Self‐Propelled, High‐Crystalline Hydrogen‐Bonded Enzymatic Framework Assembled by Bottom‐Up Strategy

Biomimetic engineering presents an insightful strategy to access fascinating biocomposites integrating biological, chemical, and material functions. Herein, a new self‐propelled hydrogen‐bonded enzymatic framework through a biomimetic bottom‐up strategy is reported. The metal‐free, mesoporous, and photoactive hydrogen‐bonded organic framework (HOF) exoskeleton is in situ grown around catalase (CAT), an enzyme well known for its ability for the biocatalytic O2 generation. This HOF biomimetic method affords the ultrahigh encapsulation efficiency of CAT, yet well preserves the high crystallinity and periodically arranged mesochannels of HOF. The resultant hydrogen‐bonded enzymatic framework enables the self‐propelled motion with the help of a biocatalytic O2 bubble. Given the exquisite architecture, the HOF shell can serve as the dynamical sorbent for pollutant removal. More than that, it is showcased that the intrinsic photoactivity of HOF can be improved by the self‐propelled motion.

Biomimetic integration of MOFs and protein represents a rapidly emerging research area for stimulus-responsive material design, however, several ongoing challenges remain. First, most of MOFs require a harsh crystallization environment (high temperature, strong acid, a mass of organic solvents, etc.), [35] in which the protein will lose its native activity. As far as we know, ZIF-8 is the mainstream framework for bottom-up assembly. [6][7][8][9][10][11][12][13][14] Unfortunately, ZIF-8 shell has a narrow aperture (%3.4 Å). [36] It not only impedes the diffusion of the guest molecule, but also restrains the subsequent applications, such as pollution elimination and drug loading/delivery. Second, this ZIF-8 biomimetic method highly relies on the electrostatic interactions between the protein and metal ions, [8,37,38] but many metal ions show high biotoxicity and may disturb the native conformation of a protein (Scheme 1A). [39,40] Third, the acid-susceptible nature of the ZIF-8 shell inevitably restricts the working scenarios of the resultant ZIF-8 bio-nanosystem. [41] Hydrogen-bonded organic framework (HOF), connected by the hydrogen-bonded interactions of discrete molecular tectons, is a new class of metal-free porous frameworks complementary to MOF. [42][43][44][45] It features the merits of mild crystallization conditions, high biosafety, and solvent processability, and has been considered the ideal carriers for protein immobilization recently. [46][47][48] We envision that the biocompatible HOF may be an ideal porous exoskeleton for biomimetic engineering and will offer new insight to access the stimulus-responsive HOF bio-nanosystem, yet still remains unexplored.
Herein, we reported a new self-propelled hydrogen-bonded enzymatic framework through bottom-up assembly using HOFs as an exoskeleton. CAT, a widely adopted enzyme for biocatalytic O 2 bubble generation, triggered the assembly of a carboxylate organic tecton into a mesoporous and photoactive HOF around its surface, without the addition of toxic metal salt (Scheme 1A). The formed CAT@HOFs bio-nanosystem reserved the long-range ordered mesopore, and has record-high biocatalyst loading. Such bio-nano-architecture enabled the self-propelling motion of HOFs with the help of biocatalytic O 2 bubble (Scheme 1B). Given this, the mesopore channels of HOFs could serve as the "pocket" to dynamically remove pollutants. In addition, the continuous oxygen production and rapid self-propulsion behaviors also enhanced the photocatalytic activity of the HOFs skeleton.

Bottom-Up Assembly of Hydrogen-Bonded Enzymatic Framework (CAT@HOF)
Previous reports have found that the successful in situ growth of ZIFs requires the electrostatic interaction between the metal ion Scheme 1. A) Schematic presentation of the reported ZIF-based biomimetic assembly and the proposed hydrogen-bonded organic framework (HOF)-based biomimetic assembly. B) Schematic presentation of catalase (CAT)@HOF-101 with self-propelling motion behavior for pollutants removal and photocatalysis.
www.advancedsciencenews.com www.small-structures.com and a given protein. [8,37,38] We note that CAT (from beef liver, %57 KD) has NH 2 -rich surface residues (highlighted in purple in Figure 1A), which may form strong interaction with carboxylate organic tecton via H-bonded interaction and electrostatic attraction and thence triggers the in situ growth of HOF without the addition of toxic metal salt. Indeed, CAT was capable of triggering the self-assembly of a carboxylate tecton named 1,3,6,8-tetra (4-carboxybenzene) pyrene (H 4 TBAPy) in the mild condition ( Figure 1B and S1, Supporting Information). The powder X-ray diffraction (PXRD) experiments showed that the obtained biohybrid has the same PXRD pattern as the simulated HOF, named PFC-1 ( Figure S2, Supporting Information), which is a well-known mesoporous HOF connected by intermolecular hydrogen bonds of H 4 TBAPy. [49] This result suggested that CAT indeed could trigger the growth of HOF (the biohybrid was denoted as CAT@HOF), since very limited precipitation was formed without the addition of CAT ( Figure 1B and S1, Supporting Information). Scanning electron microscope (SEM) observed that the CAT@HOF showed a needle-rod-like structure with a length of about 1-5 μm ( Figure 1C). The CAT loading, measured by the CAT concentration difference in the supernatant before and after this bottom-up assembly, was calculated as high as 33.7% w/w, which was the record-high value among the reported methods (Table S1 in Supporting Information). In addition, compared with the standard PFC-1, the color of CAT@HOF hybrid turned into brown, also suggesting the high CAT loading ( Figure S1, Supporting Information). Such high loading  efficiency was comprehensible, because the CAT participated in the crystallization process of HOFs ( Figure 1B) and hence resulted in a CAT-rich biohybrid. The CAT loading was further verified by the Fourier transform infrared spectra (FTIR, Figure S3, Supporting Information), wherein the newly emerging adsorption bands at 1700-1610 and 1595-1480 cm À1 in CAT@HOF were assigned to the amide I band and amide II band of the protein backbones. [50] In addition, the distinct weight loss at the temperature ranging from 250 to 450°C was observed in the thermogravimetry of CAT@HOF, and this was attributed to the thermal decomposition of CAT ( Figure S4, Supporting Information).
To demonstrate that the CAT was indeed mineralized into, rather than surface-adsorbed onto or post-permeated into the HOF crystal, we first carried out a control experiment, wherein the CAT was directly dispersed into the standard PFC-1 crystal. The ability of the surface-adsorption or post-permeation of CAT was then evaluated. Viewing from the crystallographic structure, the relatively narrow mesopore in PFC-1 (%2.0 nm) was not insufficient to accommodate bulky CAT with 4.9 Â 4.4 Â 5.6 molecular dimension ( Figure S5, Supporting Information). As we expected, the surface-adsorption or post-permeation of CAT by HOF was negligible ( Figure S6, Supporting Information), as also evidenced by the FTIR results ( Figure S7, Supporting Information). Next, further insight into the internalization of CAT was evaluated by the N 2 adsorption isotherm experiment at 77 K. As illustrated in Figure 1D and Table S2 in Supporting Information, compared with the standard PFC-1, the Brunauer-Emmett-Teller (BET) specific surface area and pore volume of CAT@HOF were significantly reduced respectively, suggesting that the CAT was internalized and occupied the porosity of the frameworks. In addition, the confocal laser scanning microscope (CLSM) images also demonstrated that the CAT (labeled by blue dyes) was uniformly dispersed within the biohybrid framework ( Figure 1E). These results together confirmed that the CAT@HOF biohybrid, with ultrahigh enzyme loading, was successfully synthesized through this biomimetic bottom-up assembly, and it might serve as the multifunctional nanoplatform with self-propelling motion.

Biocatalytic O 2 Production
CAT is able to decompose H 2 O 2 into H 2 O and O 2 in the atmospheric environment with high efficiency, and the generated O 2 bubbles are desirable driving forces for designing intelligent material with self-propelling motion. [51][52][53] Therefore, the ability of biocatalytic O 2 production was then surveyed. To illuminate the advantage of our CAT@HOFs for O 2 production, the previously reported ZIFs analog (denoted as CAT@ZIF-8) was also fabricated as the benchmark (detail seen in the method section and Figure S8-S10, Supporting Information). The CAT loading in ZIF-8 was only 3.8% (w/w), and this loading was line with the value reported before. [54] As observed in Figure 2A, when H 2 O 2 was introduced into CAT@HOF, a mass of the O 2 bubble was produced. In addition, the generation of oxygen increased in the function of H 2 O 2 "fuel" both for CAT@HOF ( Figure 2B) and CAT@ZIF-8 ( Figure S11, Supporting Information). However, at the same "fuel" concentration and same material dosage, the oxygen production rate of CAT@HOF significantly outperformed the CAT@ZIF-8 ( Figure S12, Supporting Information), attributing to the much high CAT loading in HOF (33.7% w/w) compared to that in ZIF-8 (3.8% w/w).
Besides the different loading efficiency, the effect of crystallographic structure on the efficiency of O 2 production was also investigated. We first identified the micro-structure of CAT@HOF and CAT@ZIF-8 using low-electron-dose cryo-EM. As shown in Figure 2C, the highly crystalline information was directly witnessed in the CAT@ZIF-8 and CAT@HOF, respectively. For CAT@ZIF-8, viewing from the [111] projection under cryo-EM, the CAT@ZIF-8 had periodic cavities (%12 Å), which were formed by Zn clusters and 2-methyl imidazole (the upper part in Figure 2C). This cavity had %3.4 Å 6-ring window. For CAT@HOF, the channels with %20 Å window, formed by the intermolecular H-bonded interaction and layer-by-layer π-π stacking of H 4 TBAPy, were orderly arranged throughout the nanomotors when viewing from the [011] projection under cryo-EM (the below part in Figure 2C). The larger opening window of CAT@HOF (20 Å) compared to CAT@ZIF-8 (3.4 Å) should accelerate the diffusion of the H 2 O 2 "fuel" as well as the transport of the produces, and both of these would enhance the biocatalysis process. Such speculation was supported by the following catalytic kinetic study. The constant of Michaelis-Menten (K m ) represents the substrate concentration ([S]) when the enzymatic reaction reaches half of the maximum catalytic rate (V max ), which is widely used to judge the affinity between the enzyme and its substrate. [55] The fitting K m value of CAT@HOF was 4.66 mM, while the K m value of CAT@ZIF-8 was as high as 17.37 mM ( Figure 2D and Table S3 in Supporting Information). This indicated that the CAT@HOF displayed a stronger affinity toward H 2 O 2 , confirming the high accessibility of CAT within HOF owing to the long-range ordered mesopores. As a result, the catalytic efficiency, reflected by the value of k cat /K m , [56] was calculated to be 0.8391 and 0.2397 S À1 M À1 for CAT@HOF and CAT@ZIF-8, respectively. The former was 3.5 times that of the latter, indicating that the catalytic efficiency of the designed CAT@HOF was much higher.

Chemical Stability and Storage Stability
The cryo-EM identified that CAT@HOF was assembled by intermolecular H-bonded interaction of discrete molecular tectons, in which the layer-to-layer spacing was determined to be 0.36 nm ( Figure 2C). This indicated the strong π-π stacking effect, which might afford the CAT@HOF with high structural stability. To verify this point, CAT@HOF and CAT@ZIF-8 were both soaked in PBS buffer with pH 4-7 for 30 min, respectively, and then the morphology, structure, and catalytic activity of the materials were tested. Figure 3A shows that after acidic treatment, the original dodecahedral frame structure of CAT@ZIF-8 gradually collapsed. As a comparison, CAT@HOF maintained an intact framework after soaking in these acid conditions ( Figure 3B), which is also supported by the PXRD results ( Figure S13A, Supporting Information). Importantly, the rates of catalyzing the decomposition of H 2 O 2 to produce oxygen by CAT@HOF-101 treated with different pH buffers kept a similar efficiency as the untreated one, indicating that CAT@HOF has excellent structure and catalytic stability in the pH range of 4-7 ( Figure 3C). In contrast, the catalytic rate of CAT@ZIF-8 after pH = 6 acid treatment dropped to 48.8% compared to the rate recorded in pH = 7 ( Figure 3C), because the structural collapse ( Figure S13B, Supporting Information) could lead to the leaked-out of immobilized CAT.
The storage stability of CAT@HOF was also investigated. As shown in Figure 3D, the CAT@HOF retained % 80% activity after 3 days of storage at room temperature, while the free CAT dropped to %27% activity. In addition, free CAT almost completely lost its activity after 5 days of storage, but the CAT@HOF still maintained %25% activity. These results indicated that the HOF skeleton shell could shield against the denaturation of CAT, and this protective effect was also observed in the previous emzyme@MOFs systems. [11][12][13]

Motion Behavior of CAT@HOF
Given the enhanced efficiency for O 2 bubble production as well as the desirable chemical stability, the self-motile behavior of the CAT@HOF and CAT@ZIF-8 were tracked by using the nanoparticle tracking analyzer (the videos are provided in Movie S1 and S2 in the Supporting Information). Figure 4A showed frames from the video of the suspensions of CAT@ HOF in the presence of 0.06% H 2 O 2 (Movie S1 in the Supporting Information). It was found that the needle-rod-like CAT@HOF underwent a migration accompanied by self-rotation in the horizontal plane, which is consistent with the relationship between the rotational diffusion and the length of the rod-like nanoparticle proposed by previous work. [57] Besides, a migration against the gravitational force was observed in the vertical direction (Movie S3 in the Supporting Information). Thus, the motion behavior of the  CAT@HOF resulted from the resultant of the gravitational force, the buoyancy force, and the driving force of its enzyme-powered self-propulsion ( Figure 4B). Figure 4C displays the motion trajectories of CAT@HOF under different concentrations of H 2 O 2 , respectively. And with a similar method, we characterized the motion behavior of CAT@ZIF-8, of which the tracking trajectories without and with H 2 O 2 fuel of varied concentrations are presented in Figure 4D. It is well known that the mean square displacement (MSD) curve of Brownian motion satisfied Equation (1) [52,53,58] MSD ¼ 4DΔt (1) where D is the rotational diffusion coefficient and Δt is the time interval.
In the absence of H 2 O 2 , the MSD curves of CAT@ZIF-8 and CAT@HOF fitted a linear relationship with Δt respectively and the average motion rates were 1.11 AE 0.28 μm s À1 for CAT@HOF and 1.49 AE 0.28 μm s À1 for CAT@ZIF-8 (see Movie S1 and S2, Supporting Information). These data revealed that the motion trajectories of both CAT@HOF and CAT@ZIF-8 exhibited typical characteristics of random Brownian motions without H 2 O 2 fuel. After the addition of H 2 O 2 , the oxygen bubbles generated by the H 2 O 2 decomposition provided a driving force on the materials, which helped to overcome the Brownian motion and enabled them to move in a certain direction for a long distance ( Figure 4C,D). In the existence of H 2 O 2 fuel, the MSD curves deviated from a linear fit to parabolic characteristics ( Figure 4E,F). The velocity (v) was obtained by applying the parabolic component of the Stokes-Einstein equation (Equation (2)) Specifically, under the H 2 O 2 concentration of 0.03 and 0.06 wt%, the average movement rates of the CAT@HOF were 5.65 AE 0.12 and 8.07 AE 0.45 μm s À1 , respectively ( Figure S14A and Movie S1, Supporting Information). In contrast, the average movement rates of CAT@ZIF-8 at 0.03 and 0.06 wt% H 2 O 2 were only 2.35 AE 0.14 and 3.09 AE 0.12 μm s À1 , respectively ( Figure S14B and Movie S2, Supporting Information). Obviously, increasing the concentration of H 2 O 2 did not enhance the movement rate of CAT@ZIF-8 as significantly as that of CAT@HOF, and only under the high H 2 O 2 concentration of 0.06 wt% did CAT@ZIF-8 exhibit a directional movement trajectory. These results indicated that the CAT@HOF could effectively self-propel with a low concentration of H 2 O 2 as "fuel." Table 1 compared CAT@HOF with several CAT-based robots reported in recent years. It can be found that the CAT@HOF required lower fuel concentration, yet displayed a faster motion rate than most of them.

Pollutant Removal and Photoactive Evaluation
The periodically arranged mesochannels allow the CAT@HOF with opening "pocket," and may serve as the dynamic sorbent for pollutant removal ( Figure 5A). As a proof of concept,     the CAT@HOF was applied to remove methylene blue (MB, %1.44 nm Â 0.60 nm Â 0.18 nm). We first explored the effect of the self-propulsion capability of CAT@HOF and CAT@ZIF-8 on the MB capture. As shown in Figure 5B,C and S15, Supporting Information, the effect of different "fuel" concentrations on the capture performance of the bionanosystem was first explored. Without the addition of H 2 O 2 , CAT@HOF settled down and agglomerated under gravity, which could not realize self-diffusiophoresis. The access of MB diffusing into pores of CAT@HOF and CAT@ZIF-8 was limited, leading to both the negligible removal of MB ( Figure 5B,C). After adding "fuel," CAT@HOF rapidly catalyzed the decomposition of H 2 O 2 to generate oxygen bubbles to push the movement of itself, overcoming the limitation of gravity to self-propel in the solution. The removal efficiency of MB by CAT@HOF was about 50% in 5 min with the H 2 O 2 concentration as low as 0.06% ( Figure 5B). Meanwhile, as illustrated in Figure 5D,E and S16, Supporting Information, with the extension of adsorption time, the removal of MB by CAT@HOF correspondingly increased. As a comparison, the CAT@ZIF-8 displayed negligible removal efficiency under the same fuel dosage, even though prolonging the treating time to 10 min ( Figure 5D,E), because 1) the motion rate of CAT@ZIF-8 was limited at this low concentration fuel, and no obvious self-diffusiophoresis was observed ( Figure S17, Supporting Information); 2) the narrow opening window (0.34 nm) excluded the relative large MB molecules.
The photosensitive pyrene core in the molecular tectons endows CAT@HOF with photocatalytic activity. [59,60] We envision that the continuous generation of O 2 not only enables the self-propelling motion, but also may promote the production of highly active singlet oxygen ( 1 O 2 ) that enhanced the photocatalysis efficiency ( Figure 5F). To verify this concept, we chose 3,3',5,5'-tetramethylbenzidine (TMB) as the photocatalytic substrate. TMB could be oxidized into oxTMB (a blue product with ultraviolet absorption at 652 nm) by reactive oxygen species, such as 1 O 2 . [61] To demonstrate the role of O 2 generation, photocatalysis was first operated in an N 2 atmosphere. As presented in Figure 5G and S18A, Supporting Information, compared with the group with the absence of H 2 O 2 , the 0.03 wt% H 2 O 2 addition displayed a 2-fold catalytic efficiency for TMB oxidization under light irradiation (7.0 W m À2 , 5 min), indicating CAT@HOF www.advancedsciencenews.com www.small-structures.com could facilitate the photocatalytic reaction by generating O 2 . In addition, electron paramagnetic resonance (EPR) verified that the 1 O 2 , captured by the 2,2,6,6-tetramethylpiperidine (TEMP) regent, was the main ROS generated by CAT@HOF ( Figure S19, Supporting Information). In addition, no •OH was identified using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the capturing regent ( Figure S19, Supporting Information). Besides the effect of O 2 , we also designed another experiment to confirm the positive role of the self-propelling behavior. In this experiment, O 2 gas was pre-bubbled into the sealed bottle to create an oxygen-saturated photocatalytic environment, which could minimize the effects of O 2 generation. As Figure 5H and S18B, Supporting Information show, the group with H 2 O 2 addition exhibited %1.6-fold catalytic efficiency than the group without H 2 O 2 addition, demonstrating the selfpropulsion of CAT@HOF also promoted the catalytic efficiency.

Conclusion
In summary, we pioneered to engineer the multifunctional enzyme@HOF bio-nanosystem through a biomimetic bottomup strategy. This strategy endowed the prepared hydrogenbonded enzymatic framework with a record-high enzyme package, while avoiding the utilization of toxic metal salt. Importantly, the exquisite composite structure with wellarranged mesochannels guaranteed the high accessibility of the interior enzyme. Such bio-nanoarchitecture was chemically stable and exhibited higher biocatalytic efficiency for O 2 bubble generation, which enabled the superior self-propulsion rate under a low "fuel" concentration. Combined with the opening mesoporous "pocket," the enzyme@HOF could be used as the dynamic sorbent for efficient pollutant removal. In addition, O 2 bubble-powered motion was also able to promote the photocatalytic efficiency of the HOF framework, attributing to both the high O 2 generation rate and self-propelling performance. We believe that this enzyme@HOF bio-nanosystem holds huge potential for different applications such as acidic wastewater treatment, in vitro biorecognition, in vivo tumor targeting, etc.

Experimental Section
Detailed experimental procedures were provided in the Supporting Information.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.