6-pentafluorophenyl)borate; tris[4-(4-acetylphenyl)sulfanyl phenyl] sulfonium, or Irgacure™ 1700 (a mixture of 25 % bis(2,6-dimethoxybenzoyl)-2,4,4-trimethyl pentylphosphineoxide and 75 % 2-hydroxy-2- methyl- 1 -phenyl-propan- 1 -one)).

5. A method as defined in claim 3 or claim 4, wherein the protein is engineered to contain a functional group to allow crosslinking of the protein to the hydrogel matrix, wherein the functional group optionally comprises a light-inducible functional group, and wherein the light-inducible functional group optionally comprises an allyl, vinyl, lactone, thiol, epoxide, amino, azide, alkyne, alkene or aldehyde functional group.

6. A method as defined in any one of claims 2 to 5, wherein the protein comprises a

membrane protein, and wherein the step of immobilizing the protein in the hydrogel matrix comprises:

stabilizing the membrane protein with a membrane protein stabilizing agent; and cross-linking the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix or with the hydrogel matrix.

7. A method as defined in claim 6, wherein the step of cross-linking the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix or with the hydrogel matrix comprises using light to react a light-inducible functional group on the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix, wherein the light-inducible functional group optionally comprises an allyl, vinyl, lactone, thiol, epoxide, amino, azide, alkyne, alkene, or aldehyde functional group.

8. A method as defined in claim 7, wherein the suitable monomers are cross-linked to form the hydrogel matrix concurrently with the membrane protein stabilizing agent being cross-linked to the suitable monomers.

9. A method as defined in any one of claims 6 to 8, wherein obtaining the membrane protein comprises expressing the membrane protein in a suitable host cell using molecular biology techniques and purifying the membrane protein.

10. A method as defined in any one of claims 6 to 9, wherein the membrane protein

stabilizing agent comprises functionalized beta-sheet peptides or functionalized amphipols.

11. A method as defined in claim 10, wherein the functionalized beta-sheet peptides

comprise a sequence containing, from N-terminal to C-terminal: at position 1 : octyl(Gly) or octyl(Ala);

at position 2: Ser, Thr or Cys;

at position 3: Leu, He or Val;

at position 4: Ser, Thr or Cys;

at position 5: Leu, He or Val;

at position 6: Asp, Asn, Glu, Gin or His;

at position 7: octyl(Gly) or octyl(Ala);

at position 8: Asp, Asn, Glu, Gin or His;

optionally comprising additional amino acids at either or both of the N-terminal and C- terminal ends of the functionalized beta-sheet peptides.

A method as defined in claim 10, wherein the functionalized beta-sheet peptides have of the following s

35

or

(IX)

wherein Ri and R₂ are independently a lactone group, an epoxide group, an azide group, an alkyne group, an alkene group, a thiol group, an aldehyde group, an allyl group, or a vinyl group.

13. A method as defined in any one of claims 6 to 12, wherein the functional group on the functionalized beta-sheet peptides comprises lactone, epoxide, vinyl or allyl.

14. A method as defined in any one of claims 6 to 13, wherein the step of cross-linking the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix comprises using blue light to selectively localize at least one membrane protein within the functional construct, wherein:

the blue light has a wavelength in the range of 365 to 445 nm, optionally approximately 405 nm;

the suitable monomers comprise a lactone, optionally propiolactone having the structure

the membrane protein stabilizing agent comprises a lactone functional group, optionally a propiolactone group; and

optionally a photoinitiator is used to initiate polymerization, wherein the photoinitator optionally has the structure

15. A method as defined in any one of claims 6 to 14, wherein the step of cross-linking the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix comprises using green light to selectively localize at least one biological molecule within the functional construct, wherein:

the green light has a wavelength in the range of 485 to 565 nm, optionally approximately 525 nm;

the suitable monomers comprise a combination of epoxy functionalized oligomers and amine functionalized oligomers, optionally having the following structures

the membrane protein stabilizing agent comprises an epoxide functional group; and optionally a photoinitiator is used to initiate the polymerization.

16. A method as defined in any one of claims 6 to 15, wherein the step of cross-linking the membrane protein stabilizing agent with the suitable monomers used to form the hydrogel matrix comprises using red light to selectively localize at least one biological molecule within the functional construct, wherein:

the red light has a wavelength in the range of 595 to 675 nm, optionally approximately 635 nm;

the suitable monomers comprise acrylic monomers, optionally having the following structures

the membrane protein stabilizing agent comprises a vinyl or allyl functional group; and

optionally a photoinitiator is used to initiate the polymerization, wherein optionally the photoinitiator comprises 2,4,5, 7-tetraiodo-3-hydroxy-6-fluorone.

17. A method as defined in any one of claims 6 to 16, wherein two or all of red light, blue light and green light are used to selectively localize at least two biological molecules to produce a functional construct.

18. A method as defined in any one of claims 6 to 16, wherein two or all of red light, blue light and green light are used to selectively localize at least two biological molecules to produce a functional construct, wherein the red light is applied according to the method defined in claim 16, the blue light is applied according to the method defined in claim 14, and the green light is applied according to the method defined in claim 15.

19. A method of producing a functional construct comprising first and second proteins, the method comprising:

using a method as defined in either one of claims 14 or 15 to provide a first layer of the functional construct incorporating the first protein; and

using a method as defined in claim 16 to provide a second layer of the functional construct incorporating the second protein,

wherein optionally the second layer is provided before the first layer.

20. A method of producing a functional construct comprising first and second proteins, the method comprising:

using a method as defined in either one of claims 14 or 16 to provide a first layer of the functional construct incorporating the first protein; and

using a method as defined in claim 15 to provide a second layer of the functional construct incorporating the second protein,

wherein optionally the second layer is provided before the first layer.

21. A method of producing a functional construct comprising first and second proteins, the method comprising:

using a method as defined in either one of claims 15 or 16 to provide a first layer of the functional construct incorporating the first protein; and

using a method as defined in claim 14 to provide a second layer of the functional construct incorporating the second protein,

wherein optionally the second layer is provided before the first layer.

22. A method as defined in any one of claims 2 to 21, further comprising incorporating an inorganic catalyst or other non-biological molecule into the hydrogel matrix.

23. A method as defined in claim 22, wherein incorporating an inorganic catalyst or other non-biological molecule into the hydrogel matrix comprises physically entraining the inorganic catalyst or non-biological molecule in the hydrogel matrix, or functionahzing the inorganic catalyst or non-biological molecule and then covalently coupling the inorganic catalyst or non-biological molecule to the hydrogel matrix.

24. A functional construct made by the method as defined in any one of claims 2 to 23.

25. A composite construct comprising a protein and a catalyst that is produced by three- dimensional printing.

26. A composite construct as defined in claim 25, wherein the protein comprises

bacteriorhodopsin and the catalyst comprises silver.

27. A composite construct as defined in claim 26, further comprising carbon, optionally in the form of glassy carbon or carbon nanotubes.

28. A method of producing hydrogen (H₂) by irradiating a construct as defined in any one of claims 24 to 27 with light.

29. A method as defined in claim 27, wherein the carbon acts as an electrode in a hydrogen fuel cell.

30. A functional construct for producing hydrogen (H₂) from water (H₂0), the functional construct comprising:

bacteriorhodopsin stabilized by a functionalized beta-sheet peptide and immobilized within a hydrogel matrix;

silver nanoparticles distributed within the hydrogel matrix; and glassy carbon or carbon nanotubes within the hydrogel matrix.

31. A functional construct as defined in claim 30, wherein the functionalized beta-sheet peptide comprises the following structure:

32. A functional construct as defined in either one of claims 30 or 31, wherein the hydrogel matrix comprises cross-linked acrylic monomers.

33. A functional construct made by the method of any one of the preceding claims

comprising a microfluidic chip configured for carrying out a plurality of sequential reactions, wherein the sequential reactions optionally comprise a metabolic cascade that is optionally used to screen potential drug candidates for potential toxicity.

34. A functional construct made by the method of any one of claims 1 to 22, wherein the biological material comprises an ATPase, wherein the functional construct is adapted to synthesize ATP.

35. A functional construct made by the method of any one of claims 1 to 22, wherein the biological material comprises at least one glycosidase, wherein the functional construct is adapted to break down one or more specific sugars that are substrates for the at least one glycosidase.