Bio-inspired self-healing structural color hydrogel
生物启发的自我修复结构色水凝胶

Biologically inspired self-healing structural color hydrogels were developed by adding a glucose oxidase (GOX)- and catalase (CAT)-filled glutaraldehyde cross-linked BSA hydrogel into methacrylated gelatin (GelMA) inverse opal scaffolds. The composite hydrogel materials with the polymerized GelMA scaffold could maintain the stability of an inverse opal structure and its resultant structural colors, whereas the protein hydrogel filler could impart self-healing capability through the reversible covalent attachment of glutaraldehyde to lysineresidues of BSA and enzyme additives. A series of unprecedented structural color materials could be created by assembling and healing the elements of the composite hydrogel. In addition, as both the GelMA and the protein hydrogels were derived from organisms, the composite materials presented high biocompatibility and plasticity. These features of selfhealing structural color hydrogels make them excellent functional materials for different applications.
通过将葡萄糖氧化酶（GOX）和过氧化氢酶（CAT）填充的戊二醛交联的BSA水凝胶添加到甲基丙烯酸化明胶（GelMA）反蛋白石支架中来开发生物学启发的自愈合结构颜色水凝胶。具有聚合的GelMA支架的复合水凝胶材料可以保持反蛋白石结构的稳定性及其所得的结构颜色，而蛋白质水凝胶填料可以通过戊二醛与BSA的赖氨酸残基和酶添加剂的可逆共价连接而赋予自我修复能力。通过组装和修复复合水凝胶的元素，可以产生一系列前所未有的结构色材料。此外，由于GelMA和蛋白质水凝胶均来自生物体，因此复合材料具有高生物相容性和可塑性。自愈合结构色水凝胶的这些特征使其成为用于不同应用的优异功能材料。

colloidal crystal|self-healing|inverse opal|structural color|hydrogel
胶体晶体|自愈合|反蛋白石|结构色|水凝胶

Structural colors, arising from intrinsic periodic nanostructures and resulting in the interaction of light with these photonic nanostructures, have attracted much interest because of the fascination associated with the display of various brilliant examples . The creatures displaying brilliant structural colors are prevalent in nature, and their life, including communication, shielding, and other biological functions, is closely linked with these structural colors . Researchers, marveling at these miracles, have devoted their work to bio-inspired structure colors materials.Structuralcolor hydrogels are one of the most important examples and have been widely studied and used in switches , optical devices , sensing materials , and wearable electronics , etc . However, because the deterioration and accumulation of damage of these materials during applications are inevitable, to achieve next-generation materials for both fundamental research and practical applications, the creation of bio-inspired structural color materials with increased survivability is still necessary.
由固有的周期性纳米结构引起并导致光与这些光子纳米结构相互作用的结构颜色引起了很大的兴趣，因为与各种辉煌实例的显示相关的魅力。显示出明亮结构色彩的生物在自然界中普遍存在，它们的生命，包括交流，屏蔽和其他生物功能，与这些结构色彩紧密相连。研究这些奇迹的研究人员将他们的工作投入到生物灵感的结构色材料中。结构色水凝胶是最重要的例子之一，并被广泛研究并用于开关，光学器件，传感材料和可穿戴电子等。然而，由于这些材料在应用过程中的损坏的恶化和累积是不可避免的，为了实现用于基础研究和实际应用的下一代材料，仍然需要产生具有增加的生存能力的生物激发的结构色材料。

Coincidentally, for the purpose of increasing the survivability and lifetime of an organism, the function of self-healing that can spontaneously heal injury and recover functionality in creatures is ubiquitous in nature . Inspired by these creatures, researchers have developed numerous self-healing hydrogel materials that fuse together with their homogeneous hydrogels for various important applications . However, the fusion process of the self-healing hydrogels could also happen in their neighboring nanostructures; this would cause the destruction of the periodic photonic scaffolds. Therefore, a self-healing hydrogel with stable structural color has not yet been reported, and its construction remains a challenge.
巧合的是，为了提高生物体的生存能力和寿命，自然愈合的功能可以自然地治愈伤害并恢复生物的功能，这种功能在自然界中无处不在。 受这些生物的启发，研究人员开发了许多自我修复水凝胶材料，这些材料与其均匀水凝胶融合在一起，可用于各种重要应用。 然而，自愈合水凝胶的融合过程也可能发生在它们相邻的纳米结构中; 这会导致周期性光子支架的破坏。 因此，尚未报道具有稳定结构颜色的自愈合水凝胶，其构造仍然是一个挑战。

In this paper, we present the desired self-healing structural color hydrogels by constructing them with a composite nanostructure, as indicated in Fig. 1. This nanostructure was composed of a methacrylated gelatin(GelMA) hydrogel inverse opal scaffold and a filler of glutaraldehyde cross-linked BSA hydrogel with enzyme additives of glucose oxidase (GOX) and catalase (CAT). The polymerized GelMA hydrogel scaffold in the composite materials could guarantee the stability of both the inverse opal structure and its resultant structural colors,whereas the protein hydrogel filler could impart the materials with self-healing capability through the reversible covalent attachment of the glutaraldehyde to lysine residues of BSA and the enzyme additives. As both the GelMA and protein hydrogels are derived from organisms, the composite materials had high biocompatibility and plasticity. It was demonstrated that a series of new structural color materials with one-dimensional (1D) linear microfiber, 2D pattern, and 3D photonic path structures could be developed by assembling and healing the composite structural color hydrogel elements. These features make our self-healing structural color hydrogels highly promising for different applications, such as counterfeit prevention, integrated optics, and biomedical engineering.
在本文中，我们通过用复合纳米结构构建它们来呈现所需的自修复结构色水凝胶，如图1所示。该纳米结构由甲基丙烯酸化明胶（GelMA）水凝胶反蛋白石支架和戊二醛交联填料组成。连接的BSA水凝胶，含有葡萄糖氧化酶（GOX）和过氧化氢酶（CAT）的酶添加剂。复合材料中聚合的GelMA水凝胶支架可以保证反蛋白石结构及其所得结构颜色的稳定性，而蛋白质水凝胶填充剂通过戊二醛可逆共价附着于BSA赖氨酸残基和酶添加剂，使材料具有自愈能力。由于GelMA和蛋白质水凝胶均来自生物体，因此复合材料具有高生物相容性和可塑性。结果表明，通过组装和修复复合结构色水凝胶元件，可以开发出一系列具有一维（1D）线性微纤维，2D图案和3D光子路径结构的新型结构色材料。这些特性使我们的自愈结构颜色水凝胶非常适用于不同的应用，例如防伪，集成光学和生物医学工程。

Results and Discussion

In a typical experiment, the GelMA hydrogel inverse opal scaffolds were fabricated by replicating silica colloidal crystal templates. These colloidal crystal templates were prepared by the self-assembly of silica nanoparticles in silica capillaries or on glass slides, which closely packed and finally formed an ordered structure during dehydration (Fig. 2A). This ordered packing of the nanoparticles endowed the colloidal crystals with interconnected nanopores throughout the templates, which enabled infiltration of the GelMA pregel solution. After the pregel solution penetrated the nanopores and filled all of the voids of the templates by capillary action, the solution was polymerized to form a hydrogel by UV light. Finally, the inverse opal scaffolds were obtained by etching the silica nanoparticles, leaving an inverse opal GelMA hydrogel scaffold (Fig. 2B). This kind of scaffold displays various brilliant structure colors (Fig. S1),which is an important feature of the materials.
在典型的实验中，通过复制二氧化硅胶体晶体模板制备GelMA水凝胶反蛋白石支架。 这些胶体晶体模板通过二氧化硅纳米粒子在二氧化硅毛细管或玻璃载玻片上的自组装制备，其在脱水过程中紧密堆积并最终形成有序结构（图2A）。 这种有序的纳米颗粒填充赋予胶体晶体整个模板中相互连接的纳米孔，这使得GelMA预凝胶溶液能够渗透。 在预凝胶溶液渗透纳米孔并通过毛细管作用填充模板的所有空隙后，通过UV光使溶液聚合以形成水凝胶。 最后，通过蚀刻二氧化硅纳米粒子获得反蛋白石支架，留下反蛋白石GelMA水凝胶支架（图2B）。 这种脚手架显示出各种明亮的结构颜色（图S1），这是材料的一个重要特征。

To impart to the structural color hydrogel the capability of self-healing, the glutaraldehyde cross-linked BSA hydrogel with enzyme additives of GOX and CAT was filled into the inverse opal scaffold. In this process, the pregel was first prepared with a protein concentration of 13.5 wt% (GOX, CAT, and BSA for 0.2%, 0.8%, and 12.5%, respectively). The concentration of glutaraldehyde was 0.5 wt%, and the pH of the solution was adjusted to 7.0. To fully fill the protein hydrogel into the nanopores of the inverse opal, the structure color hydrogels should be dehydrated and then immersed in the pregel solution in a vacuum environment. After these steps, the structure color hydrogels were transferred to a closed environment with a certain humidity for the polymerization of the infiltrated pregel in the inverse opals (Fig. 2C). Finally, a hybrid inverse opal hydrogel with brilliant structure color was achieved.
为了赋予结构色水凝胶自愈合能力，将戊二醛交联的BSA水凝胶与GOX和CAT的酶添加剂一起填充到反蛋白石支架中。 在该方法中，首先制备蛋白质浓度为13.5wt％的前凝胶（GOX，CAT和BSA分别为0.2％，0.8％和12.5％）。 戊二醛的浓度为0.5重量％，溶液的pH调节至7.0。 为了将蛋白质水凝胶完全填充到反蛋白石的纳米孔中，应将结构颜色水凝胶脱水，然后在真空环境中浸入预凝胶溶液中。 在这些步骤之后，将结构色水凝胶转移到具有一定湿度的封闭环境中，用于反蛋白石中浸润预凝胶的聚合（图2C）。 最后，获得了具有明亮结构颜色的混合反蛋白石水凝胶。

The formation of the structural colors of the hydrogels was ascribed to their orderly arranged nanostructure, which imparts to the inverse opal hydrogel and its derived hybrid hydrogel a unique photonic band gap (PBG). This PBG leads light with certain wavelengths or frequencies to be located in and reflected instead of propagating through the materials. As a result, the colloidal crystal templates and the inverse opal scaffold, together with the hybrid hydrogel, all showed vivid colors and possessed characteristic reflection peaks.Under normal incidence,the main reflection peak position λ of these materials can be estimated by Bragg’s equation, λ = 2d111naverage, where d111 is the interplanar distance of the (111) diffracting planes, and naverage refers to the average refractive index of the materials. Although the infiltration of the protein hydrogel into the inverse opal could increase the naverage of the hybrid material, the d111-related scaffold has also shrunk during the polymerization of the filler, and thus the structural color and the reflection peak of the hybrid material have blue shifted (Fig. S1). In addition, by using different sizes of silica nanoparticles for the colloidal crystal templates, a series of GelMA hydrogel inverse opals and resultant hybrid hydrogels with different diffraction peaks and structural colors could also be obtained (Fig. 2D and Fig. S2).
水凝胶的结构颜色的形成归因于它们有序排列的纳米结构，其赋予反蛋白石水凝胶及其衍生的混合水凝胶独特的光子带隙（PBG）。该PBG引导具有某些波长或频率的光位于并反射而不是通过材料传播。结果，胶体晶体模板和反蛋白石支架与混合水凝胶一起显示出鲜艳的色彩，并具有特征反射峰。在垂直入射时，这些材料的主反射峰位置λ可以用布拉格方程估算， λ= 2d111naverage，其中d111是（111）衍射平面的晶面间距，naverage是指材料的平均折射率。虽然蛋白质水凝胶渗透到反蛋白石中会增加杂化材料的naverage，但d111相关支架在填料聚合过程中也会缩小，因此混合材料的结构颜色和反射峰具有蓝色转移（图S1）。此外，通过使用不同尺寸的二氧化硅纳米粒子作为胶体晶体模板，还可以获得一系列GelMA水凝胶反蛋白石和具有不同衍射峰和结构颜色的所得混合水凝胶（图2D和图S2）。

In the GelMA and proteins hybrid hydrogel system, the feature of reversible imine covalent attachment of the glutaraldehyde to lysine residues of BSA, GOX, and CAT proteins imparts to the inverse opal filler protein hydrogel the self-healing function,which uses GOX and CAT to adjust the pH of the system by adding extra traces of glucose (Fig. S3). In this process, GOX assists the glucose to be oxidized to gluconolactone,which is then hydrolyzed to gluconic acid to decrease the pH value of the protein hydrogel. The by-product H2O2 of the glucose oxidation will decompose into H2O and O2 by the CAT enzyme to avoid the imine bonds being oxidized and to support the cyclic reaction of glucose oxidation. Finally, with the regulation of pH, the imine bonds provide the opportunity to heal the protein hydrogel. As a benefit from the reversible binding of the inverse opal filler protein hydrogel, the GelMA and proteins hybrid structural color hydrogel system would also be imparted with a selfhealing function.
在GelMA和蛋白质杂化水凝胶系统中，戊二醛与BSA，GOX和CAT蛋白的赖氨酸残基的可逆亚胺共价连接的特征赋予逆蛋白石填料蛋白水凝胶自我修复功能，其使用GOX和CAT来实现。通过添加额外的葡萄糖来调节系统的pH值（图S3）。在该过程中，GOX帮助葡萄糖被氧化成葡糖酸内酯，然后水解成葡糖酸以降低蛋白质水凝胶的pH值。葡萄糖氧化的副产物H 2 O 2将通过CAT酶分解成H 2 O和O 2，以避免亚胺键被氧化并支持葡萄糖氧化的循环反应。最后，随着pH的调节，亚胺键提供了愈合蛋白质水凝胶的机会。作为逆蛋白石填料蛋白水凝胶的可逆结合的益处，GelMA和蛋白质杂合结构颜色水凝胶系统也将赋予自愈合功能。

To investigate the self-healing property of GelMA and the proteins’ hybrid structural color hydrogel system, hybrid hydrogel microfibers with the same inverse opal nanostructures and composite protein materials were fabricated and cut into segments. Then, the segments of the structural color hydrogels were brought together slightly to ensure that their surfaces were in full contact. It was found that by simply connecting two of the segments, they could not adhere to each other and remained independent. However,with the addition of glucose, the two segments could adhere tightly to each other and form an integrated microfiber (Fig. 3A). Although the repaired traces could not be hidden and the reflection peak width at the fracture increased slightly (Fig. S4), the self-healing structural color microfiber maintained its vivid structural color through the whole body. In addition, the self-healing microfibers show elasticity as good as their original elasticity. Thus,the enzyme mediated hybrid structural color hydrogel exhibits excellent selfhealing properties with high recovery and reversibility.
为了研究GelMA和蛋白质的混合结构色水凝胶系统的自愈合性质，制备具有相同的反蛋白石纳米结构和复合蛋白材料的混合水凝胶微纤维并切割成片段。然后，将结构色水凝胶的片段稍微放在一起以确保它们的表面完全接触。发现通过简单地连接两个区段，它们不能彼此粘附并保持独立。然而，通过添加葡萄糖，两个区段可以彼此紧密粘附并形成整合的微纤维（图3A）。尽管修复的痕迹不能被隐藏并且裂缝处的反射峰宽略微增加（图S4），但自愈合结构色微纤维通过整个身体保持其生动的结构色。此外，自修复微纤维显示出与其原始弹性一样好的弹性。因此，酶介导的混合结构色水凝胶表现出优异的自愈合特性，具有高恢复性和可逆性。

It is noteworthy that our strategy could even heal the microfiber segments with different structural colors. To demonstrate this, hybrid hydrogel microfiber segments with blue, green, and red structural colors were assembled together. The joints of these microfiber segments were simply treated with glucose. It was found that although having different structural colors, neighbor segments could still adhere together tightly and form an integrated microfiber. The combined microfiber inherited the multiplex structural colors of each segment and preserved the good elasticity of the original microfibers (Fig. 3B). This indicated that the enzyme-mediated hybrid structural color hydrogel was suitable for different kinds of inverse opal scaffolds with different nanometer aperture sizes. Therefore, a new assembly strategy for the construction of a multiplex structural color hydrogel was developed.
值得注意的是，我们的策略甚至可以治愈具有不同结构颜色的微纤维片段。 为了证明这一点，将具有蓝色，绿色和红色结构颜色的混合水凝胶微纤维片段组装在一起。 这些微纤维段的关节简单地用葡萄糖处理。 结果发现，虽然具有不同的结构颜色，但相邻的链段仍然可以紧密地粘在一起并形成一体化的微纤维。 组合的微纤维继承了每个片段的多重结构颜色，并保留了原始微纤维的良好弹性（图3B）。 这表明酶介导的混合结构色水凝胶适用于具有不同纳米孔径尺寸的不同种类的反蛋白石支架。 因此，开发了用于构建多重结构色水凝胶的新组装策略。

To demonstrate the versatility of the self-healing structural color hydrogels in assembling other shapes besides cylindrical, three pieces of different hybrid hydrogel films were used for structural color pattern construction. By adding glucose to the intersection line, these films were stitched together to form an
indexed pattern of an integrated film with blue, green, and red structural colors (Fig. 4A). To investigate the practical value of the self-healing hybrid hydrogel materials, a tensile test was performed to quantitatively evaluate the mechanical stability of the repaired sample. It was found that the self-healing film could keep its integrated structures not only in the assembled units but also in the repaired section (Fig. 4B). Thus, the self-healing structural color film is sufficiently flexible to resist an external tensile force (Fig. S5). In each assembled unit, it could be observed that all of them showed an equal ratio of stretching with the whole film, which caused the blue shift of their structural colors. These colors’ blue shift should ascribe to the gradual decreasing of the interplanar distance d111 of the (111) diffracting planes during the stretching of the inverse opal materials. In addition to the simple indexed pattern of structural colors, a much more complex 2D pattern, such as Chinese Taiji (Fig. 4C), could also be constructed by using the same selfhealing assembly strategy.
为了证明自修复结构色水凝胶在组装除圆柱形之外的其他形状中的多功能性，将三片不同的混合水凝胶膜用于结构颜色图案构造。通过向交叉线添加葡萄糖，将这些薄膜缝合在一起形成一个具有蓝色，绿色和红色结构色的集成胶片的索引图案（图4A）。为了研究自我修复的混合水凝胶材料的实际价值，进行拉伸试验以定量评估修复的样品的机械稳定性。发现自愈膜不仅可以在组装单元中保持其整体结构，而且可以在修复部分中保持其整体结构（图4B）。因此，自修复结构彩色薄膜足够柔韧以抵抗外部拉伸力（图S5）。在每个组装的单元中，可以观察到它们都显示出与整个薄膜相同的拉伸比，这导致它们的结构颜色的蓝移。这些颜色的蓝移应归因于在反蛋白石材料拉伸期间（11​​1）衍射平面的晶面间距d111的逐渐减小。除了结构色的简单索引图案之外，还可以通过使用相同的自愈合装配策略来构造更复杂的2D图案，例如中国太极图（图4C）。

Besides the 2D patterns, the self-healing assembly strategy could also be used for the development of 3D structural color materials that have potential values in the areas of art creation, counterfeit prevention, and 3D integrated optics, etc. To demonstrate these concepts, a GelMA and proteins hybrid yellow structural color hydrogel film was cut into pieces of different sizes. These pieces were stacked together from large to small (Fig. 5A). Because of the complete porous inverse opal structure of these pieces, the filler self-healing protein hydrogels in the inverse opal scaffolds from the surface of the pieces could touch each other. Thus, these pieces could form an integrated 3D pyramid structure by a self-healing assembly strategy (Fig. 5B).
除了2D图案之外，自我修复装配策略还可以用于3D结构色材的开发，这些材料在艺术创作，防伪和3D集成光学等领域具有潜在价值。为了演示这些概念， 将GelMA和蛋白质杂合黄色结构色水凝胶膜切成不同大小的片。 这些碎片从大到小堆叠在一起（图5A）。 由于这些碎片的完全多孔反蛋白石结构，来自碎片表面的反蛋白石支架中的填料自愈合蛋白水凝胶可以相互接触。 因此，这些部件可以通过自我修复组装策略形成集成的3D金字塔结构（图5B）。

With the same method, we could also construct 3D structural color objects in a hydrogel block. In this process, hybrid blue structural color hydrogel pieces with different 2D green triangle patterns were first prepared by using the above process. Then, the pieces were stacked together and treated with glucose (Fig. 5C). Finally, a transparent blue hydrogel block containing a 3D triangle-stacked structural color object was generated (Fig. 5D). By designing the objects with more complex shapes and structural colors, advanced counterfeit prevention tags could be achieved. By using slender structural color microfibers instead of the triangle pattern, a 3D integrated photonic path could be developed in the hydrogel block (Fig. 5 E and F). Because of the existence of the PBGs in the hydrogel block and its encapsulated photonic paths, the hybrid hydrogel could show excited 3D green or invisible optical paths under green or blue light irradiation, respectively (Fig. 5 G and H). This implies that the materials could be used as new carriers for a 3D optical waveguide or optical communication.
使用相同的方法，我们还可以在水凝胶块中构建3D结构颜色对象。在该过程中，首先使用上述方法制备具有不同2D绿色三角形图案的混合蓝色结构色水凝胶片。然后，将这些碎片堆叠在一起并用葡萄糖处理（图5C）。最后，产生包含3D三角形堆叠结构颜色对象的透明蓝色水凝胶块（图5D）。通过设计具有更复杂形状和结构颜色的物体，可以实现先进的防伪标签。通过使用细长的结构颜色微纤维代替三角形图案，可以在水凝胶块中开发3D集成光子路径（图5E和F）。由于水凝胶块中PBG的存在及其封装的光子路径，混合水凝胶可分别在绿光或蓝光照射下显示激发的3D绿光或不可见光路（图5G和H）。这意味着这些材料可以用作3D光波导或光通信的新载体。

As both the GelMA hydrogel inverse opal scaffold and the filler of the protein hydrogel were derived from organisms, the self-healing structural color hybrid hydrogels should have the same high biocompatibility. To demonstrate this, the hybrid hydrogels before and after self-healing were all used for a hepatocellular carcinoma (HepG2) cell culture, respectively. The inverse opal GelMA hydrogel scaffold was also cultured with cells for the control group. It was found that the HepG2 cells could adhere and grow on the surface of the hybrid hydrogels irrespective of the repair process. These cells formed tight cell– cell connections both on the hybrid hydrogel film and on the healing section of the film after 24 h culture, as shown in Fig. 6 A–D and Fig. S6. The cell viabilities on the structural color hybrid hydrogels before and after self-healing, as well as on the commercial multiwell and on the inverse opal GelMA hydrogel scaffold, were also investigated quantitatively by using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, as presented in Fig. 6E. It could be observed that the viability of the HepG2 cells shows no obvious differences on these hydrogel substrates. Thus,the self-healing hybrid hydrogels were suitable for cell culture and reproduction. It is noteworthy that in many cases tissue engineering required hydrogels have no self-healing function, which greatly limits their application. However, with our strategy, both non- and self-healing hydrogels could be combined as a hybrid together through cross-linking nanoscaffolds, and the resultant hydrogels could be endowed with the self-healing function to remedy the restrictions of the hydrogel biomaterials in biomedical applications.
由于GelMA水凝胶反蛋白石支架和蛋白质水凝胶的填充剂均来自生物体，因此自愈合结构颜色混合水凝胶应具有相同的高生物相容性。为了证明这一点，自愈合之前和之后的混合水凝胶分别全部用于肝细胞癌（HepG2）细胞培养。逆蛋白石GelMA水凝胶支架也与细胞一起培养用于对照组。发现HepG2细胞可以在混合水凝胶的表面上粘附和生长，而与修复过程无关。如图6A-D和图S6所示，这些细胞在培养24小时后在混合水凝胶膜上和膜的愈合部分上形成紧密的细胞 - 细胞连接。通过使用3-（4,5-二甲基噻唑-2-基）定量研究结构颜色混合水凝胶在自我修复之前和之后以及在商业多孔和反蛋白石GelMA水凝胶支架上的细胞活力。 ）-2,5-二苯基四唑溴化物（MTT）测定，如图6E所示。可以观察到HepG2细胞的活力在这些水凝胶底物上没有显示出明显的差异。因此，自我修复的杂合水凝胶适合于细胞培养和繁殖。值得注意的是，在许多情况下，组织工程所需的水凝胶没有自我修复功能，这极大地限制了它们的应用。然而，根据我们的策略，非愈合和自愈合水凝胶可以通过交联纳米支架组合成杂化物，所合成的水凝胶具有自愈功能，可以弥补水凝胶生物材料在生物医学应用中的局限性。

Conclusion

In summary, we have developed self-healing structural color hydrogels with a GelMA inverse opal scaffold and BSA protein filler with GOX and CAT enzyme additives. The GelMA scaffold in the hybrid hydrogels guaranteed the stability ofthe inverse opal structure and the resultant structural colors, whereas the protein filler could impart the hydrogels’ self-healing capability through the reversible covalent attachment of the glutaraldehyde to lysine residues of proteins. We have demonstrated that a series of unprecedented structural color materials with 1D linear structures, 2D patterns, and 3D counterfeit-prevention objects and photonic path structures could be created by assembling and healing the hybrid hydrogel elements. In addition, the biocompatibility and biological applicability of the GelMA and proteins hybrid structural color hydrogels were also demonstrated. These features of the self-healing structural color hydrogels indicated their versatile values in different areas.
总之，我们开发了具有GelMA反蛋白石支架的自愈合结构色水凝胶和具有GOX和CAT酶添加剂的BSA蛋白填料。 混合水凝胶中的GelMA支架保证了反蛋白石结构的稳定性和由此产生的结构颜色，而蛋白质填充剂可通过戊二醛与蛋白质赖氨酸残基的可逆共价连接赋予水凝胶自愈合能力。 我们已经证明，通过组装和修复混合水凝胶元件，可以产生一系列具有一维线性结构，2D图案和3D防伪物体以及光子路径结构的前所未有的结构色材料。 此外，还证明了GelMA和蛋白质杂合结构色水凝胶的生物相容性和生物学适用性。 自修复结构色水凝胶的这些特征表明它们在不同区域的通用价值。

Methods

Materials.

Eight kinds of SiO2 nanoparticles with sizes 200nm,210nm,230nm, 250 nm, 270 nm, 290 nm, 300 nm, and 320 nm were purchased from Nanjing Dongjian Biological Technology Co., Ltd. The GelMA hydrogel was selfprepared. Gelatin (from porcine skin), methacrylic anhydride, and MTT were purchased from Sigma Aldrich. Calcein-AM (molecular probe) was purchased from Life Technologies, and glutaraldehyde was derived from Aladdin. BSA, GOX, and CAT were acquired from Sigma Aldrich. Cellulose dialysis membranes [molecular weight cutoff (MWCO) = 8,000–14,000] were acquired from Shanghai Yuanye Biotechnology Corporation. HepG2 cells were from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, Shanghai, China. Dulbecco’s modified Eagle ’s medium (DMEM) and FBS were purchased from HyClone. Penicillin–streptomycin was obtained from Gibco. Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore) with resistivity higher than 18 MΩ•cm.
从南京东健生物科技有限公司购买8种尺寸分别为200nm，210nm，230nm，250nm，270nm，290nm，300nm和320nm的SiO2纳米颗粒。自制的GelMA水凝胶。明胶（来自猪皮），甲基丙烯酸酐和MTT购自Sigma Aldrich。钙黄绿素-AM（分子探针）购自Life Technologies，戊二醛衍生自Aladdin。 BSA，GOX和CAT均购自Sigma Aldrich。从上海原野生物技术公司获得纤维素透析膜[分子量截止值（MWCO）= 8,000-14,000]。 HepG2细胞来自中国科学院生物化学与细胞生物学研究所，中国上海。 Dulbecco改良的Eagle's培养基（DMEM）和FBS购自HyClone。青霉素 - 链霉素从Gibco获得。使用Milli-Q Plus 185水净化系统（Millipore）纯化所有实验中使用的水，电阻率高于18MΩ·cm。

Preparation of Inverse Opal Scaffold.

The inverse opal scaffolds were fabricated using a sacrificial template method. The colloidal crystal templates were obtained at invariant temperature and humidity by a vertical deposition method. In brief, the colloidal crystal templates were prepared with the selfassembly of silica nanoparticles in silica capillaries or on glass slides. The SiO2 nanoparticles (50 wt%) with a variety of particle sizes (200 nm, 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, 300 nm, and 320 nm) were dispersed in water, showing a good monodispersity. For the preparation of colloidal crystal fiber templates, the SiO2 solution was injected into silica tubes (d = 1.56 mm) and formed an ordered fiber cluster structure during the dehydration procedure at 40 °C for 15 d. Then the fiber templates were calcined at 750 °C for 5 h to improve their mechanical strength. Finally, the silica tubes were removed and the free templates were obtained. The colloidal crystal film templates (with thickness of about 0.5 mm) were also prepared to obtain different patterns under the same condition. The silica nanoparticles self-assembled on glass slides with a silica solution (ethyl alcohol) concentration of 20 wt% at 4 °C for 3 h, and then the glass were calcined at 450 °C for 5 h to improve their mechanical strength. The inverse opal structural color hydrogels scaffold was obtained based on these colloidal crystal templates. The GelMA pregel solution (0.2 g/mL) was infiltrated into the silica templates by capillary force, and the solution was polymerized to form a hydrogel (with refractive index about 1.387) by exposure to UV light. Finally, the inverse opal scaffold was obtained by etching (4 wt% hydrofluoric acid) the silica nanoparticles, leaving an inverse opal GelMA hydrogel scaffold. These inverse opal scaffolds with different patterns could also be obtained by exposure to UV light with mask templates.
使用牺牲模板方法制造反蛋白石支架。通过垂直沉积法在恒定的温度和湿度下获得胶体晶体模板。简而言之，用二氧化硅纳米粒子在二氧化硅毛细管中或在载玻片上自组装制备胶体晶体模板。将具有各种粒径（200nm，210nm，230nm，250nm，270nm，290nm，300nm和320nm）的SiO 2纳米颗粒（50wt％）分散在水中，显示出良好的单分散性。为了制备胶体晶体纤维模板，将SiO 2溶液注入二氧化硅管（d = 1.56mm）中，并在脱水过程中在40℃下形成有序的纤维簇结构15天。然后将纤维模板在750℃下煅烧5小时以改善其机械强度。最后，除去二氧化硅管并获得游离模板。还制备胶体晶体膜模板（厚度约0.5mm）以在相同条件下获得不同的图案。二氧化硅纳米粒子在玻璃载玻片上自组装，二氧化硅溶液（乙醇）浓度为20wt％，在4℃下3小时，然后将玻璃在450℃下煅烧5小时以改善其机械强度。基于这些胶体晶体模板获得反蛋白石结构色水凝胶支架。通过毛细管力将GelMA预凝胶溶液（0.2g / mL）渗透到二氧化硅模板中，并通过暴露于UV光下使溶液聚合以形成水凝胶（折射率约1.387）。最后，通过蚀刻（4wt％氢氟酸）二氧化硅纳米粒子获得反蛋白石支架，留下反蛋白石GelMA水凝胶支架。这些具有不同图案的反蛋白石支架也可以通过用掩模模板暴露于UV光来获得。

Preparation of Bio-Inspired Self-Healing Structural Color Hydrogels.

The bioinspired self-healing structural color hydrogels were prepared based on the enzyme additives of the GOX and CAT. The glutaraldehyde (0.5 wt%) cross-linked BSA(12.5wt%)hydrogel with GOX(0.2wt%) and CAT(0.8wt%) was filled into the inverse opal scaffold. In this process, the pH of the pregel solution was adjusted to 7.0.The inverse opal scaffold was dehydrated for 2h at 35 °C and quickly filled with the pregel solution (with refractive index about 1.352) in a vacuum environment for 20 min. After these steps, the structure color hydrogels were transferred into a closed environment with a certain humidity at 4 °C for another 3 h for polymerization of the infiltrated pregel in the inverse opals. Finally, the hybrid structural color hydrogels with good visibility and brilliant structural colors were prepared. In addition, by using different sizes of silica nanoparticles, a series of hybrid hydrogels with different diffraction peaks and structural colors could also be obtained. The optical microscopy images of the colloidal crystal templates, inverse opal scaffold, and hybrid hydrogels were obtained under the same conditions by a digital camera (Canon5D Mark II). The reflection spectra of these samples were recorded at a fixed glancing angle, using an optical microscope equipped with a fiber-optic spectrometer (Ocean Optics; USB2000-FLG).
基于GOX和CAT的酶添加剂制备生物启发的自愈合结构色水凝胶。将具有GOX（0.2wt％）和CAT（0.8wt％）的戊二醛（0.5wt％）交联的BSA（12.5wt％）水凝胶填充到反蛋白石支架中。在该过程中，将预凝胶溶液的pH调节至7.0。将反蛋白石支架在35℃下脱水2小时，并在真空环境中快速填充预凝胶溶液（折射率约1.352）20分钟。在这些步骤之后，将结构色水凝胶转移到具有一定湿度的4℃的封闭环境中另外3小时，以使反相蛋白石中的渗透预凝胶聚合。最后，制备了具有良好可见度和明亮结构颜色的混合结构色水凝胶。此外，通过使用不同尺寸的二氧化硅纳米粒子，还可以获得具有不同衍射峰和结构色的一系列混合水凝胶。通过数码相机（Canon5D Mark II）在相同条件下获得胶体晶体模板，反蛋白石支架和混合水凝胶的光学显微图像。使用配备有光纤光谱仪（Ocean Optics; USB2000-FLG）的光学显微镜以固定的掠射角记录这些样品的反射光谱。

The Construction Process of Structured Structural Color Hydrogels.

The selfhealing property of cross-linked protein hydrogel systems was investigated by cutting the hybrid structural color hydrogels into two segments.Then,two segments of the hybrid structural color hydrogels were brought together slightly to ensure the two surfaces were fully contacted and stimulated with external glucose (0.1 mg) for 3 h under a closed condition at 4 °C. Finally, the enzyme-mediated hybrid structural color hydrogels, exhibiting excellent self-healing properties, were prepared. In another experiment, three kinds of hybrid hydrogel fibers with different structure colors were assembled together under the same conditions. The 2D pattern and 3D photonic path structures could also be developed by assembling and healing the composite structural color hydrogel elements. Obtained by using mask templates of UV light, these inverse opal scaffolds in different shapes were first dehydrated for 2 h at 35 °C and quickly filled with the prepared solution in a vacuum environment for 20 min. Then, the 2D pattern and 3D photonic path structures could also be assembled together with an external glucose for 3 h under a closed condition at 4 °C. The optical microscopy images of the samples were obtained under the same conditions by a digital camera (Canon5D Mark II).
通过将混合结构色水凝胶切割成两个区段来研究交联蛋白水凝胶系统的自愈合性质。然后，将两段混合结构色水凝胶稍微放在一起以确保两个表面完全接触并用外部葡萄糖刺激。 （0.1mg）在4℃的封闭条件下保持3小时。最后，制备了具有优异自愈合性能的酶介导的杂合结构色水凝胶。在另一个实验中，在相同条件下将三种具有不同结构颜色的混合水凝胶纤维组装在一起。还可以通过组装和修复复合结构颜色水凝胶元件来开发2D图案和3D光子路径结构。通过使用UV光的掩模模板获得，将这些不同形状的反蛋白石支架首先在35℃下脱水2小时，并在真空环境中快速填充所制备的溶液20分钟。然后，2D图案和3D光子路径结构也可以在4℃的封闭条件下与外部葡萄糖一起组装3小时。通过数码相机（Canon5D Mark II）在相同条件下获得样品的光学显微镜图像。

Cell Culture.

Cells were regularly cultured and passaged with DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a humidified incubator at 37 °C with 5% CO2. The structural color hydrogels were first disinfected by exposure to UV light for 2 h and rinsed with sterile PBS solution three times before cell culture. Then the HepG2 cells, cultured on the surface of the structure color hydrogels, were treated in traditional ways. The cells were seeded on the surface of hydrogel films (1 cm2) in a six-well tissue culture plate (2 × 105 cells per well) for 24 h. The viability of HepG2 cells cultured in different structural color hydrogels was analyzed. Briefly, cells were first cultured on the surface of the structure color hydrogel films for 24 h, then MTT/PBS solution (5 μg/mL) was added, and the cells were incubated for another 4 h. Then the cell viability was quantified by the MTT assays according to the manufacturer’s instructions. To test different cell viabilities under the same conditions, the cells cultured on the tissue culture plate were set as control experiments. The mean value and SD of five paralleled assays for each sample were recorded. The morphology of cells was also observed. After being cultured on the surface of the structure color hydrogel films for 24 h, the cells were stained with calcein AM (2μg/mL, 2 mL per well) for 20 min at 37 °C, followed by being rinsed twice with PBS and fixed with glutaraldehyde (2.5%, 2 mL per well) for 6 h at 4 °C. Finally, the cells were observed using an inverted fluorescence microscope.
定期培养细胞并用补充有10％FBS和1％青霉素 - 链霉素的DMEM在潮湿的培养箱中于37℃，5％CO 2下传代。首先通过暴露于UV光下对结构色水凝胶进行消毒2小时，并在细胞培养之前用无菌PBS溶液冲洗三次。然后以传统方式处理在结构颜色水凝胶表面上培养的HepG2细胞。将细胞接种在六孔组织培养板（每孔2×10 5个细胞）中的水凝胶膜（1cm 2）表面上24小时。分析了在不同结构色水凝胶中培养的HepG2细胞的存活率。简而言之，首先将细胞在结构色水凝胶膜的表面上培养24小时，然后加入MTT / PBS溶液（5μg/ mL），并将细胞再培养4小时。然后根据制造商的说明通过MTT测定法定量细胞活力。为了在相同条件下测试不同的细胞活力，将在组织培养板上培养的细胞设定为对照实验。记录每个样品的五个平行测定的平均值和SD。还观察到细胞的形态。在结构色水凝胶薄膜表面培养24小时后，用钙黄绿素AM（2μg/ mL，每孔2 mL）在37°C下染色细胞20分钟，然后用PBS冲洗两次并固定用戊二醛（2.5％，每孔2mL）在4℃下保持6小时。最后，使用倒置荧光显微镜观察细胞。

Characterization.

Reflection spectra were obtained at a fixed glancing angle, using an optical microscope equipped with a fiber-optic spectrometer(Ocean Optics; USB2000-FLG). SEM images of samples were taken by a scanning electron microscope (Hitachi S-3000N). Microscopy images of the samples were obtained with an optical microscope (Olympus BX51) equipped with a CCD camera (Media Cybernetics Evolution MP5.0) and a digital camera (Canon5D Mark II). The stiffness of the hydrogel materials was characterized by Single Column Table Top Systems (5943; Instron).
使用配备有光纤光谱仪（Ocean Optics; USB2000-FLG）的光学显微镜以固定的掠射角获得反射光谱。 通过扫描电子显微镜（Hitachi S-3000N）拍摄样品的SEM图像。 用配备有CCD相机（Media Cybernetics Evolution MP5.0）和数码相机（Canon5D Mark II）的光学显微镜（Olympus BX51）获得样品的显微镜图像。 通过Single Column Table Top Systems（5943; Instron）表征水凝胶材料的刚度。