Hinokitiol-fueled disks form exclusionary zones in the presence of iron

Abstract

Stimuli-responsive, directional motions, such as chemotaxis, are vital for the development of sophisticated synthetic systems with autonomous motility. Here, we demonstrate that disks containing hinokitiol exhibit directional self-propelled motion on water in response to metal ions, particularly Fe(III) ion. The self-propelled motion arises from surface tension gradients at the air-water interface, generated by the asymmetric release of hinokitiol, which induce Marangoni flows that propel the disks. Upon contact with Fe(III), hinokitiol forms a highly surface-active complex that locally lowers the surface tension and establishes a persistent interfacial gradient. This localized accumulation of the iron complex acts as a chemo-repulsive signal, directing the disks away from iron-rich regions and leading to the formation of exclusionary zones that influence the trajectories of subsequent disks. These findings demonstrate how self-secreted chemical signals can generate interfacial memory and communication in macroscopic active systems, providing a molecular design principle for life-like collective behavior.

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Introduction

Directional locomotion in response to chemical stimuli represents an essential biological process known as chemotaxis.¹ Motion away from chemo-repellent species, termed negative chemotaxis, serves as a communication mechanism between individuals that not only enables organisms to migrate from unfavorable conditions,² but is also vital for the assembly of neural networks through the creation of exclusionary zones.³ Reproduction of such chemo-repulsive behaviors in synthetic systems provides opportunities to model physical parameters of biological systems which could serve as a framework for next generation responsive materials that exhibit memory and patterned self-assembly.⁴ To this end, Marangoni flow has emerged as a popular avenue by which synthetic, self-propelling objects can be achieved through interfacial surface tension gradients, which are typically generated by the release of surface-active “fuel” species.⁵ Some Marangoni motors can be made to display rudimentary chemotactic behaviors for communication and maze-solving when used in conjunction with chemical reactions,⁶ pre-existing gradients,⁷ or solute-mediated interactions.⁸ Despite these advances, examples of macroscopic-level communicative behaviors through self-secreted signals remain rare—particularly in the realm of chemo-repulsive systems.

Herein, we demonstrate that disks containing hinokitiol (HT) autonomously exhibit negative chemotaxis toward aqueous iron, generating exclusionary zones. Inspired not only by the biochemical⁹ and self-propelling¹⁰ properties of HT, but also by its ability to bind and transport a range of metal ions,¹¹ we envisioned that HT could be utilized as a potential chemotactic fuel enabling synthetic systems to respond to metal ions. Furthermore, building on our previous reports that disks composed of fuel and a polymer scaffold can undergo consistent and tunable self-propelled motion on aqueous surfaces, we employed polystyrene-polyisoprene elastomer (SIS) as a scaffold to construct symmetrical disks, thereby eliminating shape-dependent effects on motion.^10,12 While examples of changes in autonomous behaviors induced by metal ion complexation have been reported,¹³ this system represents a unique example of a macroscopic object displaying negative chemotaxis by producing a chemo-repellent that is more surface-active than its own fuel (Fig. 1a).

Fig. 1 (a) Reaction between HT and aqueous FeCl₃ to yield the surface-active complex, FeHT₃. (b) UV-vis absorption spectral changes of HT (30 µM, blue) upon addition of FeCl₃ to form a 3 : 1 complex (pink) in distilled water at 25 °C. Inset shows the plot of the absorbance at 425 nm vs. [FeCl₃].

Results and discussion

The complexation of HT with ferric ions (Fe³⁺) has been reported to proceed in a 3 : 1 molar ratio in buffer solution (pH 7.0).^11b To confirm this coordination behavior under our conditions, we carried out titration experiments using UV-vis absorption spectroscopy. An aqueous solution of HT (30 µM) at pH 7 was titrated with FeCl₃·6H₂O at 25 °C. As shown in Fig. 1b, the absorption band of HT at 241 nm gradually decreased and red-shifted, accompanied by the emergence of a new absorption band at 425 nm. The intensity of the 425 nm band reached saturation upon the addition of 10 µM Fe³⁺. These results indicate that HT and Fe³⁺ form a 3 : 1 complex (FeHT₃). The absorbance at 425 nm slightly decreased at concentrations of FeCl₃ above 10 µM, probably due to partial precipitation of FeHT₃ during the UV-vis absorption titration measurements (over 30 min). Such precipitation behavior may also obscure a precise Job plot analysis for confirming the stoichiometry under the present conditions. Nevertheless, 3 : 1 complex formation was further supported by mass spectrometric analysis (see Fig. S1) as well as previously reported single-crystal X-ray structural analyses obtained under analogous conditions.^11b Given that complex formation was essentially quantitative even near the lowest concentrations accessible by UV-vis absorption titration, the stability constant of FeHT₃ is estimated to be greater than 10¹⁸ M⁻³.

Also, HT is known to form metal complexes with various stoichiometries, including 1 : 2 complexes with divalent metal ions.^11b,14 First, to examine whether the identity of the counter-anion affects the complexation behavior of HT with Fe³⁺, UV-vis absorption titration was performed using Fe(NO₃)₃·9H₂O under conditions analogous to those used for FeCl₃ (Fig. S2a). Although the concentration of Fe(NO₃)₃ required to reach saturation was slightly higher than that of FeCl₃, the final absorption spectrum obtained after saturation was essentially identical to that observed in the presence of FeCl₃, indicating the formation of the same FeHT₃ complex. Next, we examined the complexation behavior of HT with FeCl₂·4H₂O and CuCl₂·2H₂O under conditions analogous to those used for FeCl₃. In both cases, complex formation was observed (see Fig. S2b and S2c). Although a direct quantitative comparison is not straightforward due to differences in coordination modes between trivalent and divalent metal complexes, the FeHT₃ complex formation is particularly stable and exhibited a simple and well-defined change in the UV-vis absorption spectrum.

To investigate the effect of FeHT₃ formation on the self-propelled motion of HT at the air-water interface, the swimming behavior of pristine HT clumps and HT-SIS disks was monitored in a circular Petri dish on a homogeneous solution of aqueous FeCl₃ at different concentrations (see SI for the experimental setup). Overall, pristine HT clumps displayed rapid continuous motions on aqueous FeCl₃ solution (Fig. S3; SI Movie 1). HT-SIS disks exhibited similar self-propelled behavior before coming to a halt as they became confined by an increasingly shrinking barrier in the presence of FeCl₃ (SI Movie 2). Notably, the distinct switching behavior from continuous to oscillatory motion observed in the absence of FeCl₃ became much less apparent.¹⁰ Compared to pristine HT clumps, HT-SIS disks, which represent a lower supply of HT, continued to swim longer than pristine HT clumps. The motion was maintained the longest at low iron concentrations (10 µM, Fig. S4a). As the concentration of FeCl₃ was increased to 50 µM, the duration of the self-propelled motion decreased (Fig. S4b): a trend that continued at high iron concentrations (100 µM, Fig. S4c). After coming to a halt on a 100 µM FeCl₃ solution, a HT-SIS disk transferred to fresh iron solution resumed motion while a second HT-SIS disk placed on the original solution did not move (Fig. S5). These observations indicate that the accumulation of FeHT₃ on the surface can hinder self-propelled motion by trapping the disk.

To assess the possible influence of counter-anions on motion behavior, self-propelled motion of HT-SIS disks was studied using Fe(NO₃)₃ instead of FeCl₃. The resulting self-propelled behaviors were comparable to those observed for FeCl₃ (Fig. S6a), indicating that the observed chemotactic response is largely insensitive to the nature of the counter-anion under the present conditions. Similarly, self-propelled motion was observed using FeCl₂ and CuCl₂ as alternative metal ion sources. The behavior of disks in the presence of FeCl₂ showed no obvious deviation from that observed for FeCl₃ (Fig. S6b). By contrast, although HT is expected to form a complex with Cu²⁺ that may influence the interfacial properties, no self-trapping of the disk was observed (Fig. S6c). These results highlight the importance of the strong and surface-localized formation of an iron-HT complex, especially FeHT₃, in generating sustained chemotactic confinement.

To understand the mechanism underlying the change in self-propelled behavior upon FeHT₃ formation, concentration-dependent surface tension (γ) measurements were conducted using the Wilhelmy plate method (Fig. 2a). As the concentration increased, the surface tension of FeHT₃ solutions dropped much more steeply than that of HT solutions, eventually reaching 57 mN m⁻¹. This value is lower than the saturated surface tension observed for HT aqueous solutions, which plateaued at 62 mN m⁻¹ above 2.5 mM.¹⁰ A linear approximation in the low-concentration region (γ = γ₀ − a[C], where γ₀ is the surface tension of distilled water, 72 mN m⁻¹, and C is the solute concentration) yielded an a value of 368 N m⁻¹ M⁻¹ for FeHT₃, in contrast to 4.8 N m⁻¹ M⁻¹ for HT, indicating a markedly higher surface activity of the FeHT₃ complex. Meanwhile, the surface tension of FeCl₃ aqueous solutions remained nearly unchanged under the conditions used in this study. These results indicate that both HT and FeHT₃ act as surface-active species responsible for the observed self-propelled motion via Marangoni flow. Importantly, although FeHT₃ exhibits higher surface activity than HT, its primary role is not to enhance propulsion but to define the directionality of motion. While HT is released continuously from the disk and generates transient surface tension gradients that sustain motion, FeHT₃ is produced locally upon contact between HT and Fe³⁺ and remains strongly adsorbed at the air-water interface, generating persistent, localized reductions in surface tension (Fig. 2b). This leads to the formation of a relatively persistent, spatially localized low surface tension region. Consequently, the disk is subjected to a sustained Marangoni force directed toward regions of higher surface tension, leading to consistent repulsion from iron-rich areas rather than altered propulsion dynamics.

Fig. 2 (a) Concentration-dependent surface tension of FeCl₃ (red), HT (blue), and FeHT₃ complex (green) in distilled water at 25 °C. (b) Schematic illustration of the self-confinement and chemo-repulsion mechanisms due to the surface accumulation of FeHT₃.

Because the high surface activity of FeHT₃ enables it to function as a chemo-repulsive signal, we examined the chemotactic behavior of HT-SIS disks in an environment where iron is not homogenously available (Fig. 3a). A 20 µL droplet of aqueous FeCl₃ was placed along the left edge (0 cm position) of a rectangular container (5 × 20 cm) filled with water to a depth of 3 mm. The first HT-SIS disk was placed in the center of the dish (10 cm from the left edge) 5 min later. UV-vis spectroscopy indicated a sharp Fe³⁺ concentration gradient localized near the 0 cm position (Fig. 3b). The first disk exhibited stochastic, negative chemotaxis away from the iron source (Fig. 3c). In the first 120 s the disk moved ballistically across the entire dish, briefly slowing as it passed through the iron-rich region (SI Movie 3). Within the next 120 s, the disk appeared to become trapped for several seconds near the 2 cm position before bursting free and transitioning to oscillatory motion, now avoiding the furthermost left side of the dish (SI Movie 4). Finally, after becoming trapped at 4 cm, the disk cycled between periods where it began to shake in place, becoming increasingly more violent before moving slightly toward the 20 cm position (SI Movie 5).

Fig. 3 (a) Schematic illustration of the aqueous conditions during the chemotactic motions of the first and second HT-SIS disks. (b) UV-vis absorption spectra recorded at different positions from the Fe³⁺ source, measured 5 min after placing aqueous FeCl₃ at the 0 cm position. The inset shows the calculated FeCl₃ concentrations based on the absorption coefficient at 300 nm (1.07 × 10⁴ M⁻¹ cm⁻¹). (c) Trajectories of the first HT-SIS disk on an Fe³⁺ gradient. (d) UV-vis absorption spectra showing the band assigned to FeHT₃ at different positions after the first disk had been allowed to swim for 600 s. The inset shows the calculated FeHT₃ concentrations based on the absorption coefficient at 425 nm (1.20 × 10⁴ M⁻¹ cm⁻¹). (e) Trajectories of a second HT-SIS disk in the presence of the FeHT₃ barrier generated by the first disk. In (c) and (e), the color bar represents the average speed (mm s⁻¹) of the disk. The approximate starting points of the first and second disks are indicated with an x.

After 600 s, the first disk was removed, and a second disk was placed in the center of the dish. UV-vis absorption spectral analysis of the aqueous phase at different positions following the motion of the first HT-SIS disk for 600 s confirmed the formation of an FeHT₃ concentration gradient (Fig. 3d). The second disk appeared to rebound from an invisible barrier (Fig. 3e, SI Movie 6), indicating that an exclusionary zone had been established by the chemo-repulsive FeHT₃ generated through the first disk's contact with iron.

The trajectories of both the first and second HT-SIS disks tended to shift to the right over time, suggesting diffusion of FeHT₃. As a result, the system developed a persistent surface tension gradient, which also enabled a disk composed solely of SIS to migrate toward the right when placed on the surface after 600 s (Fig. S7). Similarly, when a third HT-SIS disk was introduced within the exclusionary zone, it was directed away from the iron source; meanwhile, the second disk remained outside (Fig. S8). These results demonstrate that environmental modification by the first disk can direct both fueled and fuel-free objects through interfacial gradient formation.

Notably, the size of the exclusionary zone appeared to depend strongly on the behavior of the first disk. Brief iron exposure resulted in the formation of FeHT₃ on the disk surface which could provide a strong driving force for ballistic motion across the dish. Repeated returns to the iron-rich region of the gradient ensured the continued production of FeHT₃ which could eventually trap the HT-secreting disk near the iron source as exhibited in Fig. 3c, when the first disk remained near the 5 cm position for ca. 380 s. As a result, complexation could occur continuously, causing the surface pressure to increase and the exclusionary zone to expand to the 10 cm position. Conversely, in the case of the second HT-SIS disk in Fig. 3e, the barrier did not shift significantly because HT was not released in an iron-rich environment. On the other hand, when the first disk was trapped for ca. 190 s at 5.5 cm, the exclusionary zone did not expand beyond the 6 cm position (Fig. S9). Thus, the environment is characteristically modified by the behavior of the first disk, allowing it to direct itself and others away from the iron source. This behavior demonstrates that HT-SIS disks can exhibit rudimentary memory effects and indirect, environment-mediated interactions with other Marangoni motors in a manner that is conceptually analogous to living systems.

Conclusions

In conclusion, we have demonstrated that self-propelled, HT-fueled disks on the surface of water can respond to aqueous iron through the formation of the surface-active complex FeHT₃. The interplay between HT release, FeHT₃ formation, and their distinct surface activities governs the emergence of negative chemotaxis, enabling the disks to direct their self-propelled motion via Marangoni flows. Importantly, the locally produced FeHT₃ functions as a chemo-repulsive signal that not only directs and confines the motion of the disk but also influences subsequent disks through the generation of exclusionary zones. These findings reveal how self-secreted chemical species can mediate communication and environmental memory in self-propelled systems. The present study thus provides a molecular design framework for the development of advanced responsive materials capable of adaptive, life-like motion.

Author contributions

Lara Rae Holstein: conceptualization, investigation, writing – original draft, writing – review & editing. Megan S. Santamore: investigation. Asahi Tsukamoto: investigation. Masayuki Takeuchi: writing – review & editing. Nobuhiko J. Suematsu: software, writing – review & editing. Atsuro Takai: conceptualization, supervision, funding acquisition, writing – original draft, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra01403h.

Acknowledgements

We are grateful to Ms. Izumi Matsunaga (NIMS) for her assistance with the experiments. This work was supported by a Grant-in-Aid for Scientific Research on KAKENHI (Grant Numbers: JP21H01004, JP23K03347, JP24K01475, and JP23K04725), a Grant-in-Aid for Transformative Research Areas (A) “Materials Science of Meso-Hierarchy” (Grant Number: JP24H01734), and the Inamori Foundation. We also thank support from ARIM of MEXT (JPMXP1224NM5109 and JPMXP1225NM5064) and MEXT's Promotion of Distinctive Joint Usage/Research Center Support Program (JPMXP0724020292).

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