Method for controlled isotropic shrinking of hydrogel substrates ensuring NANO precision of internal 3D structures

Abstract

The invention relates, in part, to methods for three-dimensional nanofabrication. Certain aspects of the invention include methods for isotropically shrinking patterned hydrogels, dehydrating the hydrogels after shrinking, and fixing the dehydrated hydrogel resulting in highly stable micro and nanostructures in patterned hydrogels.

Description

[0001] METHOD FOR CONTROLLED ISOTROPIC SHRINKING OF HYDROGEL SUBSTRATES ENSURING NANO PRECISION OF INTERNAL 3D STRUCTURES

[0002] Related Applications

[0003] This application claims benefit under 35U.S.C.§119(e) of U.S. Provisional application serial number 63 / 660,009 filed June 14, 2024 the disclosure of which is incorporated by reference herein in its entirety.

[0004] Government Support

[0005] This invention was made with government support under R01EB024261 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] Field of the Invention

[0007] The invention relates, in part, to methods of nanofabricating hydrogel scaffolds.

[0008] Background of the Invention

[0009] Nanometer-scale accuracy and precision of 3D fabrication is important in that it unlocks unique material properties, enhances performance characteristics, and opens up new applications. Previous methods, for example Implosion Fabrication (ImpFab, U.S. Patent Application Publication No. 2017 / 0081489), the shrinking of the matrix hydrogel is necessary to achieve higher resolution in the fabricated 3D patterns within the matrix. Implosion Fabrication, as well as related works on shrinking and dehydrating hydrogels for optical fabrication to achieve 3D nanostructures with nanoscale precision, a critical issue of nano precision remained unresolved. With prior methods, the fabricated structures could not guarantee uniformly precise shrinking across all dimensions, leading to 3D structures with nanoscale deviations. Moreover, ensuring the entire matrix material remains flat during the dehydration process was challenging. Additionally, structures with high aspect ratios tended to collapse due to the softness of the matrix during the shrinking process. Furthermore, the stability of the micro and nanostructures in the post-fabrication material could not be guaranteed upon rehydration, posing numerous challenges for constructing multilayered, complex 3D micro and nano devices, and making stable functionalization difficult to achieve.

[0010] Summary of Elements of the Invention According to an aspect of the invention, a method for isotropic shrinking of a hydrogel is provided, the method including: (a) preparing an expandable hydrogel; (b) expanding the prepared hydrogel; (c) contacting the expanded hydrogel with a patterning solution; (d) contacting the contacted expanded hydrogel with a laser light, wherein the laser light contact patterns the expanded hydrogel; (e) isotropically shrinking and dehydrating the patterned expanded hydrogel; and (f) fixing the shrunk and dehydrated hydrogel. In some embodiments, the method also includes contacting the hydrogel with a stabilizing solution including isopropylamine prior to contacting the expanded hydrogel with a patterning solution. In some embodiments, the method also includes incubating the expanded hydrogel in a solution including one or more photosensitizers. In certain embodiments, the stabilizing solution also includes isopropylamine and / or hydrogen peroxide. In certain embodiments, the method also includes contacting the hydrogel with O2 gas for a period between 2 and 10 minutes. In some embodiments, the hydrogel is contacted with O2 gas for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes. In certain embodiments, the laser light contact includes three- dimensional laser light contact. In certain embodiments, the laser light is generated by a multiphoton laser. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the method also includes removing the patterning solution following the patterning and before the shrinking. In some embodiments, the method also includes washing the patterned hydrogel with a wash solution that includes isopropylamine. In certain embodiments, the wash solution also includes MgCh. In certain embodiments, the wash solution also includes Ca2+ions. In some embodiments, the isotropic shrinking includes chelation-induced shrinking and dehydration hardening of the patterned expanded hydrogel. In some embodiments, the chelation-induced shrinking of the patterned expanded hydrogel includes incubating the patterned hydrogel in one or a plurality of ionic solutions. In certain embodiments, the patterned hydrogel is incubated in a series of increasingly ionic solutions, wherein the increasingly ionic solutions have increasing chelation effects. In some embodiments, the shrinking includes incubating the patterned hydrogel in a first solution that includes isopropylamine and MgCh and a second solution that includes isopropylamine and Ca2+ions. In some embodiments, after the shrinking the hydrogel is dehydrated. In certain embodiments, the dehydrating includes contacting the hydrogel with an organic solvent. In some embodiments, the organic solvent includes one or more of methanol, ethanol, and acetone. In certain embodiments, the contacting of the hydrogel with the organic solvent includes incubating the hydrogel in a series of organic solvent solutions, wherein the contacting transitions the hydrogel to an organic state. In some embodiments, the method also includes removing the organic solvents from the hydrogel in the organic state. In some embodiments, the removing includes freeze-drying the hydrogel in the organic state. In certain embodiments, the fixing includes contacting the dehydrated hydrogel with one or more crosslinkers that react with functional groups within the dehydrated hydrogel. In certain embodiments, the functional groups are one or more of: carboxyl, amino, and imino; and are within the hydrogel. In some embodiments, the one or more crosslinkers include one or more of: adipic acid dihydrazide, Bisphenol A, and N,N,N',N'-tetraglycidyl-4,4'- diaminodiphenylmethane (TGDDM). In certain embodiments, following the fixing the method also includes modifying the surface of the hydrogel to be hydrophobic. In some embodiments, the modifying includes contacting the surface of the hydrogel with a coating reagent that includes silane. In certain embodiments, the coating reagent that includes silane is Trichloro(octyl)silane or perfluorooctyltriethoxysilane.

[0011] Brief Description of the Drawings

[0012] Fig. 1A-D provides a confocal images and graphs illustrating results of certain studies that included resolution of nanoscale pattern in lateral. Fig. 1A shows single-pixel-width lines patterned in fully swelled hydrogel through confocal imaging. Fig. IB shows single-pixel- width lines after complete post-processing through scanning electron microscope (SEM) imaging. Fig. 1C-D are graphs showing full width at half maximum (FWHM) values of these vacant lines are 50.8 ± 6.9 nm (mean ± standard deviation (SD) across samples, n = 3) for 12- fold shrinkable scaffolds.

[0013] Fig. 2A-D provides a confocal images and graphs illustrating results of certain studies that included resolution of nanoscale pattern in axial. Fig. 2A shows stepped structures (the number of steps: 10; initial height for each step: 250 nm) after patterning process through confocal imaging. Fig. 2B shows stepped structures after complete post-processing through atomic force microscope (AFM) measurement. Fig. 2C-D are graphs showing average height values of these steps are 21.1 ± 2.8 nm (mean ± SD across samples, n = 3) for 12-fold shrinkable scaffolds.

[0014] Fig. 3A-C provides comparative images of different 3D structures built on hydrogels, before and after the shrinking process. Fig. 3 A shows fluorescent images of hydrogels featuring square porous, linear, and various height pillar structures. Fig. 3B shows scanning electron microscope (SEM) images post the controlled shrinking and dehydration-hardening steps, showing nano-scale structures that closely resemble the micro-scale structures of the hydrogel when swollen. Fig. 3C provides SEM images of structures that were directly shrunk and air-dried without undergoing controlled shrinking and dehydration-hardening, displaying various degrees of deformation at the nanoscale.

[0015] Fig. 4 provides a schematic diagram of polymer matrix crosslinking and surface modification. After dehydration, the polymer undergoes a cross-linking reaction to form a denser and more solidified structure. Subsequently, surface treatments are applied to isolate moisture, further stabilizing the polymer's structure, and preventing deformation caused by water absorption.

[0016] Fig. 5A-D provides images and a graph demonstrating meta surface for optical application of diffractive optical element. Fig. 5 A shows partial area of design and Fig. 5 B shows the experimental results of the visible computing device via AFM measurement. Fig. 5C is a schematic diagram of the cross-sectional view of the vacancy features with 8 different height level. Fig. 5 D is a graph of experimental results indicating a strong correspondence to the design and simulation results, with apparent height gradients in different positions.

[0017] Fig. 6A-D provides images of holographic meta surface with 8 gray scale. Fig. 6A shows the design of the meta surface. Fig. 6B is a fluorescent image of the patterned hydrogel before shrinking. Fig. 6C is an atomic force microscope (AFM) image and Fig. 6D is a scanning electron microscope (SEM) image of the final 3D nano structure.

[0018] Detailed Description

[0019] The invention, in part, provides methods of preparing a hydrogel, wherein the method comprises shrinking and dehydration methods that are isotropically uniform, resulting in final 3D structures that closely align with the initial designs. In some embodiments, a method of the invention comprises a controlled shrinking process, characterized by high precision and accuracy. Methods of the invention can be used to prepare micro and nano-devices using ImpFab technology that are improved versus previous methods and provide improved function versus devices prepared with previous methods.

[0020] Previous methods, termed Implosion Fabrication (ImpFab), enable three-dimensional nanofabrication by isotropic shrinking of laser-patterned and material-deposited hydrogels (see U.S. Patent Application Publication No. US 2017 / 0081489, the content of which is incorporated by reference herein). ImpFab methods can be used in conjunction with embodiments of methods of the invention for preparing improved structures, such as but not limited to micro- and nano-devices. In ImpFab, photo-activatable chromophores are excited with two-photon lasers to conjugated to specific sites in a poly aery late hydrogel, and chromophores of different functional groups can be selectively placed throughout the hydrogel in a deterministic manner. Next, materials bearing complementary moieties to the functional groups on the chromophores can be efficiently deposited to the patterned areas, forming the architectures with designated geometry and morphology'. Finally, the polyacrylate hydrogel is dialyzed with acidic or cationic solutions, leading to isotropic shrinkage by a predetermined factor. Using these previous methods, a 3D object with selected material composition is obtained.

[0021] It has now been discovered that ImpFab methods used in conjunction with methods of the invention comprising chelation-induced shrinking and dehydration hardening of the hydrogel, which may be simultaneous, result in an increase in the stability of constructed 3D micro and nanostructures, enabling the creation of more complex structures without collapse. This includes structures with high aspect ratios exceeding 100, such as pillar or branched suspending nanostructures. Moreover, by substituting the system with organic solvents, it has been identified that the material matrix may be modified to result in a more stable, crosslinked hydrophobic polymer form. This modification ensures the micro and nano devices we construct are stably preserved and maintain their functionality over the long term.

[0022] Certain Elements of Fabrication Process

[0023] Methods of the invention may be used to prepare hydrogel scaffolds comprising 3D micro and nanostructures. Methods described herein provide improvements to nanofabrication methods including, but not limited to chelation-induced shrinking and dehydration hardening of a patterned hydrogel, which results in a hydrogel that comprises complex internal micro and nano structures that has greater stability and precision than patterned hydrogels fabricated using alternative methods. The term “hardening” in used interchangeably herein with the term “curing.” The discovery of novel shrinking and dehydration strategies used in embodiments of methods of the invention, permit fabrication of improved 3D micro and nanostructures, enabling the creation of more complex structures with improved stability and utility. Generally, methods of the invention include preparing an expandable hydrogel; expanding the prepared hydrogel; contacting the expanded hydrogel with a patterning solution; patterning the contacted expanded hydrogel by contacting the hydrogel with a laser light; isotropically shrinking and dehydrating the patterned expanded hydrogel; and fixing the shrunk and dehydrated hydrogel.

[0024] Hydrogel Preparation and Expansion

[0025] Expandable hydrogels may be used in methods of the invention to fabricate a patterned hydrogel comprising 3D hollow or vacant patterning within the hydrogel. A nonlimiting example of a type of expandable hydrogel that can be used in certain embodiments of methods of the invention is a poly(acrylate-co-acrylamide) hydrogel. Details for preparing expandable hydrogels are provided herein, and additional methods of preparing expandable hydrogels suitable for use in certain methods of the invention are known in the art. Hydrogels may be prepared using art-known methods, including but not limited to methods such as those disclosed in U.S. Patent Publication No. 2017 / 0081489.

[0026] Methods of the invention utilize expandable hydrogels and hydrogel expansion is performed prior to patterning the hydrogel. In some embodiments, an expandable hydrogel is contacted with (also referred to as “incubated in”) a solution such as solvent or liquid. The solvent or liquid is absorbed by the expandable hydrogel material, resulting in expansion of the hydrogel. In some embodiments, the prepared hydrogel is contacted (incubated) in an expansion fluid, which in some embodiments, comprises water. In some embodiments, the expansion fluid comprises water and isopropylamine. The incubation of the hydrogel may be repeated 1, 2, 3, 4, 5, 6, 7, 8, or more changes of the expansion fluid, to allow the hydrogel to reach an expanded status. Methods of hydrogel expansion are described herein and additional art-known methods may be used in conjunction with methods of the invention to expand a hydrogel.

[0027] An expanded hydrogel or expandable hydrogel (prior to expansion) of the invention may be contacted with an expansion fluid comprising isopropylamine prior to patterning of the hydrogel. In some embodiments, the expansion fluid comprises a 500 pM concentration of isopropylamine. In certain embodiments, the expanded hydrogel is incubated in an expansion fluid comprising isopropylamine at a concentration between 200 pM and 600 pM, between 300 pM and 600 pM, 400 pM and 600 pM, 400 pM and 700 pM, or 400 pM and 800 pM in the solution.

[0028] Prior to patterning, an expanded hydrogel is incubated in a photosensitizer solution comprising one or more photosensitizers. Non-limiting examples of photosensitizers that may be used in methods of the invention are: Rhodamine dyes (B, 6G, and 123), octadecyl Rhodamine B chloride (R18), Methyl Blue (MB), and sulfo-Cyanine dyes (3, 5, and 7). Additional photosensitizers know in the art are suitable for use in embodiments of methods of the invention. In some embodiments of methods of the invention, a photosensitizer solution may comprise one of more photosensitizers and isopropylamine. In some embodiments of methods of the invention, a photosensitizer solution may comprise one of more photosensitizers and hydrogen peroxide. In some embodiments of methods of the invention, a photosensitizer solution may comprise one of more photosensitizers, isopropylamine, and hydrogen peroxide. In addition to incubating the hydrogel in a photosensitizer solution, in some embodiments of methods of the invention, the incubation also includes gassing the hydrogel with O2 for at least a portion of the length of the incubation period. In some embodiments, the gassing of the hydrogel is done for a period of between two and ten minutes during the hydrogel incubation with the photosensitizer solution. In some embodiments, the gassing of the hydrogel is done for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes.

[0029] Patterning

[0030] Methods of the invention include patterning in which the scaffold of an expanded hydrogel is cleaved, forming one or more 3D hollow structures within the hydrogel. Scaffold cleavage occurs due to contacting a photosensitizer present in a hydrogel with light. In some embodiments, the light is a laser light. It will be understood that other types of light may also be used in patterning methods of the invention. Patterning of an expanded hydrogel comprises a three-dimensional exposure of the hydrogel to a light. Exposure of the hydrogel to a light is also referred to herein as “contacting” the hydrogel with a light. In certain embodiments of methods of the invention, the hydrogel to be patterned is contacted with a laser light. In some embodiments of methods of the invention, the laser light is generated with a multiphoton laser, a non-limiting example of which is a two-photon laser. When contacting a hydrogel with a laser light, elements such as laser power, dwell time, number of repetitions, etc. of the contact of the hydrogel by the laser are considered.

[0031] In some embodiments of methods of the invention, contacting with a laser light comprises contacting with a laser light having an average power of 5 - 200 mW. In some embodiments of methods of the invention, the power of the laser light contacting the hydrogel is between 5mW and 200mW (inclusive), 5mM and lOmW (inclusive), 5mW and 15mW (inclusive), 5mW and 20mW (inclusive), 5mW and 25mW (inclusive), 5mW and 30mW (inclusive), 5mW and 35mW (inclusive), 5mW and 40mW (inclusive), 5mW and 45mW (inclusive), 5mW and 50mW (inclusive), 5mW and lOOmW, lOmW and lOOmW (inclusive), or lOmW and 50mW (inclusive).

[0032] With respect to dwell times, in certain embodiments of methods of the invention, a hydrogel is contacted with a laser light at a dwell time of 2 - 20 ps per contact. In some embodiments of methods of the invention, the dwell time of the laser light contacting the hydrogel is between 1 and 10 ps per contact (inclusive), 2 and 10 ps per contact (inclusive), 5 and 10 ps per contact (inclusive); 5 and 20 ps per contact (inclusive); 5 and 30 ps per contact (inclusive), or 1 and 30 ps per contact (inclusive).

[0033] For patterning, a hydrogel may be contacted with the light once or a plurality of times. AS used herein the term “plurality” means more than one. In some embodiments of methods of the invention, a hydrogel is contacted with a light 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

[0034] In some embodiments of methods of the invention, the patterning of the hydrogel is not performed with the hydrogel under deoxygenated conditions.

[0035] A hydrogel that has been patterned using a method of the invention may be washed with distilled water to remove the chemicals and polymer fragments. In some embodiments, after patterning the hydrogel is washed 2, 3, 4, 5, or more times with distilled water. In some embodiments, following patterning of a hydrogel, the method of the invention includes shrinking the patterned hydrogel. Art-known methods may be used to shrink the patterned hydrogel.

[0036] Shrinking, Hardening, and Protecting

[0037] Following patterning of the hydrogel with light, methods of the invention include removing the photosensitizer solution. This removal may be done following patterning and prior to shrinking and dehydrating the patterned hydrogel. In some embodiments, removal of the photosensitizer solution comprises washing the patterned hydrogel with a wash solution comprising isopropylamine. In some embodiments, the wash solution further comprises water. In certain embodiments of methods of the invention, the wash solution further comprises one or both of MgCh and Ca2+ions. It has been determined that incubation of a patterned hydrogel in a wash solution, results in the isotropic shrinking of the patterned hydrogel due to chelation-induced shrinking.

[0038] Embodiments of methods of the invention for shrinking a patterned hydrogel may include incubating the patterned hydrogel in a series of ionic solutions. For example, the patterned hydrogel may be incubated in a series of increasingly ionic solutions, which results in increasing chelation effects on the patterned hydrogel. In some embodiments of methods of the invention, a patterned hydrogel is incubated in a series of solutions comprising isopropylamine and increasing concentrations of MgCh and then may be incubated in a series of solutions comprising isopropylamine and increasing concentrations of Ca2+. In a nonlimiting example, a patterned hydrogel is incubated in a first series of solutions followed by incubation in a second series of solutions. Each of a first series of solutions comprises isopropylamine (concentration 66.7 pM) and an amount of MgCh, wherein each successive solution in the first series includes a higher amount of MgCh than the preceding solutions in the series. For example, though not intended to be limiting, the first, second, and third solutions in the first senes may comprise 0.1 M, 0.3 M, and 0.5 M MgCh so the patterned hydrogel is incubated in increasing concentrations of MgCh as it moves through the first series. Following the first series the patterned hydrogel moves through a second series of solutions. The second series comprises isopropylamine (concentration 66.7 pM) and an amount of Ca2+wherein each successive solution in the second series includes a higher amount of Ca2+than the preceding solutions in the series. For example, though not intended to be limiting, the first, second, and third solutions in the second series may comprise 0.1 M, 0.3 M, and 0.5 M Ca2+so the patterned hydrogel is incubated in increasing concentrations of Ca2+as it moves through the second series.

[0039] Following shnnking of the patterned hydrogel, methods of the invention include dehydrating the hydrogel. In some embodiments of methods of the invention, dehydrating a hydrogel comprises contacting the hydrogel with an organic solvent. Contacting the hydrogel with an organic solvent may also be referred to as incubating the hydrogel in an organic solvent. Non-limiting examples of organic solvents that may be used in methods of the invention are methanol, ethanol, and acetone. The incubating of the hydrogel in one or more organic solvents may include incubating the hydrogel in a series of organic solvent solutions, and through the series, the hydrogel transitions to a hydrogel in an organic state. When the hydrogel is in an organic state, that is, all fluids in and / or around the hydrogel are organic fluids, the organic solvents or fluids may be removed from the hydrogel. A non-limiting method of the invention by which organic solvents or fluids are removed from a hydrogel is freeze-drying the hydrogel that is in the organic state.

[0040] Following removal of organic solvents or fluids from the hydrogel, methods of the invention may include fixation of the dehydrated hydrogel. In some embodiments of methods of the invention, fixation or fixing the dehydrated hydrogel comprises contacting the dehydrated hydrogel with one or more crosslinkers that react with functional groups within the dehydrated hydrogel. Non-limiting examples of one or more crosslinkers that may be used in a fixation method of an invention are: adipic acid dihydrazide, Bisphenol A, and N,N,N',N'-tetraglycidyl-4,4'-diaminodiphenylmethane (TGDDM). Non-limiting examples of functional groups with which the one or more crosslinkers may react in the hydrogel are carboxyl, amino, and imino functional groups.

[0041] Following the fixation of a patterned hydrogel of the invention the patterned hydrogel may be treated to modify the surface of the hydrogel to be hydrophobic. Some embodiments of methods of the invention to modify the surface of the hydrogel comprise contacting the surface of the hydrogel with a coating reagent comprising silane. Non-limiting examples of coating reagents that may be used to modify the surface of the hydrogel to be hydrophobic are Tnchloro(octyl)silane and perfluorooctyltriethoxysilane.

[0042] Commercial applications (economic potential, etc.)

[0043] 3D nanofabrication revolutionizes industries by enabling the creation of highly precise, complex nanostructures that lead to breakthroughs in medicine, electronics, photonics and materials science, promising transformative advancements and economic growth. Methods of the invention, used in conjunction with certain elements of Implosion Fabrication, exhibit superior capabilities, enabling the construction of micro and nano devices that are unachievable with current techniques such as Direct Laser Writing (DLW) and photolithography. Embodiments of methods of the invention comprise controlled isotropic shrinking of hydrogel substrates and are used to prepare nano precision of 3D structures. 3D structures prepared using embodiments of methods of the invention, (non-limiting examples of which are micro and nano devices) have significantly enhanced functionalization and stability compared with 3D structures prepared with previous methods. Certain embodiments of methods of the invention can be used to prepare structures, such as but not limited to a mold and / or a device, which has stable 3D nanostructure for application of medicine, electronics, photonics and materials science.

[0044] Methods of the invention comprise controlled isotropic shrinking of hydrogels while maintaining internal 3D structure nanoscale precision and accuracy. The 3D structures constructed via previous methods such as laser direct writing were only able to achieve micron-scale resolution, greatly limiting their applications. In addition, it has now been recognized that if structures built by laser direct writing could be isotropically shrunk by ten times or more, it would be possible to overcome the diffraction limit and achieve nanoscale resolution. Previous methods of dehydrating hydrogels could not ensure nanoscale accuracy of the 3D structures, but embodiments of methods of the invention result in nanoscale precision and accuracy for the first time.

[0045] Certain embodiments of methods of the invention include selecting ions and salt solutions with varying chelating abilities to the hydrogel's functional groups and contacting a hydrogel with the selected ions and salt solutions, thereby dehydrating and hardening the polymer matrix of the hydrogel. In certain embodiments of methods of the invention, this is followed by the gradual addition of one or more organic solvents to the hydrogel, further dehydrating and hardening the polymer. Subsequent cross-linking and surface modification reactions in the organic solvents solidify and stabilize the polymer structure, making it hydrophobic to prevent water absorption and deformation. Methods of the invention can be used to design and prepare (also referred to herein as “to fabricate" or “to construct”) highly conforming, high-precision free-form 3D micro and nanostructures while ensuring the structures long-term stability. Preparing a structure, such as but not limited to a micro and / or nano device, using an embodiment of a method of the invention results in a structure with significantly enhanced functionalization and stability.

[0046] Studies have been performed to investigate and to select suitable solutions that result in effective isotropic shrinking of shrinkable hydrogels. Initially, ionic solutions were used that had varying chelating abilities with the hydrogel's functional groups, transitioning from solutions with the weakest to the strongest chelating ability. It was discovered that by gradually increasing the concentration of the ionic solution, the chelation process was coupled with the polymer's dehydration and hardening. This method ensured that the constructed 3D structures uniformly shrank in every dimension, preventing structural collapse.

[0047] Subsequently, studies were performed in which organic solvents such as methanol, ethanol, and acetone were gradually introduced into the thoroughly shrunk and dehydrated polymer matrix system, eventually transitioning to a system composed entirely of organic solvents. Crosslinkers and / or molecules with other functional groups that only reacted in organic solvents were added to the system. Removing the organic solvents through freeze- drying at the critical point, resulted in a stable, highly hydrophobic polymer matrix material that no longer swelled or distorted. Embodiments of methods of the invention may be used to prepare structures such as but not limited to micro and nano-devices, with structure that precisely match the design on which they are based. Methods of the invention permit preparation of more complex 3D structures than could be prepared with previous methods. In addition, methods of the invention may be used to prepare complex 3D structures that maintain long-term stability.

[0048] Examples

[0049] Example 1

[0050] Studies were conducted in which controlled isotropic shrinking of hydrogel substrates was used to prepare 3D structures with nano precision and accuracy.

[0051] Materials and methods

[0052] Starting materials for scaffolds included sodium acrylate (Gelest), acrylamide. N,N'- methylenebisacrylamide (MBAA), ammonium persulfate (APS), tetramethylethylenediamine (TEMED), agarose, methacrylated alginate (Advanced Biomatrix, Carlsbad, CA), methacrylated gelatin (Advanced Biomatrix, Carlsbad, CA), 2-hydroxy-l-[4-(2- hydroxyethoxy) phenyl] -2-methyl-l -propanone (Irgacure D-2959). The photosensitizers included Rhodamine dyes (B, 6G, and 123), octadecyl Rhodamine B chloride (R18; Thermo Fisher Scientific), Methyl Blue (MB), sulfo-Cyanine dyes (3, 5, and 7) (Lumiprobe). The chemicals for patterning, shrinking, fixation, and solvent exchange included isopropylamine, magnesium chloride (MgC12), calcium chloride (CaCb), adipic acid dihydrazide (AAD), 2- (N-morpholino)ethanesulfonic acid (MES), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; Thermo Fisher Scientific, Waltham, MA), N-hydroxysulfosuccinimide (Sulfo-NHS; Thermo Fisher Scientific, Waltham, MA), and ethanol.

[0053] Hydrogel Synthesis

[0054] The synthesis of poly(acrylate-co-acrylamide) hydrogels with a shrinkage factor of 12 began with dissolving sodium acrylate (26.0 wt%), acrylamide (7.5 wt%), and PBS (l x; 16.7 vol%) in ultrapure water to form solution A. Dissolving MBAA (2.0 wt%), APS (10.0 wt%), and TEMED (10.0 wt%) in ultrapure water formed solution B, C, and D, respectively. Mixing solution A (600 pL), B (13 pL), and ultrapure water (347 pL), followed by bubbling N2 for 3 min to remove most of the dissolved O2, resulted in a solution E. Further mixing solution E (192 pL), D (4 pL), and C (4 pL) in sequence and filling the solution in to a hydrophobic mold finished the hydrogel synthesis. The synthesis of poly(acrylate-co- acrylamide) hydrogels with different shrinkage factor was similar with only difference in solution A and solution E. The new solution A contained sodium acrylate (39.0 wt%), acrylamide (11.3 wt%), and PBS (lx; 16.7 vol%) in ultrapure water. Mixing solution A (600 pL), B (120 pL). and ultrapure water (240 pL), followed by bubbling N2 for 3 min to remove most of the dissolved 02, led to the new solution E.

[0055] Patterning

[0056] The process began with soaking the hydrogel scaffolds in isopropylamine solution (concentration: 66.7 pM; pH: 9.5) for three times (each time for 30 min) for maximum expansion, and then soaking in photosensitizer solution (concentration: 150 pM; O2 gassing for 5 min) with isopropylamine (concentration: 500 pM) to remain expanded status and with hydrogen peroxide (concentration: 10 mM) to increase the photosensitizer efficiency. Three- dimensional exposure utilized a two-photon laser system (Mai Tai Ti: Sapphire laser; wavelength: 780 nm; pulse width: 100 fs; frequency: 80 MHz). The pixel dimension of photomasks remained as 580 x 580 nm. The average power (5 - 200 mW), dwell time (2 - 20 ps), Z-step (0.1 - 2.5 pm), and the number of repetitions (1 - 10) were adjusted according to different application scenanos. Multiple water immersion objectives (CFI75 Apochromat LWD 20XC with a working distance (WD) of 2.80 mm and a numerical aperture (NA) of 1.00; CFI Apochromat Lambda S 40XC with a WD of 0.18 mm and aNA of 1.24) were used in the patterning process.

[0057] Shrinking

[0058] After laser exposure, washing the scaffolds by isopropylamine solution (concentration: 66.7 pM; pH: 9.5) for five times (each time for 30 min) fully removed the residual photosensitizers. Taking an example of the 12-fold shrinkable scaffolds, first-step shrinkage of the scaffolds utilized MgCh solution, with a concentration of 0. 1 M, 0.3 M, and 0.5 M in sequence, all with isopropylamine (concentration 66.7 pM) (shrinkage factor in this step is around 5). Continuously using Ca2+ions (0.1 M, 0.3 M, and 0.5 M in sequence, all with isopropylamine (concentration 66.7 pM)), which have higher binding affinity to carboxylic acid groups compared to Mg2+, further shrank the scaffolds (shrinkage factor in this step is around 10). All these MgCh and CaCh solutions with isopropylamine were filtered before usage.

[0059] Structural characterization

[0060] During the post-processing, the status at each step was checked and characterized by wide-field optical microscopy and confocal microscopy. The photosensitizers anchored at the surfaces and edges of the inner void structures served as an indicator to characterize the void structures with associated integrity and distortion.

[0061] Lateral resolution

[0062] Single-pixel-width lines were patterned in 10-fold shrinkage scaffold using a 40x objective (CFI Apochromat Lambda S 40XC WI); above the line pattern, a 200-pm-thick trench was created, using the same photosensitizers, to expose these lines for characterization. After post-processing, a scanning electron microscope (Zeiss Gemini 360 FE-SEM SEC), with a pre-coated Pd / Pt layer (thickness: 10 nm; EMS 150T ES Sputter / Carbon Coater) characterized the minimum dimension in lateral.

[0063] Axial resolution

[0064] Stepped structures (initial height for each step: 200 nm) were created in 10-fold shrinkage scaffolds using a 20x objective (CFI75 Apochromat LWD 20XC W); above the stepped structures, a 200-pm-thick trench was created, using the same photosensitizers, to expose these stepped patterns for characterization. After post-processing, atomic force microscopy (Cypher VRS AFM) characterized the minimum dimension in axial.

[0065] Phase measurement

[0066] To confirm the optical function, especially the phase shift from input wavefront by the patterned sample, the step-like shape was patterned. To evaluate the phase shift by the patterned structure, the sample was imaged by Diffraction Phase Microscopy (DPM; / / opg.optica.org / ol / abstract.cftn?uri=ol-31-6-775) and the interference fringes in the step-like patern were recorded. For the fringe images, a reconstruction algorithm ( / / github.com / matlockCISL / DPM-basic) was applied for extracting phase and absorption from an interferogram. In certain studies, the DPM worked with 633 nm wavelength.

[0067] Results and discussion:

[0068] I. High Resolution and precision of 3D nanoscale structure

[0069] To validate the minimum feature size of the topological nanostructures in scaffolds, create single-pixel-width vacant lines structure were designed and created to characterize the lateral resolution. Because the shrinking features lead to structures below the optical diffraction limit, SEM and atomic force microscopy (AFM) were used to assess the structure resolution. For lateral resolution, single-pixel-width vacant lines were created, with the expanded status by confocal microscopy shown in Fig. 1A. After the complete postprocessing procedures for isotropic shnnk, the structures by SEM appear in Fig. 2B. The full width at half maximum (FWHM) values of these vacant lines are 50.8 ± 6.9 nm (mean ± standard deviation (SD) across samples, n = 4 lines from 3 samples) for 12-fold shrinkable scaffolds.

[0070] The stepped structures (the number of steps: 10; initial height for each step: 250 nm) serve as a representative example to test the resolution in axial direction. The initial status after patterning process appear in Fig. 2A by confocal microscopy. After the complete postprocessing procedures, the average height values of these steps are 21.1 ± 2.8 nm (mean ± SD across samples, n = 3) for 12-fold shrinkable scaffolds (Figure 2b-d), measured by AFM.

[0071] In summary, the topological engineering in scaffolds is capable of achieving 20-50 nm nanoprecise lateral and axial resolution.

[0072] 2 Maintenance of 3D structure with nano accuracy

[0073] Accuracy control in lateral and axial direction after dehydration is not feasible using previous shrinkage strategies due to the limitations in the dehydration method. Using methods of the invention, regardless of whether the structure was porous, linear, or irregularly columnar, it could maintain a stable 3D structure supported by surrounding water after patterning, as shown in Fig. 3 A. However, during the shrinking and dehydration process, the method of the invention comprises simultaneous shrinking and curing, which ensured isotropic reduction of the 3D structure, maintaining nano accuracy. Without this, the structure may experience varying degrees of deviation, distortion, or collapse (Fig. 3B-C).

[0074] Additionally, after dehydration, the polymer required fixation and modification as depicted in Fig. 4 to ensure long-term structural stability. This involved highly cross-linking the polymer matrix by utilizing cross-linkers (such as, but not limited to adipic acid dihydrazide, Bisphenol A, and TGDDM) to react with functional groups (such as but not limited to carboxyl, amino, imino, etc.) within the polymer. Subsequently, the polymer surface was modified hydrophobic through coating reactions using agents like Trichloro(octyl)silane and Perfluorooctyltriethoxysilane to prevent local swelling and deformation caused by moisture in the air.

[0075] 3. High nano precision structural Applications and Demonstrations

[0076] Due to the ability to precisely shrink 3D structures created by Direct Laser Writing (DLW) from the micron scale to the nanoscale isotropically, methods of the invention could be used to construct highly complex nano-scale 3D meta-surfaces and metamaterials for applications in medicine, electronics, photonics, and materials science. Methods of the invention have been used to construct a diffractive optical element featuring eight different height levels, with a final step size of 56 nm and a lateral feature size of 200 nm. As shown in Fig. 5, images from atomic force microscopy closely match the design, illustrating high fidelity. The height variations created by different depths of vacancies in the z-axis result in experimental outcomes that highly align with design simulations, confirming that the structure reconstruction method of the invention ensured nanoscale precision and accuracy, critical for the functionality and stability of nanophotonics.

[0077] Furthermore, as depicted in Fig. 6, methods of the invention have been used to construct a holographic metasurface for achieving reconstructed far-field imaging. Fluorescence images show that the patterned hydrogel structures matched the design precisely. After shrinking and dehydration-hardening processes, both atomic force microscopy and scanning electron microscopy confirmed that the designed metasurface structure, featuring eight gray scales axially and a 100 nm feature size laterally, achieved nano precision, thereby ensuring high-density information retention in 3D nanophotonics.

[0078] Example 2 Nanofabrication

[0079] Hydrogels were prepared and patterned, shrunk, dehydrated, and fixed using methods set forth in Example 1 and elsewhere herein. The following provides details of certain methods used to prepare micro and nanofabricated structures in hydrogels.

[0080] (i) a patterning method begins with soaking the hydrogel scaffolds in isopropylamine solution (concentration: 66.7 pM; pH: 9.5) for three times (each time for 30 min) for maximum expansion, and then soaking in photosensitizer solution (concentration: 150 pM; O2 gassing for 5 min) with isopropylamine (concentration: 500 pM) to remain expanded status and with hydrogen peroxide (concentration: 10 mM) to increase the photosensitizer efficiency. Three-dimensional exposure utilized a two-photon laser system (Mai Tai Ti:Sapphire laser; wavelength: 780 nm; pulse width: 100 fs; frequency: 80 MHz). The pixel dimension of photomasks remained as 580 x 580 nm. The average power (5 - 200 mW), dwell time (2 - 20 ps), Z-step (0.1 - 2.5 pm), and the number of repetitions (1 - 10) were adjusted according to different application scenarios. Multiple water immersion objectives (CFI75 Apochromat LWD 20XC with a working distance (WD) of 2.80 mm and a numerical aperture (NA) of 1.00; CFI Apochromat Lambda S 40XC with a WD of 0.18 mm and a NA of 1.24) were used in the patterning process.

[0081] (ii) following contacting the hydrogel with the laser, the scaffolds are washed with an isopropylamine solution (concentration: 66.7 pM; pH: 9.5) for five times (each time for 30 min) fully removed the residual photosensitizers. Taking an example of the 12-fold shrinkable scaffolds, first-step shrinkage of the scaffolds utilized MgCh solution, with a concentration of 0.1 M, 0.3 M, and 0.5 M in sequence, all with isopropylamine (concentration 66.7 pM) (shrinkage factor in this step is around 5). Continuously using Ca2+ions (0.1 M, 0.3 M, and 0.5 M in sequence, all with isopropylamine (concentration 66.7 pM)), which have higher binding affinity to carboxylic acid groups compared to Mg2+, further shrank the scaffolds (shrinkage factor in this step is around 10). All these MgCh and CaCh solutions with isopropylamine were filtered before usage.

[0082] Results of studies described above herein demonstrated controlled isotropic shrinking of hydrogel substrates with nano precision of internal patterned 3D structures.

[0083] Equivalents

[0084] Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplar}' and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0085] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.

[0086] All references, patents and patent applications and publications that are cited or referred to in this application are incorporated by reference in their entirety herein.

[0087] What is claimed is:

Claims

Claims1. A method for isotropic shrinking of a hydrogel, the method comprising:(a) preparing an expandable hydrogel;(b) expanding the prepared hy drogel;(c) contacting the expanded hydrogel with a patterning solution;(d) contacting the contacted expanded hydrogel with a laser light, wherein the laser light contact patterns the expanded hydrogel;(e) isotropically shrinking and dehydrating the patterned expanded hydrogel; and(f) fixing the shrunk and dehydrated hydrogel.

2. The method of claim 1, further comprising contacting the hydrogel with a stabilizing solution comprising isopropylamine prior to the contacting of the expanded hydrogel with the patterning solution.

3. The method of claim 1, further comprising incubating the expanded hydrogel in a solution comprising one or more photosensitizers.

4. The method of claim 3, wherein the stabilizing solution further comprises isopropylamine and / or hydrogen peroxide.

5. The method of claim 3, further comprising contacting the hydrogel with Ch gas for a period between 2 and 10 minutes.

6. The method of claim 5, wherein the hydrogel is contacted with Ch gas for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes.

7. The method of claim 1, wherein the laser light contact comprises three-dimensional laser light contact.

8. The method of claim 1, wherein the laser light is generated by a multiphoton laser.

9. The method of claim 8, wherein the multiphoton laser is a two-photon laser.

10. The method of claim 1, further comprising removing the patterning solution following the patterning and prior to the shrinking of the hydrogel.

11. The method of claim 1 , further comprising washing the patterned hydrogel with a wash solution comprising isopropylamine.

12. The method of claim 11, wherein the wash solution further comprises MgCh.

13. The method of claim 11, wherein the wash solution further comprises Ca2+ions.

14. The method of claim 1, wherein the isotropic shrinking comprises chelation-induced shrinking and dehydration hardening of the patterned expanded hydrogel.

15. The method of claim 14, wherein the chelation-induced shrinking of the patterned expanded hydrogel comprises incubating the patterned hydrogel in one or a plurality of ionic solutions.

16. The method of claim 15, wherein the patterned hydrogel is incubated in a series of increasingly ionic solutions, wherein the increasingly ionic solutions have increasing chelation effects.

17. The method of claim 1, wherein the shrinking comprises incubating the patterned hydrogel in a first solution comprising isopropylamine and MgCh and a second solution comprising isopropylamine and Ca2+ions.

18. The method of claim 14, wherein after the shrinking the hydrogel is dehydrated.

19. The method of claim 18, wherein the dehydrating comprises contacting the hydrogel with an organic solvent.

20. The method of claim 19, wherein the organic solvent comprises one or more of methanol, ethanol, and acetone.

21. The method of claim 19, wherein the contacting of the hydrogel with the organic solvent comprises incubating the hydrogel in a series of organic solvent solutions, wherein the contacting transitions the hydrogel to an organic state.

22. The method of claim 21, further comprising removing the organic solvents from the hydrogel in the organic state.

23. The method of claim 22, wherein the removing comprises freeze-drying the hydrogel in the organic state.

24. The method of claim 1, wherein the fixing comprises contacting the dehydrated hydrogel with one or more crosshnkers that react with functional groups within the dehydrated hydrogel.

25. The method of claim 24, wherein the functional groups are one or more of: carboxyl, amino, and imino; and are within the hydrogel.

26. The method of claim 24, wherein the one or more crosslinkers comprise one or more of: adipic acid dihydrazide, Bisphenol A, and N,N,N',N'-tetraglycidyl-4,4'- diaminodiphenylmethane (TGDDM).

27. The method of claim 1, wherein following fixing the hydrogel, the method further comprises modifying the surface of the hydrogel to be hydrophobic.

28. The method of claim 27, wherein the modifying comprises contacting the surface of the hydrogel with a coating reagent comprising silane.

29. The method of claim 28, wherein the coating reagent comprising silane is Trichloro(octyl)silane or perfluorooctyltriethoxysilane.

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