ZnO-based micromotors fueled by CO2: the first example of self-reorientation-induced biomimetic chemotaxis

Abstract Synthetic chemotactic micro/nanomotors are envisioned to actively ‘seek out’ targets by following specific chemicals, but they are mainly powered by bioincompatible fuels and only show pseudochemotaxis (or advanced chemokinesis) due to their weak self-reorientation capabilities. Here we demonstrate that synthetic ZnO-based Janus micromotors can be powered by the alternative biocompatible fuel of CO2, and further provide the first example of self-reorientation-induced biomimetic chemotaxis using them. The ZnO-based micromotors are highly sensitive to dissolved CO2 in water, which enables the corrosion of ZnO to continuously occur by providing H+ through hydration. Thus, they can autonomously move even in water exposed to air based on self-diffusiophoresis. Furthermore, they can sense the local CO2 gradient and perform positive chemotaxis by self-reorientations under the phoretic torque. Our discovery opens a gate to developing intelligent micro/nanomotors powered by, and sensitive to, biocompatible atmospheric or endogenous gaseous chemicals for biomedical and environmental applications.


INTRODUCTION
Biological chemotaxis refers to the ability of those living organisms that are of an advanced level of evolution to move toward or away from specific chemicals [1,2]. To achieve chemotaxis, the living organisms are equipped with delicately coordinated sensing, signal processing and propelling systems, endowing them with the capability to adjust their motion direction on demand by evaluating the direction of chemical gradients at different times (e.g. the temporal comparison adopted by Escherichia coli) or perceiving signal intensity differences across their body (e.g. the spatial comparison adopted by neutrophils) [3,4]. Chemotaxis not only regulates the development of multicellular systems such as tissue development and cancer metastasis, but also enables many unicellular organisms to find nourishments, avoid toxins and coordinate collective motions [5][6][7].
Inspired by biological chemotaxis, researchers strive to create synthetic chemotactic micro/nanomotors (MNMs). They are envisioned to achieve self-navigation and self-targeting in complex or dynamic environments by actively following specific chemical cues, and thus may bring revolutionary changes to targeted drug delivery, microsurgeries, microfactories, etc [8]. Nonetheless, synthetic MNMs are generally too small to incorporate sophisticated sensing-processing-propelling systems and have a low sensitivity to chemical gradients. Thus, they only show a pseudochemotaxis (or macroscopic chemotaxis and advanced chemokinesis) based on a purely statistical effect rather than chemotaxis of individual MNMs because of the stochastic nature in motion for each individual [8,9]. With the pseudochemotaxis, they show negligible reorientation capabilities, and can only accumulate near (or away from) the chemoattractant (or chemorepellent) source, or shift toward (or away from) its stream in microfluidic channels due to their spatial-dependent mobility (orthokinesis) in the local chemical gradient field [8,[10][11][12][13]. This statistically average movement uphill (or downhill) to a chemical gradient based on orthokinesis undermines the self-targeting performance of MNMs, and is far from the biological chemotaxis found in C The Author(s) 2021. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. (i and ii) Trajectories of ZnO/SiO 2 MMs in 9 s in the (i) air-exposed water (Air) and (ii) CO 2 -eliminated water, including the freshly boiled water (Bo), Ar-gassed water (Ar), O 2 -gassed water (O 2 ) and N 2 -gassed water (N 2 ). Scale bars, 10 μm. (iii) Trajectories of a ZnO/Pt MM in the air-exposed water without (red curve) and with (green curve) UV irradiation. Scale bars, 5 μm. (D) MSD of the MMs in different water media. Solid color lines are fitting curves using a quadratic function. (E) Self-propulsion of a typical ZnO/SiO 2 MM within a lifetime of 90 min. Red curves depict its trajectories in 9 s at different time intervals. Scale bar, 5 μm.
nature. On the other hand, chemically powered MNMs have been predicted, in theory, to achieve chemotaxis through their self-reorientation based on the spatial comparison of chemical signal intensity across their bodies, like neutrophils [14,15], but it has not been experimentally confirmed.
In this work, we demonstrate ZnO-based Janus micromotors (MMs) powered by an alternative fuel of CO 2 , and further provide the first example of self-reorientation-induced biomimetic chemotaxis using them. The ZnO-based MMs can autonomously move based on electrolyte self-diffusiophoresis, even in water exposed to air, as the ZnO component in the MMs is highly sensitive to CO 2 dissolved in water, which continuously produces H + through hydration to react with ZnO. In this way, the MMs can sense local CO 2 signals, and exhibit a biomimetic chemotactic behavior toward the CO 2 source due to their dynamic reorientations under a phoretic torque.
The proposed alternative fuel of CO 2 , which is a constituent gas in the air and a major product of cell respiration [16,17], has remarkable advantages, including sustained remote accessibility, low cost and excellent biocompatibility. With the excellent biomedical activities of ZnO [18], the intelligent chemotactic behaviors promise that the ZnO-based MMs will 'seek out' specific cells or pathogen microorganisms by tracking metabolic CO 2 signals emitted from them, and execute targeted biomedical and environmental operations.

RESULTS AND DISCUSSION
To experimentally achieve the biomimetic chemotaxis of artificial MNMs, we at first prepared ZnObased MMs by sputtering a thin layer of SiO 2 on the exposed surface of hexagonal wurtzite ZnO microspheres (Supplementary Fig. 1A and B), as illustrated in Supplementary Fig. 2. The scanning electron microscope (SEM) observation indicates that the prepared ZnO/SiO 2 MMs have a relatively uniform spherical shape and an average diameter (d) of 2.5 μm (Fig. 1A). Energy dispersive X-ray (EDX) mapping of Zn and Si elements confirms that the ZnO/SiO 2 MMs have a typical Janus structure (the inset in When the ZnO/SiO 2 MMs were put into water previously exposed to air (air-exposed water, see 'Materials' section in the online Supplementary Data), they exhibited an autonomous motion with an average speed (v) of 7.8 μm/s (Fig. 1C(i) and Supplementary Video 1). As ZnO is poorly reactive or soluble in pure water [19], the self-propulsion of the MMs was attributed to atmospheric CO 2 because it has a much higher solubility (34 mM at 25 o C and 1 atm) in water compared to other major constituent gases (such as N 2 , O 2 and Ar) [20]. To confirm the key role of CO 2 in the self-propulsion of the ZnO/SiO 2 MMs, we investigated their motions in CO 2 -eliminated water (see 'Materials' section in the online Supplementary Data), including freshly boiled water (pH = 7.54), Ar-gassed water (pH = 7.46), O 2 -gassed water (pH = 7.36) and N 2 -gassed water (pH = 6.67), respectively (Supplementary Video 1). As shown in Fig. 1C(ii) and D, they show an extremely slow motion in the N 2 -gassed water and only Brownian motions in other CO 2 -eliminated water, with a translational v and diffusivity (D) lower than 1.3 μm/s and 0.14 μm 2 /s, respectively. The translational v and D were extracted by fitting their mean square displacements (MSDs, Fig. 1D) using a quadratic function according to the previous report [21].
The SiO 2 cap on the ZnO/SiO 2 MMs only acts as a passive layer to break the symmetry of the surface reaction of ZnO. This can be verified by the fact that the ZnO microspheres only exhibit Brownian motions in the air-exposed water due to isotropic surface reactions ( Supplementary Fig. 4), and the MM can still be self-propelled in the same condition if the SiO 2 cap is replaced by a Pt cap ( Fig. 1C(iii)). The ZnO/SiO 2 MMs showed similar motions in lowenergy red light (20.2 mW/cm 2 ) and high-energy UV light (300 mW/cm 2 ) ( Supplementary Fig. 5), while the ZnO/Pt MMs were accelerated under the irradiation of UV light because of their high photocatalytic activity (Supplementary Video 2 and Fig. 1C(iii)). This suggests that light illumination has a negligible contribution to the self-propulsion of ZnO/SiO 2 MMs. Air-exposed water has a CO 2 concentration (C CO 2 ) of 8.54 μM (pH = 5.72), which is three orders lower than the critical concentration of the H 2 O 2 fuel (∼60 mM) required to power traditional catalytic Pt-based MNMs [22]. This suggests that the MMs are highly sensitive to dissolved CO 2 in water and can be powered at an ultralow level of the chemical fuel. Moreover, the ZnO/SiO 2 MMs exhibit a long lifetime of ∼90 min (Fig. 1E), which is higher than that of the active metal-based MMs (∼5 min), catalytic MnO 2 MMs (≤40 min) and some enzyme-based MMs (≤40 min) [23][24][25].
CO 2 exists as a stable and inert gas in air, but can remotely diffuse into various aqueous media to participate in a number of reactions after hydration, such as acidic corrosion [17,[26][27][28][29]. In detail, when water is exposed to air, CO 2 molecules, which account for ∼0.04% of the air in volume [30], can enter into the water through the interface with the atmosphere, and are partially hydrated into H 2 CO 3 , which then dissociates into HCO − 3 and H + , as described by equation (1) [31]: (1) Afterwards, the ZnO-based MMs react with the released H + according to equation (2) [19]: The acidity constant (K a ) of the dissolved CO 2 molecules is 4.3 × 10 -7 M at room temperature, indicating that only a small amount of them are converted into H + and HCO − 3 . In this way, the dis-solved CO 2 stayed as molecules can continuously supply H + ions through the local equilibrium reaction (equation (1)) when H + is consumed by ZnO, and thus CO 2 is considered as the chemical fuel powering the ZnO-based MMs according to an overall reaction expressed as equation (3): (3) Due to the different D of the released ionic products of HCO − 3 (D = 1.185 × 10 -9 m 2 /s) and Zn 2+ (D = 0.703 × 10 -9 m 2 /s) from the ZnO/SiO 2 MMs according to equation (3), HCO − 3 would diffuse faster than Zn 2+ [32,33], leading to an uneven distribution of these ions in the area near the exposed ZnO surface (Supplementary Fig. 6A and B). To maintain electroneutrality, a local diffusioelectric field (E) arises to slow down the faster-diffusing ion, and speed up the slower ion [34], as verified by the simulated electric potential ϕ depicted in the color background of Fig. 2A and the local E shown in Supplementary Fig. 6C (black triangles). This local E then induces an electro-osmotic slip (EOS) [35] in the electric double layer of the negatively charged MM (ζ = −23.7 mV). The simulated flow field (EOF) initiated by the surface EOS around a ZnO/SiO 2 MM is illustrated as black streamlines with arrows in Fig. 2A Fig. 2B.
From the propulsion mechanism (Fig. 2B), it is expected that the ZnO-based MMs can still acquire fuel from the air even if no CO 2 was pre-dissolved in water. To verify this assumption, we recorded the motion behaviors of the ZnO/SiO 2 MMs in a thin film (∼160 μm in thickness) of the freshly boiled water over time when exposed to air (Fig. 2C and Supplementary Video 4). Differently to the MMs in the air-exposed water film (C CO 2 = 8.54 μM), which show a decreasing v over time (the black curve with dots in Fig. 2D), those in the CO 2 -eliminated water display an increasing v in the first 7 min (the red curve with dots in Fig. 2D). This behavior can be verified by the different lengths of trajectories of a typical MM in 8 s (Fig. 2C) and the statistic normalized v of the MMs (Fig. 2D) at different intervals within 7 min. This increase in v reveals (D) Time-dependent normalized v of the MMs in a film of freshly boiled water when exposed to air (red dots) and the air-exposed water (black dots). Solid red and black curves are fitting results using exponential functions. (E) The simulated C CO 2 as a function of time in a CO 2 -eliminated water film (160 μm in thickness) when exposed to air. (F) The v of the MMs at different C CO 2 in water. The red line is the linear fitting line of the experimental data in the low C CO 2 regime (0-8.54 μM), and the red curve in the inset is the fitting result according to the Michaelis-Menten kinetics in the high C CO 2 regime (8.54 μM to 20.5 mM). that atmospheric CO 2 molecules can diffuse into the water as fuel to power the MMs in a sustainable and rapid manner, as verified by the numerical results of the increasing C CO 2 ( Fig. 2E and Supplementary  Fig. 9A) and H + concentration (C H + , Supplementary Fig. 9B) over time when the water film is exposed to air. In the following 7-51 min, the v of the MMs decreases gradually due to the decreasing reaction rate of ZnO and the increasing ion concentration in the water film. With the increase of C CO 2 from 0 to 8.54 μM, the v of the ZnO/SiO 2 MMs increases from 2.0 to 7.8 μm/s (Fig. 2F and Supplementary Video 5). By bubbling water with pure CO 2 gas, a higher C CO 2 compared to that in the airexposed water can be obtained, and the v of MMs can reach 41.5 μm/s when C CO 2 increases to 20.5 mM (the inset in Fig. 2F and Supplementary Video 5). The fitting results show that the v of the MMs increases linearly in the low C CO 2 regime from 0 to 8.54 μM (red line in Fig. 2F), and gradually reaches to a plateau in the high C CO 2 regime (8.54 μM-20.5 mM) following Michaelis-Menten kinetics (red curve in the inset of Fig. 2F). These results reveal that the ZnO/SiO 2 MMs are of a high sensitivity to C CO2, especially in the low C CO 2 regime. The sensitivity can be quantified by the rate of the speed change of the ZnO/SiO 2 MMs to C CO 2 , which has a value of 0.57 μm/(μM·s) (the slope of the red line in Fig. 2F) in the low C CO 2 regime, about five orders of magnitude higher than that of common H 2 O 2fueled MMs (2.72 × 10 -6 μm/(μM·s)) [36]. The appearance of the plateau of v at a high C CO 2 of 20.5 mM indicates that the maximum speed is limited by the maximum reaction rate depending on total surface reactive sites.
Benefiting from the high sensitivity to C CO 2 , the ZnO/SiO 2 MMs can sense a local CO 2 gradient and exhibit positive chemotaxis toward a local CO 2 source in water (Fig. 3A). When a micropipette filled with a CO 2 solution (C CO2 = 93.7 μM) was introduced to the water medium (C CO 2 = 0.2 μM), a gradient field of C CO 2 (∇C ) is formed (the color background in Fig. 3A). In this gradient field, the ZnO/SiO 2 MMs swarm toward the micropipette, in analogy to the positive chemotaxis of living organisms in nature ( Fig. 3B and Supplementary Video 6). The average chemotactic velocity, defined as the net displacement of the ZnO/SiO 2 MMs divided by time when they are approaching the region close to (∼20 μm) the CO 2 source, is 0.57 μm/s. In contrast, passive SiO 2 microspheres (d = 2 μm and ζ = −18.5 mV) show no chemotactic motion in the same gradient field (Supplementary Fig. 10 and Supplementary Video 7), indicating that the MMs were actively swimming toward the CO 2 source, and the possible passive diffusiophoretic attraction generated by the gradient field is negligible.
To quantify the degree of chemotaxis, we have analyzed the chemotaxis index (CI) of ZnO/SiO 2 MMs, which was defined as the ratio of the displacement in the direction of the chemical gradient to the total migration distance (i.e. as the cosine of the angle θ of the MM's motion direction with the chemical gradient) [37]. The average CI is 0.21, suggesting that they, in general, move uphill to the chemical gradient. Due to the persistent ZnOforward configurations during the propulsion (Supplementary Figs 6 and 7), the self-reorientation dynamics of the ZnO/SiO 2 MMs can be characterized by the changes of CI and the angle θ over time. When CI > 0 (i.e. 0 ≤ |θ | < π/2), the ZnO/SiO 2 MMs prefer to move up the gradient, while they tend to move down the gradient if CI < 0 (i.e. π/2 < |θ | ≤ π ). The CI and |θ | of four typical ZnO/SiO 2 MMs versus time (Supplementary Fig. 11) suggest that they move up the gradient for a longer time than moving down. The longer upgradient motion time implies that the ZnO/SiO 2 MMs prefer to move with an orientation along the upgradient direction even though their motion direction was seriously randomized by the Brownian rotational diffusions.
To further demonstrate the biomimetic chemotactic behavior, the instantaneous v, orientation and rotation of a slightly etched ZnO/SiO 2 MM, in which the slightly etched ZnO end was distinguishable under an optical microscope, were recorded and analyzed when it was approaching the CO 2 source (Fig. 3C and D). The ZnO/SiO 2 MM shows an increasing v when approaching the CO 2 source because of the increasing C CO 2 (i.e. orthokinesis of the motor [38]), as verified by the normalized instantaneous v in 0-15 s and 15-30 s (Fig. 3C), respectively. In addition, when the ZnO/SiO 2 MM deviates with ∇C because of the strong perturbations of Brownian randomizations, it dynamically reorientates itself by clockwise or counterclockwise rotations (Fig. 3D). When the MM moves with an orientation down ∇C , it rotates clockwise gradually and aligns with ∇C (0-16.5s in Fig. 3D). After a sharp turn (16.5-20 s in Fig. 3D), the MM deviates with ∇C again, and then gradually aligns with ∇C by a counterclockwise rotation (20-30 s in Fig. 3D). This self-reorientation behavior bears resemblance to that of chemotactic microorganisms which show a 'memory' of the direction of the chemical gradient and move up the gradient of chemoattractant in a 'deviating-rectifying' manner. This is the first experimental evidence showing that synthetic MNMs can achieve chemotaxis by dynamic self-reorientations in a gradient field [8]. After reaching the area close to the tip of the micropipette (CO 2 source), the MMs were wandering in this area with no preferred directions, as depicted by the curved trajectory of the slightly etched MM in 34-163 s in Supplementary Fig. 12, so that they could accumulate near the CO 2 source (Supplementary Video 6).
To decipher the chemotactic behavior, we have simulated the flow field around the ZnO-based MM when a local CO 2 source is introduced, as shown in Fig. 3E. The results show that, if the axis of the MM is aligned with the CO 2 gradient, EOSs (e.g. u s1 and u s2 in the left panel in Fig. 3E, u s1 = u s2 ) across the axis of the MM are symmetrical because of symmetric surface reactions, and the MM migrates up the gradient at this condition. However, due to the strong rotational Brownian diffusion, the symmetry axis of the MM is constantly deviating from ∇C . When misaligned with ∇C with an angle of θ , the ZnO surface close to the CO 2 source has a higher reaction rate (due to the higher C CO 2 (or C H + )) than the surface further away from the source, resulting in the asymmetric distribution of chemical products and thus unbalanced EOSs across the motor axis (e.g. u s1 and u s2 in the right panel of Fig. 3E, u s1 > u s2 ). The unbalanced EOSs then produce a phoretic torque (M p ) to induce rotation of the MM until its axis is aligned with ∇C (the right panel of Fig. 3E). With the increasing degree of deviation, the self-EOSs across the motor axis become more unbalanced, and thus M p increases accordingly for reorientation, as verified by the increasing M p with a varying θ from 0 to −π /2 and π /2 (Fig. 3F). Hence, the chemotactic behavior of the MM is attributed to dynamic self-reorientations under the phoretic torque. Due to their self-reorientations, they show a higher efficiency in chemotaxis compared to the developed (pseudo)chemotactic MNMs, which usually take hours to aggregate near the chemical source [10,39,40]. As shown in Fig. 3G, the MMs swarm to the chemical source in several minutes, as demonstrated by the increasing number (N) of MMs from 0 to 15 within 284 s in a sector region close to (80 μm) the CO 2 source. CO 2 not only exists in the air and various aqueous media (such as natural waters and biological media), but also can be produced and secreted by cells or pathogenic microorganisms during their oxidative metabolism [41][42][43][44]. For instance, the CO 2 tension (P CO 2 ) of the intracellular and extracellular fluids is ∼50 and 46 mmHg, indicating that there is a high C CO 2 (∼20.8 and 19.3 mM together with the chemically bound CO 2 ) and a steep C CO 2 gradient near tissue cells (e.g. ∼3.3 × 10 4 mol/m 4 at a distance of 20 μm to the cell surface), respectively [45]. On the other hand, ZnO micro/nanomaterials are of low toxicity and good biodegradability and have shown great potential in biomedical applications due to their excellent anti-cancer, anti-diabetic, anti-bacterial and anti-inflammatory activities [18]. Thus, the chemotactic ZnO-based MMs with proper surface modifications (e.g. polyelectrolyte ion-tolerance coating [46]) are expected to act as biomimetic microrobots, 'seeking out' specific cells or pathogenic microorganisms by actively tracking their extracellular CO 2 signals, thereby executing targeted biomedical and environmental operations.

CONCLUSION
In conclusion, we have demonstrated that ZnObased MMs can be powered by CO 2 fuels and perform intelligent positive chemotaxis by selfreorientations. We analyzed the propulsion mechanism of ZnO-based MMs and confirmed that it was the result of electrolyte self-diffusiophoresis based on the corrosion of the ZnO end by H + continuously supplied from the dissolved CO 2 . Benefit-ing from their high sensitivity to local CO 2 signals, the ZnO-based MMs can perform a biomimetic chemotactic behavior toward the CO 2 source due to their dynamic self-reorientations in the chemical gradient field. The self-reorientation of the MM is attributed to the phoretic torque produced by the unbalanced EOSs across the MM axis when it is misaligned with the chemical gradient. Due to the readily accessible biocompatible CO 2 fuel and intelligent chemotaxis, ZnO-based MMs have the potential to autonomously move in a wide range of aqueous media and execute targeted biomedical and environmental operations by actively tracking respiratory CO 2 signals emitted from cells or pathogenic microorganisms.

Preparation of ZnO-based MMs
The ZnO microspheres were synthesized according to the previous report [47]. To prepare ZnO/SiO 2 MMs, the ZnO microspheres on the glass slide were partially coated with a SiO 2 nanolayer by magnetron sputtering for 40 min (JCP500, Beijing Technol Science Co., Ltd, China). The substrate was heated to 100 o C and rotated at a speed of 30 r/min during the sputtering process. In addition, ZnO/Pt MMs and SiO 2 /ZnO MMs were also prepared by sputtering Pt on the ZnO microspheres on the glass slide for 30 s and a ZnO layer on monodispersed SiO 2 microspheres (2 μm) for 60 min, respectively.

Self-propulsion and chemotaxis
To observe the self-propulsion of ZnO/SiO 2 MMs, a 20 μL aqueous suspension of the MMs (0.03 mg/mL) was dropped into a Petri dish (d = 35 mm) with 2 ml water. The self-propulsion of the MMs was observed and recorded through an inverted optical microscope (Leica DMI 3000B). All videos were analyzed using ImageJ and Video Spot Tracker V08.01 software. More than 10 MMs were analyzed to obtain the statistical result. To investigate the MM's chemotaxis, a micropipette (inner d, 20 μm) filled with a CO 2 solution (C CO 2 = 93.7 μM) was fixed on the holder of a high-precision micromanipulator (Leica Microsystems, Germany) and then put in a water droplet (50 μL, pH = 6.52) with the ZnO/SiO 2 MMs (0.003 mg/mL).

Numerical simulations
The simulations were performed by using the diffusions, electrostatics and creeping flow modules of