Next-generation stimuli–responsive materials must be configured with local computational ability so that instead of a discrete on-off responsiveness, they sense, process and interact reciprocally with environmental stimuli. Because of their varied architectures and tunable responsiveness to a range of physical and chemical stimuli, polymers hold particular promise in the generation of such “materials that compute”. Here, we present a photopolymer cuboid that autonomously performs pattern recognition and transfer, volumetric encoding and binary arithmetic with incandescent beams. The material’s nonlinear response to incident beams generates one, two or three mutually orthogonal ensembles of white-light filaments, which respectively self-organize into disordered, 1-D and 2-D periodic geometries. Data input as binary (dark-bright) strings generate a unique distribution of filament geometries, which corresponds to the result of a specific operation. The working principles of this material that computes with light is transferrable to other nonlinear systems and incoherent sources including light emitting diodes. Some next-generation computing may be based in physical systems that respond directly and reciprocally to environmental stimuli. Here, the authors describe a photoresponsive material that autonomously performs computations with incident beams of incoherent white light. When stimulated by electromagnetic, electrical, chemical or mechanical signals, these pliant polymer architectures transition between states while exhibiting discrete changes in physical or chemical properties that can be harnessed for biosensing4, controlled drug delivery5, tuning photonic band gaps7, surface wettability and swelling8. The ultimate objective of this field is the biomimicry of intelligent responsiveness such as tactility, vision, camouflage, contractility and flight where complex natural sensors such as skin, eye and muscle seamlessly adapt to environmental stimuli through exquisitely programmed response sequences9. Artificial intelligent sensing systems could be developed by configuring local computational ability into soft polymer constructs and in this way, enabling autonomous sensing, manipulation and processing of stimuli signals without the need for external processors or electrical power. A recent and seminal example of such a polymeric “material that computes” coupled the nonlinear self-oscillations generated through the Belousov–Zhabotinsky reaction in a group of gels with piezoelectric (PZ) cantilevers to perform binary pattern recognition10.Soft polymer thin films, monolayers, colloids, fluids, gels and solids are essential for the design of stimuli–responsive systems and devices13. Binary data input into the cuboid as black and bright stripes of light precisely control the 3-D spatial distribution and self-organization of filaments and in this way, generate the result of an operation, which can be read in a single step at the cuboid output face. Because it is based on nonlinear waves, which are unified through mathematical expression and exhibit strikingly similar dynamics in disparate media14, this system provides proof of a concept that is transferrable to a wide range of nonlinear materials with photoresponses that can be tuned in terms of reversibility, speed, and magnitude16. Equally powerfully, the system functions with incoherent white light—like sunlight—comprising all visible wavelengths and is therefore also operable with compact, inexpensive and visible light-emitting diodes (LEDs), which typically emit incoherent light.Here, we present a self-contained, soft photoresponsive system—comprising only a homogeneous photopolymerizable fluid contained in a transparent cuboid and up to three beams of white incandescent light—that performs multiple operations including the recognition and transfer of binary data, single-step volumetric encoding, and two foundational operations of digital computing—the addition and subtraction of binary strings Fig. . These o−2)11 of white-light filaments confined to a polymer cuboid (~ 1 cm3). Up to three mutually orthogonal populations of filaments are generated through the modulation instability (MI)—stochastically driven filamentation18—of broad incandescent beams (Δλ = 370 nm–1000 nm) launched along the x, y, and z axes of the cuboid, which contains a photopolymerizable organosiloxane fluid20. Mechanisms underlying the MI of optical fields in this and other polymerizable systems have been detailed elsewhere22 and are briefly described below.Figure n) push the nascent system into the nonlinear regime where noise—normally insignificant and random intensity variations—becomes amplified. As these intensity modulations grow, the beam becomes unstable and collapses—typically over 20 min—into large populations of statistically equivalent filaments (see Methods). Each filament induces a high-refractive index channel along its path, populates this cylindrical waveguide as bound optical modes and travels through the medium without diverging. Because the photopolymerization process is irreversible, the waveguides are permanently inscribed in the medium.When launched into the cuboid, a broad incandescent beam with a uniform spatial intensity profile initiates free-radical polymerization along its propagation axis. Corresponding refractive index changes  of bright and dark stripes (bits). Because MI-induced generation of encoders occurs exclusively in the bright bits, the input of binary strings effectively divides the polymer cuboid into micro-volumes—voxels—in which carriers encounter 0, 1, or 2 encoder populations and therefore, respectively, adopt the 0D, 1DH, 1DV or 2D configuration  of the cuboid as a unique combination of the quaternary configurations.For the operations, we designate the filament population propagating along ion Fig. . The conN = 7) with alternating dark (intensity < threshold for MI) and bright (intensity > threshold for MI) intensity  encoders traveling along z only within these voxels; they remain disordered everywhere else , whereas Beam X remains uniform. MI occurs everywhere in Beam X to form carriers but encoders form only within the bright stripes of Beams Y and Z and divide the cuboid into quadrants or four cubic voxels. In the upper left voxel, carriers encounter encoders only along z and assume configuration 1DH, whereas in the lower right voxel, they adopt the 1DV configuration owing to equivalent interactions with encoders along y. Carriers remain disordered in configuration 0D in the lower left voxel where neither Beam Z nor Beam Y suffers MI. In the upper right voxel, all three filament populations self-organize into a cubic lattice and thereby the 2D configuration. At the yz output plane, the distinct configurations of the carrier filaments are quantitatively confirmed through FFT spectra . Conversely, the vertical stroke of “I” corresponds to the 1DV configuration of carriers, resulting from the combination of the “1” bit in Beam Y  and “0” bit in Beam Z . At the intersections of the horizontal and vertical strokes, carriers adopt the 2D configuration due to the combination of “1” bits from both Beams Y and Z . The encoded letter can be visualized in real space and through FFT, distinguished from the background 0D configuration  was initiated through the addition of 0.05 m HCl (1.1 g). The homogenized sol was then sensitized to visible light with the photoinitiator, bis(η5cyclopentandienyl) bis-phenyl) titanium(IV)  and stirred for at least 24 h. The resulting sol, which was transparent and orange in color, was protected from ambient light and filtered through a polytetrafluoroethlyene membrane (pore size = 0.2 μm) immediately prior to use.Photopolymerizable organosiloxane sols were prepared through a previously-described methodλ = 370 nm−1000 nm) were positioned along the x, y, and z axes and each collimated with a planoconvex lens (f.l. = 250 mm) prior to being launched into the photopolymer cuboid sample. Typical optical powers for the beam propagating along x was 0.5 mW and 2.5–3.0 mW for those propagating along y and z. The spatial intensity profile of the beam propagating along x was imaged through a planoconvex lens pair (f.l. = 250 mm) onto a high-resolution charge coupled device (CCD) camera. The photopolymer cuboid consisted of the organosiloxane sol contained in a transparent, glass cuvette (10 mm × 10 mm × 5 mm); the cuvette was sealed with a glass coverslip to ensure that all entrance faces were optically flat. For computing operations, vinyl amplitude masks representing binary strings were attached to the entrance faces of the cell. A typical experiment spanned ∼ 20 min, which corresponded to the time required for filaments to achieve a steady-state, stationary configuration13.All optical experiments were carried out on the custom-built assembly depicted in Supplementary Fig. Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Slowly varying systems are common in physics and control engineering and thus stability analysis for those systems has drawn considerable attention in the literature. This paper uses the “frozen time approach” to derive Lyapunov inequality conditions for the stability of a wide class of slowly varying systems. These conditions refine those developed in (Khalil in Nonlinear Systems,  Numerous techniques for solving slowly varying systems with parameters influenced by environmental conditions have been developed in )Consider a functionwhereandAssume that: The functionzis absolutely continuous on each compact interval ofThere existandsuch thatandfor almost allthat satisfyThenfor all(i)x of stable if for any (ii)x of system uniformly stable if for any (iii)x of system globally attractive if for all (iv)globally asymptotically stable if it is stable and globally attractive.The point m and n. The origin Γ are continuously differentiable with “sufficiently” small derivative (see  is satisfied.For each solutionof the system For anyeach solutionof the system (is continuable onentclass1pt{minima(ii)If Assumption (A4) of [Theorem 9.3] is satisfied Ifis the zero function andis independent of its second component (i.e. for everyis a constant function), then, for anythe originis uniformly stable and is globally asymptotically stable.We prove Results (i), (ii) and (iii) separately as follows.Proof of Result (i): For any x, h, u, and V are continuously differentiable, the functions y and z are absolutely continuous on each compact interval of entclass1pt{minimaentclass1pt{minimaees from , 10), : Assume that H is nondecreasing, we deduce by Claim z.mentclass2pt{minimEach solutionof the system  is  is Each W implies that it attains its infimum on the compact interval (i)If (ii)W is the zero function or can be strictly positive at some element in its domain. When z is the zero function, the property If  □Assume that there is some Thus Eq.  leads toe system –19) is  is \docuBy Claim Proof of Result (iii): Assume that equality  that20\mentclass2pt{minimSuppose that all assumptions of Theoremare satisfied. Ifexists finitely, thenh. □It follows by Eq. , the resWe observe that the special cases u̇. Consider the nonlinear systemThis section uses Theorem equality  is satisH being the identity function.Let e system –22) wit wit\docuequality  is satisϵ. Let δ be a positive constant such thatConsider the change of variables  by Eqs.  and 22)y(⋅)=[y1 is In this example, we have proved that the quadratic Lyapunov function satisfies all conditions of Theorem equality .Simulations: Let t versus t axis and passes through mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimu is treated as a “frozen parameter”. These conditions open the routes to further knowledge on the stability of more generic classes of systems. Numerical simulations for the nonlinear case have been carried out to illustrate the results.We have provided sufficient conditions that ensure the stability of the slowly varying system


We have found that ZnFe2O4 undergoes an intercalation-to-conversion reaction sequence, with the initial intercalation process being size dependent. Larger ZnFe2O4 particles (40 nm) follow a two-phase intercalation reaction. In contrast, a solid-solution transformation dominates the early stages of discharge when the particle size is about 6–9 nm. Using a thermodynamic analysis, we find that the size-dependent kinetics originate from the interfacial energy between the two phases. Furthermore, the conversion reaction in both large and small particles favors {111} planes and follows a core-shell reaction mode. These results elucidate the intrinsic mechanism that permits fast reaction kinetics in smaller nanoparticles.Spinel transition metal oxides (TMOs) have emerged as promising anode materials for lithium-ion batteries. It has been shown that reducing their particle size to nanoscale dimensions benefits overall electrochemical performance. Here, we use in situ transmission electron microscopy to probe the lithiation behavior of spinel ZnFe Reducing particle size of electrode materials to nanoscale dimensions is believed responsible for their enhanced reaction kinetics and electrochemical performance. Here, the authors use in situ transmission electron microscopy to study the dynamic process of the spinel zinc ferrite nanoparticles as a function of size, finding that the intercalation reaction pathway changes below a critical particle size. Reducing materials to nanoscale dimensions also leads to a high surface-to-volume ratio and reduced transport length4. Thus, there have been substantial efforts devoted to the development of nano-sized electrode materials14. Recent studies regarding lithiation of intercalation compounds have indicated that the intrinsic ionic/electronic transport behaviors can be tuned by controlling particle size18. Kobayashi et al. found that the miscibility gap of LiFePO4 cathode materials shrinks as particle size reduces, which results in a homogeneous single-phase transformation pathway for small particles (<100 nm)19. To date, a single-phase transformation pathway in nano-sized electrodes has only been observed in LiFePO4 and the relationship between particle size and reaction pathway has not been clarified in conversion-type electrode materials.There is an ever-increasing demand for lithium-ion batteries that have both large energy density and improved cycling life. With respect to anode materials, the most competitive candidates are those that exhibit alloying and/or conversion reactions because they can provide a much larger capacity than carbonaceous anode materials. A primary challenge to the adoption of these electrode materials is the huge capacity loss that they experience during cycling, which is generally attributed to the strain induced by the large volume changes that occur during lithiation and delithiation. Prior studies have shown that reducing particle size can help to relax the strain, and that this can lead to improved cyclability22. Among TMOs, Fe3O4 has received significant attention due to its nontoxicity, low cost, and high electronic conductivity25. However, reduced Fe0 is not electrochemically active with Li+. Compounds in which another electroactive transition metal ion is substituted for one iron atom in the Fe3O4 structure have recently gained attention28. Zinc ferrite (ZnFe2O4), for example, has a theoretical capacity of 1000 mAhg−1, an improvement over Fe3O4 that results from the fact that metallic Zn can further alloy with additional Li+ after the conversion reaction has occurred29. The process of lithium insertion into spinel zinc ferrite  has been studied by Waszczak et al. using X-ray diffraction30, and can be expressed as: [Zn2+]8a[Fe3+2]16dO4 + Li+ → [Li+Zn2+]16c [Fe3+2]16dO4 + 7Li+ → Zn0 + Fe0 + 4Li2O. Previous studies have reported that ZnFe2O4 powder and thin films suffer poor capacity reversibility and limited capacity when they have a particle size that is of the micron scale32. A more recent study found that even when the particles have nanoscale dimensions (<15 nm), the rate performance and cyclability of ZnFe2O4 still varied significantly with particle size33. These results indicate that in order to improve electrochemical performance it is crucial to explore the origin of particle size effects on reaction kinetics and elucidate the reaction mechanism.Spinel transition metal oxides (TMOs) have been shown to be a high specific capacity anode material for lithium-ion batteries38. Because of the absence of liquid electrolyte, the dry cell configuration enables high spatial resolution, which in turn allows real-time high-resolution TEM (HRTEM) imaging at atomic resolution. Moreover, because these HRTEM images can be subsequently converted to diffractograms using the Fast Fourier transforms (FFT) technique, in situ HRTEM imaging is capable of visualizing phase evolution and correlating it directly with morphological changes. In this work, we investigate the kinetics of small (6–9 nm) vs. large (ca. 40 nm) ZnFe2O4 nanoparticles using in situ TEM. Although both the large and small ZnFe2O4 nanoparticles undergo both an intercalation and a subsequent conversion reaction process, we find that the lithiation pathways of the intercalation process varies with particle size: the small nanoparticles show a solid-solution behavior while the large nanoparticles undergo a two-phase intercalation reaction. We explain the origin of the size-dependent kinetics using a straightforward thermodynamic theory.To this end, we utilize the in situ dry cell transmission electron microscopy (TEM) technique, an approach which allows direct observation of structural changes at the nano- and atomic scale in real time2O4, abbreviated as ZFO hereafter) nanoparticles with different crystalline sizes were synthesized  Li+ initially inserted into 16c sites (~ 1.64 V), (2) further incoming Li+ forcing the Zn2+ at the 8a site to neighboring empty 16c sites (~ 1.58 V), and (3) the generation of the final discharge product (<1 V). The major difference in the kinetic response of the small ZFO (S-ZFO) and large ZFO (L-ZFO) occurs at the lower depth of discharge (DOD), which is associated with the Li+ intercalation process. To investigate the mass transfer in detail, we utilized a galvanostatic intermittent titration technique (GITT) test at a rate of 62.5 mAg−1 to measure the open-circuit voltage (OCV) at different DOD, as depicted in the inset of Fig. 43.Zinc ferrite . This is the same as is observed in the ex situ data, which validates the approach. It is worth noting that two additional peaks appear at the lower DOD for the L-ZFO (<60 s), indicated by the white arrows in Fig. −1 and 5.08 nm−1, which are associated with 222 and 133 reflections of the ordered rock-salt phase . These are indicated by the black dashed line in Fig. + repelling [Zn2+]8a into neighboring empty 16c sites. The structure of the ordered rock-salt phase is equivalent to a standard rock-salt structure, where both kinds of cations equally occupy all of the 16c sites. Upon further lithiation, both peaks gradually decrease in intensity with the emergence of the Li2O 111 and Fe 110 / Zn 101 reflections, thereby indicating that material has undergone the conversion process. In summary, L-ZFO undergoes a two-step reaction during lithiation  whereby the ordered rock-salt intermediate phase is first formed, then subsequently decomposed. Intriguingly, in the S-ZFO, the additional reflections found at 4.04 nm−1 and 5.08 nm−1 in the L-ZFO are not present, as shown by the dotted lines in Fig. 2O phases, indicating that the S-ZFO exhibits a “direct” conversion upon lithiation . For example, the corresponding FFT pattern , incoming Li+ ions push [Zn2+]8a to nearby empty 16c sites, leading to a phase transformation from the spinel  to the ordered rock-salt phase . Further lithiation triggers the subsequent conversion reaction, during which the intermediate phase evolves into the final discharge products of ultra-small metallic Zn and Fe nanoparticles and lithium oxide. In addition, the conversion reaction is observed to generally propagate from the exterior surface  toward the particle center, whereas no preferential lithiation pathway is observed during the initial intercalation process. Based on observations in both real space and reciprocal space, we can conclude that the L-ZFO undergoes a two-step reaction, which initiates with random Li+ intercalation, followed by a “core-shell” pathway upon the subsequent conversion reaction.In order to obtain a more comprehensive understanding of the lithiation process, the microstructural evolution of L-ZFO during lithiation is investigated in real space , which is where the intersection {111} planes occurs. This broadly verifies that the {111} planes are the most preferred reaction interface, as depicted in Fig. Similarly, we investigated the lithiation of S-ZFO in real time . A similar mixture of spinel and rock-salt phases was found in ex situ sample  as the initial state of lithiation, as depicted in Fig. In addition, we conducted ex situ HRTEM imaging on ZFO at an early DOD (xε to a mixture of α phase with concentration xα and β phase with concentration xβ can happen only when the change of Gibbs free energy is negative54. The change of Gibbs free energy (ΔG) can be expressed as Eq.  is the free energy of the phase per unit volume, xn is the concentration of solute atoms of each phase, S is the surface area, σ is surface energy and γ is the energy of the interface between the α and β phases, assuming the surface energy and the volume of a particle remain the same during the phase transformation (ε → α + β). Conventionally, the surface and interfacial energy terms in Eq. (G ≤ 0), where ∆gv is the change of free energy per unit volume after the phase transformation. In other words, particles less than the critical size xZnFe2O4. This is consistent with our in situ TEM observations of the S-ZFO particles, where we only observed a direct conversion process. For the solid-solution process, the reaction kinetics are not limited by the migration speed of phase boundary: this fact may benefit the cycling performance. It is also worth noting that analysis above regarding the size-dependent interfacial energy can be extended to other intercalation oxide electrode materials. Furthermore, during the conversion process, we observed a core-shell reaction mode. The volume expansion induced by the conversion reaction (the shell) produces a compressive strain on the core which can retard further reaction55. In other words, the smaller particle size ZFO particles are, the less compressive strain they suffer. Therefore, during both intercalation and conversion processes, the reaction kinetics are enhanced by small particle sizes.The differences between the intercalation pathways of the large and small ZFO may originate from the difference in interfacial energy between the pre-existing phase and the new phase. These concepts can be explored within a well-established thermodynamic theory: the phase separation from a homogenous concentration d as Eq. :2\documes in Eq. . Thus, tR* <40 nm) the two-phase reaction pathway is suppressed and a solid-solution process dominates the intercalation reaction, which is analogous to the case of LiFePO4. Moreover, we have found that the enhanced electrochemical performance of small electrode materials is not only a result of the reduced Li+ and e− diffusion length but also benefits from the modification of both the electrochemical reaction pathway and the reduction of the strain during the reaction. In addition, we found the subsequent conversion reaction prefers to proceed via {111} planes regardless of particle size: this indicates that crystallographic structure also strongly impacts electrochemical performance and Li+ transport. These results suggest a rational approach for improving the electrochemical performance of other conversion-type electrode materials and provide a criterion for the design and selection of spinel TMOs as anode materials with enhanced rate capability and cycling life.In summary, we have investigated the lithiation pathway of spinel zinc ferrite as a function of particle size using in situ TEM techniques. We have found that below a critical size 2, Fe(NO3)3) were added concurrently to a deionized water solution containing excess triethylamine in an ice bath. The precipitate was collected and vacuum-dried. For small ZnFe2O4, the precipitate was treated hydrothermally at 120 °C for 12 h using DI water as solvent. The small ZnFe2O4 was further prepared by using graphene oxide as a template and Pluronic copolymers with 4400 molecular weight as surfactants. The large ZnFe2O4 sample is obtained by heat treatment of the precipitate at 500 °C for 6 h. The final sample is washed and vacuum-dried.ZnFeN-methyl-2-pyrrolidone and cast onto a copper foil current collector. R2032-type coin cells were assembled inside an argon-filled glove box with the as-prepared composite electrode as cathode and Li metal as anode. A Celgard 2400 monolayer polyethylene separator and 1 M lithium hexafluorophosphate (LiPF6) solution in ethylene carbonate:dimethyl carbonate (DMC) (1:1 in weight) were used as the electrolyte for coin cells. Battery testing was performed using a battery test station (Arbin BT2000) at room temperature. Each current pulse performed during the GITT measurements was followed by a 24-h relaxation period to ensure full relaxation of the OCV.The composite electrode used for electrochemical measurements were prepared with 80 wt% active material, 10 wt% carbon black, and 10 wt% polyvinylidene fluoride in 2O formed on the surface of Li anode which functions as a solid electrolyte and (3) ZnFe2O4 powder dispersed on a half TEM grid with amorphous carbon support work which functions as the positive electrode. After installation, the holder is transferred to the TEM column within a sealed, argon-filled bag in order to avoid air exposure. During operation, a constant negative DC potential was applied between the positive electrode and the Li source in a range of 3~5 V (discharge). The lithiation process was observed in real time by TEM imaging or diffraction mode. The ex situ samples after cycling were directly removed from the coin cell to a DMC solution inside an argon-filled glove box. For sample preparation, the cycled materials were sonicated and dispersed on a TEM grid. In situ and ex situ TEM characterization were done on JEOL JEM-2100F transmission electron microscope equipped with a field-emission electron gun that operated at 200 kV. Analytical EELS and high-resolution HAADF imaging were performed at a Hitachi-2700C scanning transmission electron microscope operated at 200 kV, which is equipped with cold field-emission gun and a probe aberration corrector, yielding a spatial and energy resolution down to 1 Å and 0.35 eV, respectively.The in situ dry cell was assembled into a Nanofactory STM specimen holder inside an argon-filled glove box. The electrochemical cell is composed of three parts: (1) metallic Li that is coated on a piezo-driven tungsten tip that functions as an anode, (2) LiSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


A crystal growth of hydrothermal ZnO nanowires essentially requires a concentration control within so-called “concentration window”, where the anisotropic crystal growth of ZnO nanowires preferentially occurs. Although understanding what exactly determines the width of “concentration window” is important to tailor the anisotropic crystal growth process, the fundamental knowledge as to “concentration window” is still scarce. Here we report the effect of ammonia addition on the width of “concentration window” using conventional hydrothermal ZnO nanowire growth. We found that the ammonia addition substantially narrows the width of “concentration window”. Within the narrow range of zinc complex concentration, we found a significant increase of growth rate (up to 2000 nm/h) of ZnO nanowires. The narrowed “concentration window” and the resultant increased growth rate by the ammonia addition can be understood in terms of synchronized effects of both (1) a reduction of zinc hydroxide complex (precursor) concentration and (2) a fast rate limiting process of ligand exchange between different zinc complexes. Thus, the present knowldege as to “concentration window” will accelerate further tailoring an anisotropic crystal growth of hydrothermal ZnO nanowires. The major useful feature of hydrothermal method is that whole hydrothermal processes can be performed under a relatively low-temperature range less than 100 °C7, which is hardly attainable to other conventional vapor-phase nanowire growth methods13. This low-temperature process expands the application range, particularly when integrating nanowires with other components on various substrates14. Preferential nucleation on ZnO (0001) polar plane is a key process for the anisotropic ZnO nanowire crystal growth17. Previous studies have revealed the roles of various parameters, such as pH, temperature, and ionic species on hydrothermal ZnO nanowire growth27. The origin of anisotropic crystal growth in hydrothermal ZnO nanowires has been interpreted in terms of the variations of ionic species in aqueous solutions and their electrostatic interactions with ZnO crystal planes27. Typically, these hydrothermal ZnO nanowires have been grown under an alkaline condition since divalent Zn ions (Zn2+) do not hydroxylate in acidic environments30. This is because zinc hydroxide complexes (Zn(OH)n) are necessary as precursors in solution for ZnO crystal growth25. A supplying rate of zinc hydroxide complexes to crystal growth interface on (0001) plane essentially determines a growth rate of hydrothermal ZnO nanowire growths25. Therefore, increasing a concentration of zinc hydroxide complexes in solution is effective to increase a growth rate of hydrothermal ZnO nanowires. However, there are inherent limitations for the enhancement of the nanowire growth rate within the framework of conventional strategy based on increasing a precursor concentration. First, the homogeneous nucleation in bulk solution occurs at the relatively low Zn concentration range30. This limits the supplying rate of Zn species to nanowire and the resultant growth rate of ZnO nanowires. To suppress this homogeneous nucleation in solution, an ammonia was added30. The resultant zinc ammonia complexes act as buffer for Zn species, which suppresses the homogeneous nucleation in solution even at relatively high Zn concentration range30. However, there is another inherent limitation of increasing Zn species concentration to keep an anisotropic crystal growth along [0001] direction. This is because a face selective anisotropic growth on (0001) plane emerges within a certain concentration range16. This is so-called as “concentration window”16. Above the concentration range, a crystal growth on 30. These backgrounds motivated us to study the effect of ammonia addition on the width of “concentration window”, which is an essential requirement for the anisotropic nanowire crystal growth.Hydrothermally grown ZnO nanowires have attracted significant attentions of many researchers in academia and industry due to their various functional properties, including optical and electrical properties2O3 (0001) substrate by pulsed laser deposition31. The deposition temperature and the oxygen partial pressure during film deposition are 600 °C and 1 Pa, respectively. As shown in Fig. 16, this is because our early work employed an equimolar ratio of the two, whereas the present work used the constant concentration (15 mM) of hexamethylenetetramine (HMTA) when varying the concentration of zinc nitrate hexahydrate from 0.1 to 40 mM. On the other hand, the ammonia addition lowers the critical concentrations for lateral crystal growth from 5 mM to 2 mM. In addition, as to the nanowire growth rate at the critical concentration for lateral crystal growth, the nanowire growth rate with ammonia addition is 570 nm/h, which is almost 4 times higher than the nanowire growth rate (140 nm/h) without ammonia addition. These concentration data highlight that the ammonia addition narrows the width of “concentration window” with the increased nanowire growth rate.Figure 2+, Zn(NH4)n, Zn(OH)x, ZnNO3+ are shown as the major ionic species in solution. When ammonia is not added, Zn2+ and zinc hydroxide complex ions coexist at pH values ranged from 6 to 10. The ammonia addition (500 mM) results in the increase of zinc ammonia complex ions30 via a ligand exchange process at pH values ranged from 6 to 11. These zinc ammonia complex ions tend to suppress the populations of zinc hydroxide complex ions at pH values ranged from 6 to 10, as increasing the ammonia concentration, as shown in Fig. 30, leading to the increase of critical concentration for nanowire growth as shown in Fig. We calculate the populations of existing ionic species in aqueous solution, as shown in Fig. 2+, ZnNHn, Zn(OH)2+, ZnNHn, Zn(OH)33. It has been reported that IEP value for (0001) plane of ZnO is reported to be 8.7 ± 0.2, which is lower than that for 33. In other words, the effective surface charge of 3)42+, and the major negatively charged ions are NO3−. On the other hand, for experiments without ammonia addition at pH range 6.5–8, the major positively charged ions are -Zn2+, and the major negatively charged ions are NO3−. Since the crystal planes of ZnO at pH range-10–11 should be negatively charged, the major positively charged ions-Zn(NH3)42+ should act as a counter ion, forming an electric double layer onto crystal planes. In this case, the rate limiting process for ZnO nanowire crystal growth is a ligand exchange reaction from zinc ammonia complexes surrounding the nanowire surface to hydroxide complexes. On the other hand, the major counter ions on positively charged crystal planes at pH range 6.5–8 are NO3−, which do not contain Zn. In this case, the diffusion of zinc hydroxide complex ions is the rate limiting process for ZnO nanowire crystal growth. Thus, these rather contrasting results as to a polarity infers that the critical concentration for lateral crystal growth of nanowires is reduced due to the accumulated positively charged Zn source ions at the crystal growth interface.Next we question why the critical concentration for lateral crystal growth of nanowires shifts to the lower value from 5 mM to 2 mM with the ammonia addition. This effect cannot be interpreted in terms of our above model based on reduced molar ratio of zinc hydroxide complex ions. Here we consider the difference of pH values due to the ammonia addition to explain this effect. An effective surface charge of crystal plane varies from a positive value to a negative value below and above an isoelectric point (IEP)x) concentrations. To explain this apparently contradictory observations of a reduced nanowire heterogeneous nucleation rate and an increased growth rate above the nucleation threshold, we consider the surface polarity variation and the distribution of zinc complex ion species. We propose a model based on a fast rate limiting process of a ligand exchange reaction between zinc ammonia complexes and hydroxide complexes (Zn(NH3)x + H2O → Zn(OH)x + NH3OH) from a diffusion dominant process. In the presence of excessive ammonia-500mM, the major zinc complexes are zinc ammonia complexes, as calculated in Fig. 3. Figure Next we explain how the growth rate of ZnO nanowires can be enhanced when we control the zinc complex concentration within “concentration window”. The increased nanowire growth rate seems to be contradictive since a nanowire heterogeneous nucleation rate is reduced by a reduction of precursor complex (Zn(OH)2(low), E2(high), A1(TO), A1(LO), LO(A1 + E1), E2(high)-E2(low) phonon modes, which are prominent peaks of wurtzite ZnO structure36. We utilize E2(high) peak to evaluate the crystalline quality of ZnO nanowires, which is attributed to the vibration of oxygen sublattice35. The full width half maximum (FWHM) of high frequency E2 mode (E2(high)) peaks tends to decrease for ammonia addition and pH control, as shown in Table 3) addition samples. This is due to the effect of narrowed concentration window width, since the occurrence of film growth at nanowire sidewall tends to be not negligible in such narrowed concentration window. All data consistently demonstrate that the ammonia addition and pH control increase the crystallinity of ZnO nanowires. These trends are contradictive to the previous report of which the crystallinity of hydrothermal ZnO nanowires decreases by the ammonia addition37. One possible reason to explain these contradictive results is the difference of lateral growth. Since Watanabe et al. reported that the crystal grown on 38, the decrease of crystallinity in previous study is presumably due to the promotion of crystal growth on 39, the present methodology would further promote such applications of nanowires.Finally, we examine the effect of ammonia addition on the physical properties of fabricated ZnO nanowires. Figure In summary, we show the effect of ammonia addition on the width of “concentration window” using conventional hydrothermal ZnO nanowire growth. We found that the addition of ammonia substantially narrows the width of “concentration window”. Within the narrow range of zinc complex concentration, we found a significant enhancement of growth rate (up to 2000 nm/h) of ZnO nanowires. This concept of narrowed “concentration window” would be useful especially when it is necessary to increase a growth rate of hydrothermal ZnO nanowires without any additional equipment. The narrowed “concentration window” and the resultant growth enhancement by an ammonia addition can be understood in terms of two synchronized effects of both a reduction of zinc hydroxide complex concentration and a fast rate limiting process of ligand exchange between different zinc complexes when the critical nucleation emerges.2O3 substrates by utilizing hydrothermal method. Epitaxial ZnO seed layers (33 nm thickness) are deposited onto single crystalline Al2O3 (0001) substrate by pulsed laser deposition31. The deposition temperature and the oxygen partial pressure during film deposition are 600 °C and 1 Pa, respectively. Fabricated ZnO seed layers are grown along [0001] orientation. Photoresist-AZ5206/Z5200 (2:1) , which is utilized for a mask of nanowire regular array, is coated onto ZnO/Al2O3 substrate by spin-coating at 5000 rpm for 60 s and subsequently bake it at 120 °C for 2 min. After this process, the circular hole array patterns with 520 nm diameter and 400 nm interval distances are fabricated by nanoimprint lithography. The patterned photoresist is then solidified at 120 °C for 5 min. The photoresist residuals at the bottom of patterned hole are removed by reactive ion etching process . The remained photoresist thickness on nonpatterned area is ∼50 nm. Then, we perform the hydrothermal ZnO nanowire growth experiments. Solutions for hydrothermal reactions are mixtures composed of zinc nitrate hexahydrate-Zn(NO3)2•6H2O  and hexamethylenetetramine-HMTA, (CH2)6N4  15 mM. The aqueous solution is prepared at room temperature, and the zinc nitrate hexahydrate concentration is varied from 0.1 to 40 mM to examine the concentration dependence. For the ammonia contained growth condition, 500 mM ammonia aqueous solution is added after mixing zinc nitrate hexahydrate and HMTA. The pH values are measured by using a pH meter . For the pH control condition, HNO3 is carefully added to the ammonia contained growth solution by monitoring the pH value. The patterned substrate is immersed into the solution and kept at 95 °C for a given time. Finally the regular arrays of ZnO nanowires grown along [0001] orientation are obtained. Structural characterizations of fabricated ZnO nanowires are performed by using x-ray diffraction , field emission scanning electron microscopy-SEM  and transmission electron microscopy-TEM . Photoluminescence , Raman spectroscopy  and UV-vis absorption spectra  are measured to examine the properties of nanowires. Visual MINTEQ software is employed to calculate equilibrium concentrations of various ionic species in solution at given temperature and pH ranges.ZnO nanowire regular arrays are employed to measure a growth rate of each crystal plane. The ZnO nanowire regular arrays are fabricated onto spatially patterned photoresist/ZnO seed layer/Al


To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts.CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS , primary progressive MS  and other neurological disease controls, both inflammatory  and non-inflammatory . CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available.There was an increased median CSF κ:λ free light chain (FLC) in all MS groups  but not controls . This ratio predicted Expanded Disability Status Scores (EDSS) progression at 5 years, with a lower median EDSS in the group with high (>10) CSF κ:λ FLC . CSF κ:λ FLC correlated with CSF IgG1 κ:λ  and was intrinsic to CSF plasmablasts .These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification. Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system, usually presenting as a clinically isolated syndrome (CIS). Not all patients convert to clinically definite MS, and in those who do the level of disability accumulation is highly variable.All subjects provided written informed consent, in accordance with the Declaration of Helsinki and were not on disease-modifying therapies at the time of sampling and had not received steroids for at least 3 months prior to sampling.et al.Matched peripheral blood and CSF samples were prospectively collected during routine diagnostic lumbar puncture, and blinding was achieved, with all samples analysed before the diagnosis had been formally determined. Samples were collected from patients with MS fulfilling 2010 revisions of the McDonald criteria,10.1136/jnnp-2018-317947.supp1Supplementary file 1g, 8 min). The cell-free CSF was collected and stored at −80°C, and CSF cells were stained for flow cytometry (see below). Peripheral blood was collected into EDTA-containing tubes  and peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation .CSF was obtained by non-traumatic lumbar puncture and centrifuged . CSF samples were diluted 1:1 with sample diluent buffer and concentrations calculated from a standard curve.Antibodies specific for human IgA, IgG1-Fc, IgG2-Fc, IgG3 hinge, IgG4-Fc and IgM  were coupled to carboxylated xMAP fluorescent microspheres using the xMAP Antibody Coupling Kit  according to the manufacturer’s instructions. For CSF analysis, all incubation steps were for 30 min at room temperature with agitation followed by two washes in phosphate-buffered saline (PBS). CSF samples (50 µL) were added to a mixture of antibody-coated microspheres (50 µL) and 50 µL assay buffer , followed by 50 µL of biotin goat anti-human κ or λ light chain-specific antibodies , then streptavidin R-phycoerythrin . Samples were analysed using a Luminex-200 and xMAP software, with final concentrations calculated from standard curves.RNA isolation and cDNA synthesis from CSF cell pellets were performed using the μMACS One-step cDNA Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Synthesised cDNA was eluted from the column and collected with 50 µL cDNA elution buffer. Immunoglobulin Kappa Variable (IGKV) and Immunoglobulin Lambda Variable (IGLV) sequencing of cDNA were performed by Adaptive Biotechnologies  using the immunoSEQ survey level assay.Statistical analyses were performed using GraphPad Prism V.7.0 . All data sets were determined to require non-parametric testing following use of the D’Agostino and Pearson normality test and were two tailed.Ig FLC, in addition to OCB, are present in the CSF of patients with MS and are highly sensitive and specific in supporting a diagnosis of MS.The highly variable CSF κ:λ FLC ratio in all MS groups led us to assess whether this ratio might be prognostic for disability accumulation. EDSS were determined at a 5-year follow-up time point following the diagnostic lumbar puncture. We divided the cohort into high (>10) and low (<10) CSF κ:λ FLC ratio groups based on approximately equal numbers in each group. There was a significantly lower EDSS at follow-up in the group of individuals with a high CSF κ:λ FLC ratio, with the lower ratio group accumulating greater disability . There wThere are several possible explanations for the bias towards κFLC in the CSF of patients with MS, unrelated to their antigenic specificity, including assay-dependent sensitivity for either κ or λ, in the FLC and/or intact Ig. We therefore examined the κ and λ light chains present in the intact IgG1, IgG2, IgG3, IgG4, IgA and IgM in CSF of patients with MS. It was clear that the observed bias towards CSF κFLC was recapitulated when examining IgG1κ and IgG1λ, with a strong correlation between the IgG1 κ:λ ratio and FLC κ:λ ratio . There wAlthough the past two decades have seen major advances in the sophistication and availability of diagnostic modalities used in MS (particularly MRI), the prognostic heterogeneity of MS makes temporal disability prediction extremely difficult for any given individual.25et al failed to detect any relationship to disability progression, possibly due to a lower frequency of high CSF κ:λ FLC ratio individuals in their study and/or the shorter follow-up period examined. Our data show that for patients with MS demonstrating a low CSF κ:λ light chain ratio at diagnosis, the combined profile of greater conversion from CIS, higher relapse rates, increased disability progression and greater requirement for therapeutic intervention suggest that this immunological feature of MS is highly predictive of aggressive disease and poor prognosis, although it is worth noting that only disability progression was significantly different.In this study, we demonstrated, for the first time, that the ratio of κ:λ Ig FLC measured at the time of diagnostic lumbar puncture predicts EDSS disability at 5 years postdiagnosis. This is a critical advance on previous studies indicating that the ratio of κ:λ FLC in the CSF might predict the conversion from CIS to CDMS.The presence of B cells and their secreted antibody and FLC in the CSF and brain tissue is well established.34 35The availability of a prognostic marker in the CSF at the time of diagnosis is of clear utility. There are now many highly efficacious therapies for MS, making a rational choice difficult for any individual patient, particularly as some are associated with a significant adverse event profile. MRI protocols are now well established for the diagnosis and therapeutic monitoring of patients with MS. The use of CSF κ and λ assay measurements at diagnosis may help to identify patients with a poor prognosis, justifying the use of highly effective therapies, with potential benefit on long-term outcomes. Larger independent studies would be required to validate the findings and determine the ultimate clinical utility of these measurements.


The properly designed homogeneous metasurface can be dynamically tuned from a broadband absorber to a reflecting surface due to the insulator-to-metal transition of VO2. When VO2 is in its insulating state, the metasurface can efficiently absorb the normally incident THz wave in the frequency range of 0.535–1.3 THz with the average absorption of ~97.2%. Once the VO2 is heated up and switched to its fully metallic state, the designed metasurface exhibits broadband and efficient reflection (>80%) in the frequency range from 0.5 to 1.3 THz. Capitalizing on such meta-atom design, we further extend the functionalities by introducing phase-gradients when VO2 is in its fully metallic state and consequently achieve polarization-insensitive beam-steering and polarization-splitting, while maintaining broadband absorption when VO2 is in insulating state.In this paper, we design a type of switchable metasurfaces by employing vanadium dioxide (VO By designing artificial meta-atoms and positioning them properly in a periodic or aperiodic manner, arbitrary optical responses with different local amplitudes, phases, and polarizations can be achieved. Therefore, a variety of novel physics phenomena and attractive EM devices have been demonstrated with metasurfaces, such as generalized Snell’s law14, surface waves coupling18, focusing lenses22, holograms25, absorbers27, as well as polarization generation and detection34. Besides the surface-confined configurations, compact footprints and arbitrary wavefront control, metasurfaces are capable of effectively integrating multiple distinct functionalities into one single device44, superior to the aforementioned metasurfaces with single functionality.The capability of manipulating electromagnetic (EM) waves is of critical importance to many EM-based devices and systems in both fundamental and applied sciences. However, the typical EM devices relying on the gradually accumulated phase during wave propagation are suffering from curved or spatially-distributed shapes and bulky configurations, which limit their potential applications in EM integration for the ever-increasing demands on data-storage capacity and information processing speed. Additionally, EM devices that possess multiple diversified functionalities yet with miniaturized configurations are greatly desired. In recent years, metasurfaces, the two-dimensional analogues of metasurfaces that consist of planar arrays of nanostructures, have become an emerging research area due to their unprecedented control over scattered EM fields within the compact and ultrathin geometries, thereby releasing the nano-fabrication and decreasing the loss to some extent54, vanadium dioxide (VO2)71. In spite of the significant achievements in PCMs integrated metasurfaces with dynamic responses, switchable metasurfaces exhibiting totally different functionalities that can be actively switched over a wide frequency range are still largely unexplored.However, in most of these approaches, the metasurfaces possess fixed functionalities once been fabricated, lacking the switchable ability and thus hindering practical applications. As such, it is highly desired to achieve switchable metasurfaces that are capable of integrating two or more diversified functionalities together and actively tuning different functionalities at the same time. To realize switchable metasurfaces, an effective way is to incorporate standard metasurfaces with phase-change materials (PCMs), such as chalcogenide GeSbTe(GST) alloys2, which possess tunable and diversified functionalities in the terahertz (THz) frequencies. The designed homogeneous metasurface can be dynamically tuned from a broadband absorber to a reflecting surface by thermally stimulus. At room temperature, VO2 is an insulator and the metasurface can efficiently absorb the incident THz wave at normal incidence in the frequency range of 0.535–1.3 THz with the average absorption as high as ~97.2%. If VO2 is heated up and switched to its fully metallic state, the metasurface could reflect the incident wave efficiently and the reflectivity is above 80% in the frequency spectrum ranging from 0.5 to 1.3 THz. Furthermore, we introduce phase-gradients by varying the topmost VO2 antennas of the metasurface when VO2 is in its fully metallic state and consequently realize beam-steering functionalities. Meanwhile, the metasurface can work as an efficient absorber when VO2 is in its insulating state.In this paper, we propose a type of switchable metasurfaces by employing the insulator-to-metal transition in VO2 integrated THz metasurfaces, which is composed of six different functional layers. Specifically, the functional layers from top to bottom are periodic VO2 brick-shaped antennas, a polyimide spacer layer, double chrome (Cr) square ring resonators (SRRs), a continuous VO2 layer, a second polyimide layer and a bottom Cr substrate  resonator that consists of Cr SRRs, the second polyimide spacer, and the bottom Cr substrate. Therefore, the incident THz wave can transmit through the topmost VO2 brick array and interact with Cr SRRs, resulting in highly-efficient absorption. On the other hand, once the temperature is increased above the phase-change temperature of Tc = ~340 K, VO2 undergoes an insulator-to-metal transition and gains a high conductivity56. When VO2 is in its fully metallic state, the conductivity σ can reach as high as 2 × 105 S/m61. In this case, the VO2 brick-shaped antennas interact strongly with the THz wave and the continuous VO2 film can block all the transmission, thereby forming another MIM cavity composed of VO2 brick array, the polyimide spacer and the VO2 continuous film. By tailoring the dimensions of such MIM cavity in which VO2 functions as metal, one can realize reflecting surface and consequently manipulate the reflected wave at will.Figure ate Fig. . At roomx- and y-directions, and the medium above the unit cell is chosen to be air and truncated using a perfectly matched layer (PML) to minimize reflection. An x- or y-polarized plane wave is normally incident on the top surface as the excitation source along the -z-direction. In our simulations, the relative permittivity of VO2 is described by Drude model ε∞ = 12, the plasma frequency ωp = 1.4 × 1015 s−1, the damping frequency ωd = 5.75 × 1013 s−1, and σ0 = 3 × 105 S/m61. The conductivity σ of VO2 is 200 S/m and 2 × 105 S/m in insulating and fully metallic states corresponding to the temperature of ~298 K (room temperature) and ~358 K, respectively61. The thickness of VO2 structures is t = 1 μm. The thickness of Cr SRRs is d = 0.3 μm and the conductivity of Cr is set to be 2.2 × 105 S/m72. The polyimide spacer layer is considered to be a lossy dielectric with a constant relative permittivity of ε = 2.4 + 0.005i. The other geometrical dimensions are p = 90 μm, lx = ly = 30 μm, td1 = 30 μm, td2 = 34 μm, a = 55 μm, b = 36 μm, and w = 1 μm, respectively.To begin with, we analyze a three-dimensional (3D) homogeneous metasurface composed of periodic meta-atoms shown in Fig. 2 is in different states. From Fig. 2 incorporated metasurface can efficiently absorb the normally incident THz wave over an ultra-broadband spectrum when VO2 is in its insulating state at room temperature. In particular, the calculated absorption is exceeding 90% in the frequency range of 0.535–1.3 THz with the average absorption reaching ~97.2%. Such broadband and efficient absorption is ascribed to the bottom MIM cavity formed by the Cr SRRs, the continuous VO2 film, the second polyimide spacer and the bottom continuous Cr film, which can interact effectively with the incident wave. It is worth noticing that such MIM cavity can also be regarded as an asymmetric Fabry–Perot resonator73. When the THz wave impinges on the structure, the interface between Cr SRRs and the continuous VO2 film induces significant reflection/transmission phase. Thus, the conventional Fabry–Perot resonance condition will be modified as the propagation phase within the dielectric layer is no longer dominating.Figure f = 0.757 and 1.212 THz, respectively  while the other parameters are kept fixed. As shown in Fig. 2 is in its insulating state, the reflectivity is sufficiently low (less than 2%) regardless of the varied topmost VO2 bricks with different dimensions, which is consistent with the previous discussion that the absorption is mainly determined by the SRRs-polymer-Cr cavity. In contrast to the ultra-low and nearly-constant reflectivity when VO2 is in the insulating state at room temperature, the reflectivity and phase response of each unit cell can be engineered by changing the dimensions of VO2 bricks at f = 0.8 THz when VO2 is in its fully metallic state  resonance within the VO2-polyimide-VO2 cavity. Meanwhile, the two degrees of freedom (dimensions lx and ly) allow us to vary the phases of reflected polarizations approximately within the whole phase space of ~2π44. Close to the GSP resonance, the reflection phase varies rapidly over the range of π. Away from the resonance, the reflection phase is affected by the retardation of the incident wave when being reflected by the bottom reflected or by the process of coupling into GSPs. Here it should be mentioned that for larger lx the reflection phase of x-polarized wave does depend on the value of ly, which is ascribed to the coupling between neighboring elements. Therefore, one should consider this dependence when designing phase-gradients.In addition to the switchable functionalities between broadband absorption and highly-efficient reflection with a homogeneous metasurface utilizing single meta-atom, the proposed metasurface can be further engineered with phase-gradients, thereby allowing for arbitrary beam-steering of the reflected THz wave when VOate Fig. . For exa2 bricks in a supercell to create identical linear phase-gradients for both x- and y-polarizations along the x-direction when VO2 is in its metallic state. In this case, we can achieve polarization-insensitive anomalous reflection while maintaining broadband efficient absorption when VO2 is in its insulting state. Figure p = 90 μm, where four different square-shaped meta-atoms are selected from the intersection points between the solid and dashed lines in Fig. x-direction, which is large enough to avoid generating driven surface waves15. When VO2 is an insulator at room temperature, the selected four meta-atoms have rather low reflectivity and the reflection phase plays little effect on the reflected field. Therefore, the resulting supercell can efficiently absorb the incident THz wave at the design frequency of f = 0.8 THz for x-polarization with the absorption reaching as high as 98.7%  wafer, and the process is presented as Fig. 2 layer is magneton sputtered and annealed at ~450 °C; (d) photoresist is spin coated and optical lithography; (e) 300-nm-thick Cr is deposed using e-beam evaporation; (f) the photoresist is dissolved by a lift-off process to form the Cr SRRs; (g) 34-μm-thick polyimide layer is spin coated and cured at ~300 °C; (h) photoresist is spin coated and optical lithography; (i) 1-μm-thick VO2 layer is magneton sputtered; (j) the photoresist is dissolved by a lift-off process to form the VO2 bricks and the remaining VO2 is then annealed at ~450 °C. As a final comment, it should be noted that our device would be insensitive to the proper multi-layer alignment errors during the fabrication since the proposed structure can be regarded as two independent devices isolated by the continuous VO2 film when VO2 is in different states  in the frequency range from 0.5 to 1.3 THz. Based on the meta-atom design, we further extend the functionalities by introducing phase-gradients when VO2 is in its fully metallic state and consequently achieve polarization-insensitive beam-steering and polarization-splitting, while maintaining broadband absorption in insulating state. It should be emphasized that the achieved two functionalities exhibit comparable and even superior performance once compared with the existing metasurface-based absorbers72 and beam-steerers76, in terms of efficiencies and operating bandwidth. Additionally, the fully insulator-to-metal transition makes VO2 an excellent candidate to bridge two isolated devices together, which is hard to achieve with other phase-change materials possessing limited tunability. Although VO2 only exhibits volatile switching, it is easier to realize reversible tuning between insulating and metallic states with lower power consumption, superior to GST alloys which need high power density and protective layer. The proposed switchable THz metasurfaces can enable advanced applications in THz society, such as temperature sensor and imaging.The designed homogeneous metasurface can be dynamically switched from a broadband absorber to an efficient reflector by altering the working temperature. When VO


Despite advances in directing the assembly of biomacromolecules into well-defined nanostructures, leveraging pathway complexity of molecular disorder to order transition while bridging materials fabrication from nano- to macroscale remains a challenge. Here, we present templated crystallization of structural proteins to nanofabricate hierarchically structured materials up to centimeter scale, using silk fibroin as an example. The process involves the use of ordered peptide supramolecular assemblies as templates to direct the folding and assembly of silk fibroin into nanofibrillar structures. Silk polymorphs can be engineered by varying the peptide seeds used. Modulation of the relative concentration between silk fibroin and peptide seeds, silk fibroin molecular weight and pH allows control over nanofibrils morphologies and mechanical properties. Finally, facile integration of the bottom-up templated crystallization with emerging top-down techniques enables the generation of macroscopic nanostructured materials with potential applications in information storage/encryption, surface functionalization, and printable three-dimensional constructs of customized architecture and controlled anisotropy. Despite recent progress in directed assembly of protein molecules into well-defined nanostructures, bridging materials fabrication from nano- to macroscale remains a challenge. Here, using silk fibroin as an example, the authors demonstrate templated crystallization to drive hierarchical materials growth from disordered molecules all the way up to centimeter scale. Examples of such materials include wood, bone, tendon, silk, and mussel threads, which have constantly been a source of inspiration for the fabrication of synthetic materials that mimic the structural organization of their natural counterparts to integrate, for example, augmented mechanical properties2, selective mass transport3, energy storage4, and stimuli-responsiveness5. In natural systems, hierarchical assembly of nanoscale building blocks is at the core of materials generation and often involves the use of templates to direct the assembly pathway6. The same principle underpins amyloid fibrils formation through pathogen (such as prions and misfolded Aβ oligomers) templating, which follows a nucleation-elongation model8. Beyond the fundamental studies on amyloidogenesis for treatment of various neurodegenerative diseases9, the field of functional amyloid materials10 has also expanded, with interesting applications in underwater adhesives11, enzyme sensors12, and water purification membranes13. Inspired by protein fibrils formation with kinetic control enabled by proper templates, many novel biomimetic supramolecular assembly pathways were also developed for synthetic polymers, resulting in polymers of extremely narrow polydispersity15 and complex architectures with distinct functional domains and electronic properties18.Through millions of years, the natural world has developed unique methods to direct the disorder to order transition of biomacromolecules, which then allowed generation of complex materials with a variety of structural hierarchy and functionality6, bridging materials fabrication from nano- to macroscale through a precise regulation over the disorder to order transition of biomacromolecues remains an open challenge. This is particularly true for protein-based materials, where lack of sufficient understanding of their sophisticated interactions at the molecular level makes controlled bottom–up assembly quite demanding. In addition, the delicate nature of proteins limits the use of harsh reagents and conditions commonly involved in traditional top–down nanofabrication techniques. Nonetheless, recent reinvention of structural proteins  as technical materials19 have generated some exciting progress, although the materials achieved so far still have nontrivial limitations in either the control of protein folding and assembly at the molecular level21 or the scale-up of materials fabrication to macroscale22. Here, we report a template-based bottom–up approach to guide hierarchical materials growth from disordered molecules all the way up to macroscale, a process hereby termed templated crystallization. Using silk fibroin as an example, templated crystallization refers to the use of ordered peptide supramolecular assemblies to drive a phase transformation of silk fibroin from unordered to ordered conformations, thereby enabling further assembly of the silk fibroin chains into nanostructured materials . Silk polymorphs can be engineered through templated crystallization on either homologous or heterologous peptide seeds of distinct molecular structures. Multiple parameters including the relative concentration between silk fibroin and peptide seeds, silk fibroin molecular weight (MW), and pH are investigated to fine tune the assembly kinetics and nanomechanical properties. Finally, facile integration of the templated crystallization process with emerging top–down approaches enables nanofabrication of centimeter scale nanostructured functional materials such as (i) a free-standing patterned silk film with different silk polymorphs embedded at predesigned areas, which can be used to store or encrypt information; (ii) surfaces functionalized with mesoporous and nanofibrillar silk fibroin networks obtained by epitaxial-like growth of silk fibroin on pre-deposited peptide seeds; and (iii) three-dimensional printable silk constructs with customized architecture at the macroscale and controlled anisotropy at the microscale.Despite recent progress in directed molecular assembly into well-defined nanostructuresn sequence of the hydrophobic domains of silk fibroin heavy chain 2 thereafter for brevity) was chosen owing to its ability to self-assemble into regular nanowhisker-like supramolecular oligomers of dimensions around 200 × 20 × 4 nm  show maintenance of the oligomerization state and minimal size growth of the nanowhiskers over time  forms ordered supramolecular assemblies, 2) binds to silk fibroin molecules, typically through the formation of hydrogen bonds or hydrophobic interactions, and 3) shares similar isoelectric point and pH stability with silk fibroin. In this parameter space, the highly repetitive (GAGAGS)ain Fig.  provides forms or nm Fig. . Structud24 Fig. , and the 25 Fig. . Circulater Fig.  show a p binds to shares s2 nanowhiskers with a well-defined β-sheet structure and an amino-acid sequence that composes > 40% of the silk fibroin heavy chain , a fluorescent probe that undergoes an emission blue shift when it binds to hydrophobic surfaces. As depicted in Fig. 28, and the ANS emission intensity increased when silk fibroin was exposed to nanowhiskers. This phenomenon correlates positively with the relative concentration of nanowhiskers and indicates an enhanced exposure of silk’s hydrophobic surfaces, which leads to β-sheet formation and protein assembly driven by hydrophobic interactions. Size measurements by DLS corroborated the nanowhisker-driven molecular assembly process  (GAGSGA)2 nanowhiskers caused a fast and dramatic increase in the measured hydrodynamic radius, reaching a plateau at around day 2.The (GAGSGA)ain Fig.  were fouess Fig. : compare2 nanowhiskers, depicting a continuous increase in the β-sheet content of silk fibroin until reaching an equilibrium. CD spectra were then used to evaluate the kinetics of protein assembly29. Fraction completion of silk fibroin assembly at any given time and nanowhisker concentration , plotted and fitted with an empirical logistic function30k is the growth rate, t0.5 is the time at 50% protein assembly and the lag time is calculated as31, indicating possible occurrence of molecular events during the lag phase of silk fibroin assembly that resemble the primary nucleation and surface-catalyzed secondary nucleation in the Aβ42 aggregation. Other models such as a variation of the Johnson-Mehl-Avrami-Kolmogorov equation29 can also well describe the kinetics of templated silk fibroin assembly  concentration with the same relative amount of (GAGSGA)2 nanowhiskers added. As shown in the CD spectra and corresponding atomic force micrographs in Supplementary Fig. 2 nanowhiskers) occurred only at 0.1 and 1 mg/ml  usually correlates positively with monomer concentration32  and third (I3) pyrene emission peaks (intensity averaged over 371–376 nm for I1 and over 398–403 nm for I3) has been used in silk proteins to evaluate the formation of micelle-like structures33. Here, we found that I1/I3 decreases linearly with the logarithm of silk fibroin concentration until reaching an inflection point at ~1 mg/ml where the slope of the linear relationship changes significantly 2-silk nanocomplexes at different assembly stages were measured from the TEM images and summarized in Fig. 2 nanowhiskers by adsorption of silk fibroin in the first hour of the assembly process . The quickly formed (GAGSGA)2-silk nanocomplexes ers Fig.  and thosers Fig.  corroborBombyx mori cocoons for 30, 45, and 60 mins 34, along with native silk fibroin extracted from the gland of B. mori silkworms at the 5th instar were used in templated crystallization. The MW distributions of the four silk fibroins were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), as shown in Supplementary Fig. 35, from which the MW distributions of all three regenerated silk fibroins were quantitatively determined  concentration Fig.  and silk2 solution at 80 ˚C for 20 mins results in disassembly of the nanowhiskers23, yielding separated and unstructured peptides  thereafter. Similar to (GAGSGA)2, HBSP is also able to undergo supramolecular self-assembly to form oligomers 2 counterparts in terms of width and height. The elongated HBSP oligomers also appear to be more flexible owing to the curved morphologies, as compared to the straight and thick (GAGSGA)2 nanowhiskers. CD spectra of HBSP solution over time  observed in the spectrum of silk fibroin templated by HBSP, compared with the spectrum of naturally aged silk.To investigate the structural differences of the end materials templated by (GAGSGA)eds Fig. . The speeds Fig. . The res40. For the peptide seeds deposited on mica, AM-FM mapping reported Young’s moduli of 4.18 ± 0.71 GPa  for (GAGSGA)2 nanowhiskers  films that have a similar Young’s modulus to silk fibroin -based bimodal tapping mode force microscopy, namely amplitude modulation-frequency modulation (AM-FM) mappingers Fig.  and 4.41ers Fig. , indicaters Fig. , as comp. 2 Fig. , corrobo. 2 Fig.  as the nime Fig. , which iime Fig. , indicat45. Nonetheless, the extremely small indentation depths  applied by the AFM tips makes the modulus measurement by AM-FM prone to errors and variations caused by surface effects such as roughness, adhesion, and contaminations39, so comparison of the absolute modulus values measured by different techniques should be performed with caution.Following the study of how silk fibroin chain length and polydispersity affect the templated crystallization process, mechanical properties of nanofibrils assembled from native silk fibroin and regenerated silk fibroins of different degumming time were also measured. As presented in Supplementary Fig. 2 nanowhiskers and the templated silk nanofibrils in hydrated and dried state 2-templated silk nanofibrils of GPa scale stiffness, the same materials in hydrated state become much softer with the stiffness dropping down to 1–2 MPa Templated crystallization as a bottom–up molecular assembly process can be easily combined with various top–down manufacturing techniques to generate macroscopic hierarchical materials with complex shapes and predesigned nanostructures. In particular, two different strategies were pursued: controlled deposition of peptide seeds on a solid surface allows to topographically regulate the growth of silk nanofibrils and form a nanostructured protein overlayer, in a way that resembles epitaxial growth of inorganic materials. Alternatively, suspensions or gels composed of silk nanofibrils obtained by templated crystallization can be developed as printable inks and integrated with printing techniques to fabricate three-dimensional constructs with customized architecture at the macroscale and controlled anisotropy at the microscale. The results are summarized in Fig. 2 nanowhiskers and HBSP nanoassemblies were selectively deposited on polydimethylsiloxane (PDMS) following a predesigned pattern . Representative spectra at the three asterisks in the FTIR map were included in the right panel of Fig. 2-patterned areas, HBSP-patterned areas, and non-patterned areas were measured to be 68, 51, and 16%, respectively ern Fig. . The pepern Fig.  inset, weds Fig. . The fabeds Fig. , inset weds Fig. , left wieds Fig.  left repely Fig. , right. ely Fig. . Alternaely Fig.  obtained46 to deposit nanofibrillar mats with a spatial resolution of 10 microns in the x–y plane and 30 nm in the z direction  in the silk nanofibrils templated by different peptide seeds (upper and lower panel correspond to silk nanofibrils templated by (GAGSGA)2 and HBSP, respectively). Rheological studies of the nanofibrillar gels show that both gels exhibit a shear-thinning behavior with a similar viscosity of ~ 30 Pa∙s at shear rate of 0.1 s−1 50 and air as a result of the mesoporous feature, along with the orientational arrangement of silk nanofibrils20 induced by the shear-force during the printing process.Another demonstration for scaling up nanomaterials fabrication to macroscale involves the development of printable inks that can be used to build three-dimensional nanostructured constructs with customized architecture at the macroscale and controlled anisotropy at the microscale. The process to achieve such materials encompasses (1) formation of silk fibroin nanofibrils in suspension through templated crystallization, (2) concentrating the suspension to achieve desired rheological properties, (3) use of the obtained bioink in combination with various printing techniques (4) tuning the printing parameters to achieve nanofibrils alignment at the microscale. As a first proof-of-concept, silk nanofibrils suspension was coupled with inkjet printingion Fig. . To enabion Fig. . The gelion Fig. , where ts−1 Fig. . The stoTemplated crystallization enables the design and fabrication of nanostructured silk fibroin materials through a precise regulation over protein disorder to order transition. Facile integration of templated crystallization with emerging top–down techniques allows the engineering of macroscopic hierarchical protein-based materials with control over molecular structures, surface morphology, mesoporosity, and mechanical properties. The capability to grow and print silk nanofibrils of different polymorphs on demand and into customized shapes is essential for engineering the next generation smart and compartmentalized devices. Nanomanufacturing with templated crystallization also promises to be extended to other natural or synthetic polymer systems where highly ordered molecular structures can be achieved, facilitating nanofabrication of multiscale and complex materials with programmable functions.All the peptides used in this study were synthesized by GenScript , with free N- and C termini. In brief, peptides were synthetized using standard Fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis and purified by reverse-phase high-performance liquid chromatography to a purity of 95% or higher. All peptides were dissolved in pure Milli-Q water.B. mori cocoons following established protocols. In brief, chopped silk cocoons were degummed in a boiling 0.02 m sodium carbonate solution for 30–60 mins to remove the sericin. The obtained silk fibers were then washed with Milli-Q water for several times followed by overnight drying. The dried silk fibers were dissolved in 9.3 m lithium bromide for 4 h at 60 ˚C followed by dialysis against Milli-Q water for 2 days with constant changing of water. The resulting silk fibroin solution was then purified by sequential centrifugation at 4800 × g and 20,000 × g for 25 mins each, yielding a final silk fibroin solution of ~ 7 wt%. The regenerated silk fibroin solution was then stored at 4 ˚C until use. Unless otherwise noted in the text, the silk fibroin used in all experiments were degummed for 30 mins (30 mb).The regenerated silk fibroin used in this study was extracted from B. mori silkworms at the beginning of 5th instar. In brief, silkworms were dissected for their glands, which were then transferred to cold (4 ˚C) Milli-Q water. After 5 mins, the epithelial membrane was carefully peeled off from the gland using tweezers. The posterior and anterior parts of the glands were then separated off and discarded. The middle glands were washed in Milli-Q water for another 30 mins to remove the sericin coating, after which they were stored at 4 ˚C and used within a day.Native silk fibroin was extracted from The MW distributions of native and regenerated silk fibroins were measured by SDS–PAGE using NuPAGE 3–8% Tris-Acetate Protein Gels (Invitrogen). Sample preparation followed the protocols from the gel manufacturer. HiMark Pre-stained Protein Standard (Invitrogen) was used as the ladder. The gel was run under reducing conditions for 60 mins at 150 V, stained with a Colloidal Blue staining kit (Invitrogen) and imaged by ChemiDoc (Bio-Rad). To determine the MW distributions from the gel image, pixel intensity as a function of lane position was obtained using Matlab. The reference lane containing protein standards was used to establish a conversion between pixel position and molecular weight. Pixel intensity was normalized to be in the range of 0–1 and a threshold of pixel intensity was applied, above which the pixels were considered as background.2 oligomer was constructed by threading the dodecapeptide sequence onto the structure of a poly-(Gly-Ala) β-sheet (Protein Data Bank identification code 2slk) using UCSF Chimera and Modeller. The system was then equilibrated in a transferable intermolecular potential 3 P (TIP3P) explicit water box. Simulations were run for 60 ns with a time-step of 2 fs at constant temperature (300 K) and pressure (1 bar) using GROMACS. The force field used was CHARMM27. The stability of the β-sheet assembly was verified from hydrogen bond dynamics and root mean squared deviation data obtained from the molecular dynamics trajectory.The initial model for the (GAGSGA)Scanning electron microscopy (SEM) images were obtained using a Zeiss Merlin High-resolution SEM. Samples containing liquid were first quickly frozen in liquid nitrogen to minimize changes in mesoscopic structures, followed by lyophilization at − 105 ˚C and vacuum condition using a freeze drier . All samples were sputtered coated with a thin layer of gold (5 nm) before imaging.ϕ = 10 mm, Ted Pella) and dried before imaging. All morphological characterization were performed by tapping mode in air, at a scan rate of 2.0 Hz and a resolution of 256 × 256 pixels per image, using AC160TS-R3 (Olympus) probes.AFM images were obtained using a Cypher S AFM (Asylum Research). Samples were diluted to around 30 μg/ml (for silk fibroin) and 15 μg/ml (for peptide oligomers) to have the optimal feature densities on the substrate for topography imaging. A 10 μL aliquot of diluted samples was dropped on freshly cleaved mica surface  was deposited on glow discharged continuous-film carbon-coated copper grids (Ted Pella Inc.), wicked off after 2 mins, and then stained with 5 μL Nano-W  for 1 min before being wicked off. The grid was then left to dry before imaging. Measurements of nanofibrils lengths and widths were performed using DigitalMicrograph (Gatan Inc.).Negatively stained transmission electron microscopy (TEM) images were recorded using a Tecnai GCryo-TEM imaging was performed on a JEOL 2100 FEG microscope operated at 200 kV. For sample preparation, 3 μL of each sample solution was deposited on a standard copper grid coated with a lacey carbon film and an ultrathin carbon film on top (Ted Pella Inc.), which was then plunge freezed using a Gatan Cryoplunge 3. Grids were stored in liquid nitrogen until being loaded into a pre-chilled cryogenic sample holder Gatan 626. The sample holder tip and grid were kept in liquid nitrogen throughout the transferring process and subsequent imaging. Minimum electron dose was used to avoid quick sample damage during the imaging. All images were recorded with a Gatan Ultrascan 2k × 2k CCD camera.Fluorescence and polarization microscopy images were obtained on a Nikon TE2000-E inverted microscope. For fluorescence imaging, white light was coupled with DAPI and FITC filter cube sets. For birefringence imaging, two linear polarizers (one placed at the light source and one above the sample) were used to illuminate the samples with cross-polarized light.2 silicon wafers and dried naturally on the bench. Crystalline silk films were produced by soaking the silicon-supported amorphous silk films in 80% ethanol-water for 3 mins and then quickly dried under gentle air flow. PS films were fabricated by spin coating 20 wt% PS-xylene suspension on 1 × 1 cm2 silicon wafers at 3000 rpm for 1 mins, followed by heating at 100 ˚C for an hour to complete the solvent evaporation. Each type of sample was prepared and indented in triplets to ensure good fabrication repeatability. For each sample, indentation was performed at a total of 49 points (7 × 7 grid with an increment of 20 μm in both directions) to ensure statistical reliability of the modulus measurements.Nanoindentaion measurements were performed on a Hysitron TriboIndenter with a nanoDMA transducer (Bruker). Samples were indented in load control mode with a peak force of 500 μN and a standard load-peak hold-unload function. Reduced modulus was calculated by fitting the unloading data  using the Oliver-Pharr method and converted to Young’s modulus assuming a Poisson’s ratio of 0.33 for both silk and PS. For sample preparation, amorphous silk films were produced by drop casting 6 wt% silk fibroin suspension on 1 × 1 cm2) on silicon was used as the reference sample (the Young’s modulus of which was 6.3 GPa based on the nanoindentation results) to calibrate the tip radius before each sample scan. Sample preparation, scan rate and image resolution were the same as that described for topography imaging in air. For each modulus map, histograms of the moduli at pixel positions corresponding to the peptide assemblies and silk nanofibrils were plotted and fitted with either a normal or log-normal distribution to determine the modulus average and standard deviation.AM-FM viscoelastic mapping was performed on a Cypher S AFM (Asylum Research) using AC160TS-R3 (Olympus) probes. Cantilever spring constant and sensitivity were calibrated using the built-in GetReal function of the Asylum Research software. A spin-coated PS film  and settled for 30 mins in a humid chamber before imaging. Cantilever spring constant and sensitivity in air were first calibrated using the built-in GetReal function of the Asylum Research software before immersing the cantilever in water, followed by a calculation of the cantilever sensitivity in water through capturing and fitting the thermal spectrum of the cantilever in water. All force maps were acquired at a scan rate of 0.5 Hz and a resolution of 256 × 256 pixels. Young’s modulus at each pixel was obtained by fitting the force-indentation curve with JKR model using Igor Pro (WaveMetrics Inc.). For each modulus map, histograms of the moduli at pixel positions corresponding to the peptide assemblies and silk nanofibrils were plotted and fitted with a log-normal distribution to determine the modulus average and standard deviation.Nanomechanical measurements on the peptide assemblies and silk nanofibrils in water were performed by fast force mapping mode on a Cypher S AFM (Asylum Research) using BL-AC40TS (Olympus) probes. A 50 μL aliquot of diluted samples was dropped on freshly cleaved mica surface (2 bead attached at the end of the probe tip (Novascan Technologies). Before the experiment, cantilever sensitivity in air was calibrated by indenting a hard surface (a glass slide), followed by calibration of the spring constant through capturing and fitting the thermal spectrum of the cantilever in air. Then the cantilever was immersed in water and its sensitivity in water was again obtained through the thermal method. A 6 × 6 indentation grid over an area of 20 × 20 μm was performed at each location and a minimum of six different locations per sample were indented. Data acquisition was taken at a rate of 1.0 μm/s with a withdraw distance of 1.0 μm and a deflection trigger of 100 nm. Force-indentation curves were analyzed using Hertz, JKR, and Oliver-Pharr models, assuming a Poisson ratio of 0.33.Nanoindentation of bulk silk nanofibrils gel in hydrated state was performed on a Cypher S AFM (Asylum Research) using a round-tip probe composed of a silicon nitride cantilever of 0.6 N/m nominal stiffness with a 0.6 μm diameter SiOCD spectra were recorded from 185 to 260 nm using a JASCO J-1500 spectrometer, with each spectrum averaged from three consecutive scans. Samples of a concentration higher than 0.1 mg/ml were diluted to 0.1 mg/ml and measured in a 1 mm path length quartz cuvette . To estimate the fraction completion of templated assembly at any given time, the relevant CD spectrum was fitted as a linear combination of the initial spectrum at time 0 and the final spectrum at equilibrium time point where the CD spectrum remains constant thereafter. The fraction completion of templated assembly as a function of time was then fitted with an empirical logistic function commonly used for describing the kinetics of protein fibrils growth, from which the lag time and growth rate were extracted.DLS measurements were performed on a DynaPro NanoStar Light Scatterer (Wyatt Technology). Samples of concentrations from 0.01 mg/ml to 1 mg/ml were measured in plastic cuvettes . The laser was at 658 nm and its power was adjusted automatically for samples of different concentrations to an optimized range of counts by the built-in auto-attenuation capability. The acquisition time for each data point was 5 s, and 10 data points were acquired for each sample. The autocorrelation curve for each sample was examined to make sure there was no severe aggregates.m for ANS and 0.2 μm for pyrene, respectively. Fluorescence EEM were recorded with excitation from 250 to 500 nm and emission from 265 to 600 nm in 5 nm increments. For all EEM acquisition, sample concentration was 2 mg/ml and slit widths were set to 2 nm.Fluorescence emission spectra were recorded using a FluoroLog-3 spectrofluorometer (Horiba Jobin Yvon). ANS emission spectra were collected from 420 to 600 nm with excitation at 350 nm, while for pyrene, excitation was set to 332 nm and emission was recorded from 350 to 420 nm. For all scans, both excitation and emission slit widths were 5 nm. Standard 10 mm path length spectrofluorometer cuvettes were used for loading the samples. The fluorophore concentrations used were 10 μ−1 in which the residence time at each shear rate was sufficiently long to reach the final steady state flow. Oscillatory strain sweeps at a frequency of 1 Hz and with a strain range of 10−4–1 were performed to measure the storage and loss moduli in the linear viscoelastic region. A solvent trap cover was used to minimize water evaporation during the experiments.Viscosity measurements were performed on a rheometer  at 22 ˚C with a cone plate of diameter 40 mm and cone angle 2˚. The evolution of viscosity was monitored at shear rates over a range of 0.1 to 3000 s−1, with a resolution of 4 cm−1 and an accumulation of 64 scans. For the peptides used in this study, ATR-FTIR spectra were collected on both as-synthesized powders (lyophilized format) and dried films which were drop cast from a peptide-water suspension of ~ 8 mg/ml. Mapping was performed on a 1.2 × 1.2 cm2 area of the patterned silk film with an increment of 500 μm in both directions. The relative fractions of different secondary structures of silk fibroin were determined by Fourier self-deconvolution of the Amide I band (1705–1595 cm−1) and Gaussian curve-fitting of the deconvoluted spectra using Origin.ATR-FTIR was performed on a Nicolet 6700 FTIR spectrometer (Thermo Scientific) equipped with a liquid nitrogen-cooled microscope. Spectra were collected in reflection mode with ATR correction using a germanium crystal. All spectra were collected on dried samples from 4000 to 650 cmα1 X-rays of wavelength 1.5409 Å. Samples were deposited on a thin mica window  and dried before exposing to X-rays. The thin mica windows were chosen owing to its transparency to X-rays with no characteristic peaks in the scattering range of interest. Each spectrum was collected for 5 mins.WAXS measurements were performed on a SAXSLAB instrument in transmission mode with a Dectris Pilatus3R 300 K detector set at a distance of 109.1 mm from the sample and a Rigaku 002 microfocus X-ray source producing Cu KFor the fabrication of patterned silk films with predesigned crystallinity at selected areas, a PDMS sheet was first covered by a mask and plasma-treated to introduce polar functional groups (mainly SiOH) on the exposed areas, while keeping the surface property of the covered areas unchanged. Peptide suspensions of 3 mg/ml (or higher concentrations) were then pipetted onto the exposed areas following a predesigned pattern. After drying, the peptide assemblies were assumed to be immobilized on the PDMS surface through the formation of hydrogen bonds. Silk fibroin suspensions of 10 mg/ml (or higher concentrations) were then drop cast onto the peptide-modified PDMS sheet and incubated in a humid chamber for 24 hours, followed by natural drying on the bench to produce free-standing films or freeze-drying for SEM characterization.Inkjet printing was performed on a piezoelectric inkjet printer  with cartridges of 10 pL nozzles (21 μm in diameter). Silk nanofibrils suspension was diluted to 50 μg/ml and filtered with cellulose acetate membranes of 0.45 μm pore size  before loading into the cartridges. Custom designed jetting waveforms were used to optimize the jetting trajectory. Typically, drop spacing was set to 10 μm and three layers were printed to achieve uniform coverage of the printed pattern. Silicon wafers were used as the substrate.Continuous printing of the silk gel was performed on a 3D bioprinter . Typically, straight needles with an inner diameter of 0.3 mm  and a printing speed of 5 mm/s were used. Extrusion pressures in the range of 10–50 kPa were applied and adjusted for silk gels of different concentrations. Standard glass slides were used as the substrate.Supplementary Information