Synthesis of Catalytic Microswimmers Based on Anisotropic Platinum Sorption on Melamine Barbiturate Supramolecular Structures

Herein, a straightforward synthesis approach for the formation of anisotropic microswimmers is proposed as an alternative to a top–down fabrication for asymmetric artificial swimmers. Melamine barbiturate (MBA)–self‐assembled supramolecular crystals are utilized as an asymmetric matrix for selective chemical platinum deposition. Surface functional groups of prevalent crystallographic planes for platinum sorption are presented by acidic sites of the assembly. The formed platinum catalytic layer is separated from the bulk MBA and investigated using X‐Ray photoelectron spectroscopy and scanning electron microscope energy‐dispersive X‐Ray spectroscopy analysis. Both methods confirm platinum adsorption and reduction of platinum ions during the deposition process. The MBA asymmetric swimmers show directional motion in a fuel environment, proved by the mean‐squared displacement study. Previously reported pH‐dependency and encapsulation capacity of MBA opens wide opportunities for functional materials formation.


Introduction
Recent advances in supramolecular self-assembly studies have shown wide applicability of complex molecular assemblies in medicine, [1,2] drug delivery, [3] molecular recognition, [4] and environmental remediation. [5,6] In fundamental science, they allow an unprecedented degree of ensemble architecture control. [7,8] Great number of artificially prepared supramolecular assemblies are proposed for integration into biochemical processes and enzyme catalysis due to their building blocks biocompatibility. [9] In practice, supramolecular self-assembled materials are great candidates for study due to the easy fabrication process. Moreover, simple and plain formation mechanisms involving only certain groups of molecules give a wide opportunity for modification [10] right down to the Lego constructor concept. [11] Another important property of self-assemblies is that the formed stable structures are strongly influenced by the pH or temperature of the media. [12] Supramolecular architectures as artificially fabricated matter can biomimic living systems when placed in complex media. The very common cell function of directional motion in the presence of a chemical gradient (chemotaxis [13] ) may be performed likewise by self-assembled architectures based on enzymes [14] or asymmetrically bound catalysts. [15] Similarly to cells, they can change location under external chemical or physical changes in the environment thus providing autonomous movement. [16][17][18] Furthermore, supramolecular architectures have a wide potential as a replacement for complex synthetic molecules, for example, they are great candidates for artificial motors. They can be obtained by straightforward synthesis mechanisms, where constituent parts can be modulated sophisticatedly toward a wide range of applications. At the same time, they meet the requirements for biocompatibility and simple inclusion mechanisms of molecular agents in the self-assembled structure.
Achieving control over the artificially prepared supramolecular structure as autonomously moving material requires new approaches in the design of supramolecular motors.

Melamine-Barbiturate
In our previous work, we reported on the supramolecular assembly of melamine and barbituric acid as a radical trap material. [19] The water solutions of melamine and barbituric acid form a white precipitate when mixed. The assembly of melamine and barbituric acid is held by hydrogen bonds and it is stable within a wide range of concentrations and components ratios and this stability is strongly dependent on the pH. Moreover, our research on melamine barbiturate (MBA) is showing its capability of trapping organic molecules. The MBA assembly may be effectively DOI: 10.1002/aisy.202200436 Herein, a straightforward synthesis approach for the formation of anisotropic microswimmers is proposed as an alternative to a top-down fabrication for asymmetric artificial swimmers. Melamine barbiturate (MBA)-self-assembled supramolecular crystals are utilized as an asymmetric matrix for selective chemical platinum deposition. Surface functional groups of prevalent crystallographic planes for platinum sorption are presented by acidic sites of the assembly. The formed platinum catalytic layer is separated from the bulk MBA and investigated using X-Ray photoelectron spectroscopy and scanning electron microscope energy-dispersive X-Ray spectroscopy analysis. Both methods confirm platinum adsorption and reduction of platinum ions during the deposition process. The MBA asymmetric swimmers show directional motion in a fuel environment, proved by the mean-squared displacement study. Previously reported pH-dependency and encapsulation capacity of MBA opens wide opportunities for functional materials formation.
incorporated with Rhodamine-6 G causing changes in organic crystal shape and producing highly luminescent functional material. [20] The size of the MBA composites is highly tunable, and may be controlled with external factors, including temperature, composition of solution temperature, stirring parameters, etc. The biocompatible nature of MBAs with their high sensitivity to the pH media together with sufficient resistance to external factors make them strong candidates for drug delivery platforms and biological imaging.

Micromotors
Chemically driven micro-and nanomotors require fuel for motion which they derive from a surrounding solution. The generated mechanical work helps them to move and to accomplish a task at the final destination. Several mechanisms are responsible for propulsion, including bubble generation, self-diffusiophoresis, or electrophoresis. [21] Though a variety of fuels are known, hydrogen peroxide (H 2 O 2 ) remains the most commonly used fuel among researchers in micro-and nanomotors studies. [21] H 2 O 2 is decomposed at the metal site of a motor (usually Pt, Ag or Au) into water (H 2 O) and oxygen (O 2 ), resulting in the micromotor self-propulsion by oxygen generation. The biomedical application of these motors is rather controversial due to the toxicity of H 2 O 2 fuel which leads to cell apoptosis. [22] Nevertheless, nanomolar (nM) amounts of H 2 O 2 are known to be produced by tumor cells and at inflammation sites in human bodies, [23,24] which might be sufficient for driving some catalytic nanoswimmers. The larger the tumor, the more hydrogen peroxide fuel it produces, and the catalyst availability together with sufficient H 2 O 2 concentration facilitate nanoswimmer to start moving. [25] Most existing micromotors are asymmetrically covered either with a catalytic layer or metal nanoparticles, bound at the micromotor's surface, or loaded inside the micromotor's structure. [26] Due to the asymmetry, the net force has a pronounced direction, and a particle demonstrates steady forward movement, when a regular catalyst layer over the whole particle surface may impact its directionality. Moreover, for an asymmetric catalytic particle, lower quantities of fuel are needed for propulsion. [27] In Table 1, we compare the motion mechanisms and performance of MBA-based supramolecular micromotors with the literature reports by considering the capping method, motor size, and material characterization. Our careful review on previous work revealed that there have already been reported a study of polymer single crystals with asymmetric orientation of functional groups, that determines specific one-side attachment of catalytic nanoparticles. [28] Our method in the current study utilizes the same principle, where the crystal-chemical properties of the anisotropic crystal determine specific catalyst assembly and a Janus morphology formation. The controlled molecular selfassembly of nano-and micromotors is versatile, and the motion performance of such particles is not inferior to conventional microswimmers. Many studies consider asymmetricity, and for those motors, the velocity tests show remarkable results, even at low fuel concentration. The morphology of these swimmers enables integration of functional components for delivery purpose, which in combination with external stimuli response make these motors promising functional platforms. In comparison with previous work, our method proposes one-pot heterogeneous selective Pt adsorption and does not require intermediate auxiliary-phase formation or colloid system generation. Moreover, for this synthesis, no template is implemented, as well as additional instrumentation set such as sputter-coating.
Following the idea of synthesizing the pH-dependent material with autonomous movement together with an encapsulated species, here we report on the "bottom-up" fabrication of Janus microswimmers based on the crystalline structure of the supramolecular self-assembly, which determines the orientation of the catalytic layer of the flat composite and its behavior in the fuel media. The versatile and plain formation of MBA microswimmers with low Pt consumption opens up a great opportunity for mass production of MBA motors and their future application for specified tasks.

Assembly of MBA Substrate Crystals
The synthesis procedure begins with the equimolar mixing of aqueous solutions of barbituric acid and melamine. White fine-grained powder is obtained, with the average size of the particles 20-30 μm. Optical images of MBA powder in polarized light show that it consists of well-shaped single crystals arranged in radial-radiant starry agglomerates. Each particle has several characteristic features: the growth center where all the single crystals meet and the pronounced anisotropy of the star facets (Figure 1a,b). The anisotropy is repeated in each particle and thus is thought to have a structural explanation. MBA single crystal has the shape of an elongated isosceles prism with complex sharp ends (see Figure 1d). Scanning electron microscopy (SEM) of pure MBA shows no signs of developed surface on any of the crystal's faces (Figure 1c) regardless of the fabrication method and confirms homogeneous particle shapes.

MBA Crystal Structure Studies
To correlate the shape of a crystal with its internal structure, an oriented specimen was prepared for single-crystal studies. For this purpose, high-quality single crystals were obtained by slow precipitation from the solution of mixed melamine and barbituric acid with the temperature decrease. They were then fixed to a glass fiber with epoxy resin, and their shape was accurately described using electron microscopy ( Figure S1, Supporting Information).
The correlation of the data on single-crystal diffraction of the oriented preparation with its shape made it possible to assign exact indices to the crystallographic planes. Thus, it turned out that the upper and lower surfaces of the crystal have crystallographic indices (0 0 1) and (0 0À1) and have different chemical structures. Previously, in our work, [19] it was shown that in the MBA structure, there are several types of interactions responsible for the bonding of barbituric acid and melamine molecules in different crystallographic directions. In particular, the molecular layers lying in the (0 0 1) plane are connected electrostatically due to the deprotonation of the C5 atom of barbituric acid and the protonation of the nitrogen of the melamine ring. It is known  www.advancedsciencenews.com www.advintellsyst.com that the solvation of ionized molecules is an energetically more favorable process than the formation of an electrostatic bond in a salt crystal. Thus, we can assume that the outer layer of the MBA crystal consists of melamine and barbituric acid molecules bound by hydrogen and hydrophobic bonds, but not of individual molecules of either type electrostatically attached to the surface. This hypothesis limits the possible outer functional groups for any given crystal facet and within the framework of this assumption, we proposed a model of the MBA crystal surface (Figure 2), within which, in particular, it turns out that the (0 0 1) plane on the surface is covered with barbituric acid acidic groups, while (0 0À1) has basic nitrogen atoms of melamine on the surface, the other manifested planes are (0 1 1) and (0À1 1) sides of the crystals, which have mixed surface composition with both acidic barbituric acid groups and basic NH 2 of melamine. Such a difference in the surface structure can lead to different sorption activities and, in general, to different chemical behavior of different crystal facets. Thus, it was assumed that acidic surface groups would preferentially react with metals while the basic facet would not participate in the sorption process.

Platinum Deposition
The platinum deposition is subsequently carried out by incubating the clean MBA crystals in hexachloroplatinic acid (H 2 PtCl 6 ) solution for a platinum source. After soaking in platinum solution for at least 4 days, the particles are washed, and their catalytic reactivity is proven under a microscope on a clean glass slide in a drop of 5% hydrogen peroxide solution. MBAs that do not undergo a platinum-deposition process do not show any sign of catalytic activity. When Pt-treated MBA particles are immersed in H 2 O 2 solution, they immediately provide a long chain of small oxygen bubbles, propelling the particle in an opposite direction of a catalyst site.
It was previously reported that nitrogen-contained covalent organic framework can stabilize noble metal ions (Pd 2þ ). [29]   This phenomenon may be applied in nitrogen-contained hydrogen-bonding self-assembled supramolecular structure, where active sites can lead to subsequent ion reduction and anchoring. [30] Therefore, no addition of a reducer to the system is required, since the supramolecular platform itself is a reducing agent for catalytic species. It is unclear which component of the MBA composite dominates in the reduction process, since both barbituric acid [31] and melamine [32] possess active sites for Pt reduction.
In addition, in previous work, it was reported that nitrogen-Pd complexes are great catalysts for a considerable set of compounds. [29] In this work, we assume that reduction of Pt ions together with their subsequent coordination with organic ligands form a stable and recyclable catalytic layer at the MBA particle surface.

Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy Study
Platinum as a solid catalyst was reported previously in many works dedicated to the catalytic decomposition of hydrogen peroxide. [33,34] To estimate the distribution of platinum over the Pt@MBA surface, energy-dispersive X-ray spectroscopy (EDX) was used for Pt mapping. Significant Pt content was observed in MBAs after platinum deposition whereas no Pt was found on clean MBA particles ( Figure S2, Supporting Information). As long as platinum is the heaviest element by far in the studied system, SEM in the backscattered electrons (BSE-SEM) imaging mode is an easy and convenient method for platinum-containing phase observation. The time-series analysis of BSE-SEM images of particles was carried out to trace the evidence of platinum incorporation and localize it (Figure 3a). The extended crystals' incubation with Pt ions favors the deposition process as progressive slow delivery of the precursor promotes both dissolution of the crystal and Pt adsorption. Eventually, two types of defects occur throughout depositionmacroscopic defects on the crystals' smooth surface (up to micrometers in size and depth) and the new contrasting phase formation, which preferably occupies (and slowly erodes) the (0 0 1) acidic facet of the 8-pointed MBA crystal, primarily indicated as a bright thin line at BSE-SEM. In the meantime, the bottom side of the crystals remains smooth and does not evolve visually, only being slightly damaged on the edges. Significant damage occurs gradually, and by the fourth day of the deposition, the particles seem to have sufficient platinum content for motion deposited selectively on the (0 0 1) plane. Since the platinum-deposition process is continuous, it needs to be interrupted at the time when stable motility is achieved. Due to that reason, the process of platinum plating requires daily control of the crystals, because significant defects or breakage of the particles along the edges may obstruct their further motility.
The EDX mapping for both sides of MBA showed high content of platinum on the (0 0 1) acidic facet and its total absence on the bottom base facet (Figure 3b). Apparently, chlorine allocation coincides with platinum, admittedly occupying the same slots caused by defects, and at the same time, chlorine is not present on the bottom side either. Therefore, a composite of a true Janus morphology was fabricated with a distinct orientated deposition by wet chemistry methods only.
To characterize a pure catalytic layer, the MBA matrix was dissolved with 0.5 M NaOH, when the solid dense Pt layer remained intact (Figure 4). The effect of the "snakeskin" was observed as the catalytic layer completely replicated the shape of the MBA crystal. The morphology of the catalytic film addresses the shape of the top face of the flat composite. Immersing the clean catalyst films in hydrogen peroxide solution provides active bubbling on both sides of the films, with no directional movement. The EDX map confirms the regular continuous Pt distribution over the whole surface which is crucial in terms of the catalytic process and microswimmers study (Figure 4d).

X-Ray Photoelectron Spectroscopy Studies
To reveal the platinum oxidation state as well as C, N, and O states, clean samples of Pt catalytic films were analyzed with X-ray photoelectron spectroscopy (XPS). The overall spectra ( Figure S3, Supporting Information) confirm that Pt and Cl can be detected inside the films after chloroplatinic acid treatment, as well as N, O, and C, which may be attributed to melamine or barbituric acid. An EDX maps study of chlorine showed that its allocation replicated the Pt sites, which may be associated with adsorbed Pt-Cl complexes. High-resolution Pt4f spectra of the prepared sample (Figure 5a) may be described by two doublets of Pt which suggests that platinum exists in two forms: Pt(II) with the binding energy of 73.0 eV and Pt(IV) with 74.5 eV according to the XPS databases; [35] however, the latter doublet has significantly lesser intensities. Some amount of Pt(IV) complexes are adsorbed at the MBA surface and are not changed during the deposition process. The emergence of Pt(II) and may correspond to a reduction process of Pt(IV) in [PtCl 6 ] 2À occurring at the MBA matrix. Higher content of Pt(II) to Pt(IV) may be related to the reduction of Pt(II) as a more favorable process during platinum deposition.
The oxygen peak in Figure 5b most likely stems from oxygen in barbituric acid. The peak fitting revealed two peaks for oxygen at 531.2 and 532.4 eV. Possible scenarios of the splitting may include a barbituric acid oxidation process of C-H and the emergence of a new C=O bond with a different electron density. [31] The peak of nitrogen of high symmetry in the substructed plot in Figure 5c is satisfactorily described by a single Gaussian peak with binding energy 398.9 eV. The XPS spectra for N in melamine in literature [36] are presented by two peaks of nitrogen at 399.1 eV for -NH 2 and 398.2 eV for -C=N. Concluding from the O1s spectra study, we assume that the peak for nitrogen in our spectra relates to the N-C bond in barbituric acid.
The peak fitting for the carbon component in Figure 5d shows three peaks, contributing to the resulting spectra. A relatively lower peak of carbon at 285.6 eV possibly stands for the previously characterized C-N bond in barbituric acid at the N1s scan. The peak at 284.4 eV most likely refers to the C-C bond and the peak at 287.7 eV possibly refers to the C=O bond. Considering the aforementioned, we believe that Pt-bearing catalytic layers consist primarily of platinum barbiturate forming a continuous insoluble net on the MBA surface.

Microswimmers Motion Study
The clean MBA particles show no motility in H 2 O 2 solution. The Pt@MBA particles show catalytic activity in diluted H 2 O 2 after incubation with H 2 PtCl 6 ( Figure 6a). They show directional motion originating from intensive bubbling from one side of the symmetric MBA composite. We observe upright rotation upon the addition of hydrogen peroxide into the media which addresses the same effects with round Janus microswimmers and their self-diffusiophoretic behavior. [37,38] Surface tension www.advancedsciencenews.com www.advintellsyst.com brings limitations to the bubble-driven particles, as they move close to the water-air interface, and the addition of sodium dodecyl sulfate (SDS) into the solution gives a clearer picture of the particle's motion. The presence of SDS also reduces the contribution of neighboring particles to the moving MBAs as the produced bubbles may accumulate and collapse, affecting the trajectory of motors. Nevertheless, the particle size variability (20-30 μm) and heterogeneity of the process that affect the influential external flows in the system become the main reason for the lack of uniformity in particle velocities. The particles were studied individually in solution with 0.2 wt % SDS and H 2 O 2 with a concentration range of 5-25%. A great evolution of speed is observed with the increasing H 2 O 2 concentration from 9.66 AE 2.47 μm s À1 for 5% to 42.19 AE 4.28 μm s À1 for 25% (Figure 6b). The critical concentration of hydrogen peroxide for particles to move is 2.3% when only some of them are moving. With a high concentration of 20-25% H 2 O 2 , the number of swimming particles reaches nearly 100%. For further hydrogen peroxide concentration increasing, no drastic change in velocities is observed. The mean-squared displacements (MSDs) were calculated with Equation (1) for H 2 O 2 concentration range of 5-25% and the plot is observed in Figure 6c. The greatest incline of the parabolic MSD curve refers to the hydrogen peroxide concentrations of 20-25%. The particles show directionally oriented motility at all fuel concentrations, the MSD curves for smaller concentrations indicate a smoother slope along with the average velocity values decreasing.

Conclusion
In summary, we demonstrated a bottom-up approach for the formation of artificial Pt@MBA microswimmers self-assembled from a solution. A single-crystal X-ray diffraction analysis of MBA revealed the anisotropic character of the formed MBA crystal structure, while microscopy methods confirm the high shape repeatability of its crystals. Further metal-absorption experiments www.advancedsciencenews.com www.advintellsyst.com had shown that platinum from a surrounding solution is preferably adsorbed at the selected crystallographic planes of the crystal. The XPS study showed that Pt is predominantly adsorbed in the reduced form of Pt(II), most likely interacting with barbituric acid. The obtained asymmetric catalyst is able to decompose hydrogen peroxide from a solution and move around with oxygen bubbles as a propulsion source. The isolated swimmers without external influence of surrounding bubbles show directional motion, speeding up to 40 μm s À1 . The specific sorption of the supramolecular MBA crystal may contribute to selective catalysis implementation, as well as catalytic microswimmers with the minimum set of laboratory instrumentation and a plain design. The controlled molecular self-assembly as an approach for the formation of catalytic microswimmers provides options for simultaneous mass production and requires minimum of instrumentation set and low reagents consumption.

Experimental Section
Assembly of MBA-Based Microswimmers: The initial particles were prepared by mixing previously heated to 50°C solutions of barbituric acid (1 mL, 20 mM) and melamine (1 mL, 20 mM) 1:1 in an Eppendorf flask. After 20-30 s visible white crystals of MBA precipitated in the solution. The particles were centrifuged and rinsed three times with water to eliminate the reagents from the solution. The solution of 3 Â 10 À4 M H 2 PtCl 6 was prepared and pH = 4 was controlled by the addition of some 1 M NaOH prior to the plating process. Then, 500 μL of H 2 PtCl 6 solution was added to the particles, stirred for 20 min, and aged for at least 2 days. The result of plating was visible as the initial white powder of MBA turned black after aging in H 2 PtCl 6 solution. The suspension of particles was centrifuged at 6000 rpm for 2 min, decanted and redispersed in water, and repeated four times.
Microscopy: Optical microscopy was involved at every stage of the process for particles' characterization and experiment control. Routine observations and self-propellent particle movement capturing were performed using Leica DMi8 optical microscope in transmittance mode. Polarization microscopy in both transmitted and reflected light was carried out using Leica DM4500P optical microscope (SPbU, "Geomodel" center).
SEM and EDX analysis were performed using a Hitachi S-3400 N SEM equipped with Oxford X-Max 20 EDX spectrometer. EDX mapping was performed at 5 kV accelerating voltage and 0.2 nA beam current to increase the locality of the method. All the samples were deposited on a semiconductor grade polished silicon wafer and washed with deionized water five times to remove all the water-soluble reagents and byproducts.
Single-Crystal X-ray Diffraction: Goniometric studies were carried out using Rigaku XtaLAB Synergy-S single-crystal X-ray diffractometer equipped with an area hybrid photon-counting HyPix-6000HE detector, which was operated at room temperature with monochromated microfocused CuKα radiation (λ[CuKα] = 1.54184 Å) at 50 kV and 1.0 mA. After the set of X-ray diffraction, data was collected and processed, the crystal shape was outlined in CrysAlisPro [39] program package (absolute (ABS) routine mode). Indexation of drawn crystal faces was done automatically based on the integrated data and crystal orientation.
The Pt 4f7/2,5/2 doublets, O1s, C1s, and N1s were fitted, after the subtraction of the Shirley-type background, with the Gaussian peak profiles, using the Origin Pro software package. Movement Analysis: The particle tracks observations were executed with Fiji ImageJ image-processing package. The velocities and MSDs were calculated using MATLAB. A minimum of three independent replicates were collected for quantitative data implementation. MSD for the microswimmers in different hydrogen peroxide concentration was calculated using Equation (1), where r(t) is the position of the particle at time t, and τ is the lag time between the two positions.

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