Biohybrid Magnetically Driven Microrobots for Sustainable Removal of Micro/Nanoplastics from the Aquatic Environment

RESEARCH ARTICLE www.afm-journal.de Biohybrid Magnetically Driven Microrobots for Sustainable Removal of Micro/Nanoplastics from the Aquatic Environment Xia Peng, Mario Urso, Martina Kolackova, Dalibor Huska, and Martin Pumera* The proliferation of micro/nanoplastics derived from the fragmentation of plastic waste released in the environment represents an increasingly alarming issue with adverse implications for aquatic ecosystems worldwide. Conventional approaches for mitigating such contamination are inadequate in removing plastic fragments with exceptionally tiny sizes. Therefore, it is highly urgent to develop efficient strategies to address the threats posed by micro/nanoplastics. Here, biohybrid microrobots, integrating the magnetic properties of Fe3O4nanoparticles, are investigated for the dynamic removal of micro/nanoplastics from various aquatic environments via high-precision magnetic actuation and reliable electrostatic interactions. After the surface decoration with Fe3O4nanoparticles, algae cells can achieve precise locomotion and wireless manipulation by regulating an external magnetic field. Taking advantage of this active movement, magnetic algae robots (MARs) display considerable capture and removal efficiencies for micro/nanoplastics in water with extensive application scenarios. The reusability of MARs is also investigated, proving great recyclable performance. The growth and cell viability experiments elucidate that the presence of Fe3O4 nanoparticles may result in hormesis stimulation of algae growth. Such recyclable microrobots with eco-friendly and low-cost characteristics offer an attractive strategy for sustainably tackling micro/nanoplastics pollution. X. Peng, M. Urso, M. Pumera Future Energy and Innovation Laboratory Central European Institute of Technology Brno University of Technology Purkynova 123, Brno 61200, Czech Republic E-mail: [email protected].cz M.Kolackova,D.Huska DepartmentofChemistryandBiochemistry MendelUniversityinBrno Zemedelska1,Brno61300,CzechRepublic The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.202307477 © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/adfm.202307477 1. Introduction Plastics are synthetic polymers composed of hundreds to thousands of organic subunits known as monomers, which are interconnected through robust covalent bonds.[1] Their exceptional properties, characterized by high chemical and thermal stability, have led to widespread utilization, while simultaneously presenting a significant obstacle in terms of their environmental management and elimination. Nowadays, the rampant abuse of plastics coupled with inadequate disposal measures has led to a dire situation.[2,3] Undesirably, large plastic waste can disintegrate into minute pieces, microplastics (<5mm in size) and nanoplastics (<1000 nm in size), which have become a growing global concern due to their harmful effects on aquatic organisms and human beings.[4–6] Microplastics tend to settle and accumulate on the seafloor. Nanoplastics, due to their buoyancy, remain suspended in water and disperse rapidly under the influence of M. Pumera Department of Medical Research China Medical University Hospital China Medical University No. 91 Hsueh-Shih Road, Taichung TW-40402, Taiwan M. Pumera Advanced Nanorobots & Multiscale Robotics Laboratory Faculty of Electrical Engineering and Computer Science VSB – Technical University of Ostrava 17. listopadu 2172/15, Ostrava 70800, Czech Republic M. Pumera Department of Chemical and Biomolecular Engineering Yonsei University 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, South Korea Adv. Funct. Mater. 2024,34, 2307477 2307477 (1 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de marine currents.[7] In the marine realm, the ingestion of micro/nanoplastics by fish and other aquatic organisms can result in physical harm and impede their digestive and reproductive processes. The unfortunate consequence of this phenomenon is the suffering and eventual death of these creatures.[8,9] Furthermore, the absorption of toxic substances by micro/nanoplastics, due to their high surface-to-volume ratio, can pose a risk to human health by transferring these harmful chemicals up the food chain.[4,8–11] Thus, there is a pressing need to address the pervasiveness of plastic pollution, specifically the proliferation of micro/nanoplastics in the environment. In light of this, conventional methods, such as physical removal methods (i.e., filtration and sedimentation)[12–14] and chemical degradation methods (i.e., chemical oxidation and photodegradation)[15–18] have been explored for the treatment of micro/nanoplastics in the environment, which are generally limited by low efficiencies and lack of accessible and cheap characterization means because of the tiny size of micro/nanoplastics. The advent of micro/nanorobots, which synergistically integrate the exceptional physical–chemical attributes of micro/nanomaterials with powerful locomotion and allow exactitude in positional control,[19–22] is transforming a plethora of application domains, such as biomedicine,[23–25] sensing,[26–28] imaging,[29,30] and environmental remediation.[31–34] In the past decades, diverse micro/nanorobots actuated by light,[35,36] chemical fuels,[37] and magnetic fields[38,39] have been developed to tackle the threats posed by micro/nanoplastics. Photocatalytic Au@Ni@TiO2micromotors could remove microplastics via phoretic interaction by individual micromotors in a low amount of H2O2or shoveling interaction due to the assembled microchains of several micromotors under the control of magnetic fields.[35] Light-powered multi-layered TiO2/Pt microrobots display negative photogravitaxis, leading to a strong, fuel-free motion in three dimensions when exposed to light. With a distinctive blend of self-propulsion and adjustable Zeta potential, these microrobots swiftly draw and capture nanoplastics on their surfaces, even within the gaps among stacked layers.[40] Moreover, the bubble separation of microplastics was achieved by chemically driven core–shell MnO2-based micromotors in the presence of H2O2.[37] Recently, autonomous magnetorobots consisting of ion-exchange resin spheres and paramagnetic Fe3O4nanoparticles were demonstrated for removing or separating micro/nanoplastics from nonmarine waters.[38] Similarly, well-designed adhesive PDA@Fe3O4MagRobots demonstrated robust adhesion to microplastic pollutants in moist aquatic environments, facilitating efficient capture, transport, and retrieval of microplastic contaminants under the influence of an external rotating magnetic field.[39] Nevertheless, the involvement of chemical fuels may lead to secondary contamination of the environment, and the fabrication cost of microrobots comprising noble metals such as Au and Pt is relatively expensive, making it unfavorable for large-scale production and viable implementation. More importantly, exploring inexhaustible resources from the natural world to address micro/nanoplastics elimination represents a research topic worthy of investigation. Here, we demonstrate biohybrid microrobots integrating algae platforms and magnetic nanoparticles for active capture and removal of micro/nanoplastics from aquatic environments with excellent sustainability and ease of scaling, as illustrated in Figure 1.Chlorella vulgaris (C. vulgaris), which represents one of the most important microalgae in various biotechnology fields,[41] was selected as the structural platform of the microrobots. Through decorating the algae surface with Fe3O4 nanoparticles , the resulting MARs exhibited active motion under wireless magnetic manipulation with a rapid on/off response to the applied magnetic field, without the involvement of chemical fuel and noble metals. The enhanced replication capability of algae with 0.3 mg∙mL−1Fe3O4nanoparticles revealed the hormesis effect on the algae growth rather than higher toxicity. Biohybrid MARs were applied to capture and remove micro/nanoplastics thanks to their active movement and electrostatic interactions. Improved efficiency for capturing nanoplastics than microplastics was demonstrated and attributed to the much smaller surface area of nanoplastics, allowing MARs to capture a larger number of plastic particles. Such magnetic microrobots also display practical utilization in different aquatic environments, endowing them as promising candidates for real-world applications in the future. 2. Results and Discussion 2.1. Fabrication and Characterization of MARs The proposed construction process of magnetic algae robots (MARs) is conceptually illustrated in Figure 2a. Taking advantage of negative surface charges resulting from the carboxylic (─COOH) group on the algae cytomembrane,[42] the algae surface was partially decorated with oppositely charged magnetic Fe3O4nanoparticles, leading to MARs. The morphology of bare algae and MARs was investigated and characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) spectroscopy mapping images. As shown in Figure 2b, the original alga cell possesses the characteristic spheroidal micro-sized structure, and the diameter is ≈3−5μm. After mixing the algae and Fe3O4nanoparticles suspensions (Figure S1a, Supporting Information), the SEM image depicted in Figure 2c suggests that the MAR preserved the original structural feature of the alga cell, while a small amount of Fe3O4nanoparticles asymmetrically bound to its surface. The distribution of the element Fe confirmed that MARs were successfully decorated with Fe3O4 nanoparticles. To further confirm the successful contact between Fe3O4nanoparticles and bare algae cells, X-ray diffraction (XRD) patterns of Fe3O4nanoparticles and MARs were measured. The peaks in the XRD pattern of Fe3O4nanoparticles in Figure 2d correspond to the (220), (311), (400), (422), (511), and (440) crystalline planes of Fe3O4inverse spinel structure (PDF No. 89– 0691).[43] As expected, these characteristic peaks can also be appreciated in the XRD pattern of MARs. Zeta potential measurements validated the electrostatic interaction between algae and Fe3O4nanoparticles. Fe3O4nanoparticles exhibit a positive charge due to the interaction between Fe-OH sites on the surface of Fe3O4nanoparticles and H+ions in the medium, resulting in the generation of positive (Fe-OH2+) surface charges.[44,45] As depicted in Figure 2e, the zeta potential of algae varies from −27 to −8 mV after being integrated with Fe3O4nanoparticles (18 mV). Adv. Funct. Mater. 2024,34, 2307477 2307477 (2 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 1. Biohybrid MARs for effective removal of micro/nanoplastics from water. a) Bare algae cells integrated Fe3O4nanoparticles on the surface via electrostatic absorption. b) Utilizing magnetic actuation and electrostatic interactions, MARs facilitate the removal of micro/nanoplastics from water by drawing them to their surface. 2.2. Motion Behavior of MARs Precise and controllable locomotion of microrobots is of great significance for the capture and removal of micro/nanoplastics.[34] The original C. vulgaris cells exhibit uncontrollable Brownian motion, as shown in Figure S2 and Movie S1 (Supporting Information). On the contrary, extracellular deposition of Fe3O4 nanoparticles constitutes an attractive strategy to endow the algae with excellent magnetic properties, as schematically illustrated in Figure 3a. The optimal concentration of Fe3O4nanoparticles loading on the microalgae cells’ surface was investigated as shown in Figure S3 and Movie S2 (Supporting Information). The Fe3O4nanoparticles concentration of 0.3 mg mL−1was identified as the optimal one for the preparation of MARs because only a few Fe3O4nanoparticles-free microalgae were left in the mixture, while higher concentrations led to microalgae overloading or unbound Fe3O4nanoparticles. The magnetic properties of Fe3O4 nanoparticles (Figure S1b, Supporting Information) and MARs were investigated by the hysteresis loop measured by a VSM. Figure 3b shows the measured magnetization curve for MARs, exhibiting 20.4 emu g−1with zero coercivity, which indicates a superparamagnetic behavior.[46] In addition to being easily separated from water, untethered magnetic manipulation of MARs can be achieved by the magnetic field generated by a permanent magnet, as displayed in Figure 3c and Movie S3 (Supporting Information). Using a rotating magnetic field generated by a homemade set of orthogonal coil pairs allows exploiting several parameters to tune MARs actuation precisely, such as the magnetic field intensity and rotation frequency. MARs speed shows remarkable variation under different intensities and frequencies. In particular, Figure 3d provides the evidence that the MARs speed increases proportionally with the frequency until reaching 35 μm s−1when exposed to a magnetic field intensity of 5 mT and a frequency of 30 Hz, which is commonly known as the “stepout” frequency,[47] followed by a decline since MARs cannot follow the rotation of the magnetic field at higher frequencies. The motion behavior of MARs can be easily recognized by switching on/off the magnetic field, as evidenced by the distinctive changes in the instantaneous speed over time reported in Figure 3e. The time-lapse images in Figure 3f also corroborate the controllable Adv. Funct. Mater. 2024,34, 2307477 2307477 (3 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 2. Preparation and characterization of MARs. a) Schematic diagram of the fabrication process. SEM and EDX elemental mapping images of: b) (a) abarealgaandc)aMAR.Scalebarsare1μm. d) XRD patterns of Fe3O4nanoparticles and MARs. e) Zeta potential of bare algae, Fe3O4nanoparticles, and MARs. actuation of MARs manipulated by the external magnetic field (Movie S4, Supporting Information). 2.3. Cell Viability of Microalgae The growth of C. vulgaris cells under Fe3O4nanoparticles exposure at the concentrations of 0.3 and 0.6 mg∙mL−1was studied. First, the growth was observed and expressed as OD750, a typical growth evaluation parameter.[48] In fact, C. vulgaris absorbed some Fe3O4, which caused interference in the measurement, resulting in false-negative results. Concurrently, it showed and confirmed the excellent capacity of C. vulgaris for metal accumulation, similar to previous works.[49,50] Therefore, the growth was then evaluated gravimetrically by comparing dry mass (DW) after treatment. As shown in Figure 4a, the dry mass of both Fe3O4 nanoparticles-treated C. vulgaris was comparable with the control. Notably, the treatment with 0.3 mg∙mL−1concentration of Fe3O4nanoparticles significantly favored the growth process. Indeed, Fe3O4nanoparticles may trigger the hormesis stimulation of C. vulgaris. Contrarily, a slightly reduced growth can be observed in the presence of 0.6 mg∙mL−1concentration of Fe3O4 nanoparticles, which indicates that higher doses can be more toxic, hindering the growth of microalgae.[51] To explain this behavior, different aspects and properties of the materials need to be considered since iron plays a crucial role in the cell division process.[52] Additionally, the morphology of nanoparticles or selected microalgal species may be also considered.[53] For instance, a reduced growth of C. vulgaris. was previously demonstrated in the presence of 0.1 mg∙mL−1and a higher concentration of Fe3O4nanoparticles.[49] Another study revealed the highest tolerant/sublethal dose of Fe3O4nanoparticles, specifically 0.05 mg∙mL−1, using microalgae Coelastrella terrestris.[54] Figure 4b, as a photo documentation supplement of the growth process, also displays the color change over time for the control sample and the Fe3O4nanoparticles concentration of 0.3 mg∙mL−1, consistent with the results in Figure 4a. On the contrary, the color of the sample with a concentration of Fe3O4nanoparticles of 0.6 mg∙mL−1is darker even at 0 h. Consequently, the higher amount of Fe3O4nanoparticles does not allow a straightforward comparison between the results in Figure 4a and the photos in Figure 4b for the 0.6 mg∙mL−1Fe3O4nanoparticles concentration. Although the morphology of nanoparticles may affect their toxicity in microalgal growth,[55,56] the results indicate minimal toxicity of selected Fe3O4nanoparticles despite using relatively high concentrations on the growth of C. vulgaris.It is worth noting that the 0.3 mg∙mL−1Fe3O4nanoparticles concentration corresponds to the growth condition of MARs whose motion and micro/nanoplastics removal has been investigated in this work. Adv. Funct. Mater. 2024,34, 2307477 2307477 (4 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 3. Magnetic motion of MARs. a) Schematic diagram of the motion principle of MARs under a rotating magnetic field. b) Magnetic hysteresis loop of MARs. c) Magnetic property of MARs using a permanent magnet promises precise manipulation by utilizing an external magnetic field. d) MARs speed at different intensities and frequencies of the applied rotating magnetic field. Error bars represent the standard deviation, n=10 independent replicates. e,f) MARs on/off response in terms of instantaneous speed and time-lapse micrographs by on/off switching of the applied rotating magnetic field. The scale bar is 10 μm. 2.4. Micro/Nanoplastics Capture The escalating presence of hazardous micro/nanoplastics in aquatic ecosystems poses an emergent challenge to the health of aquatic organisms and human beings.[57] Microrobots, the ground-breaking technology that integrates active interaction and precise orientation, represent a practical approach to addressing this issue.[58–60] Here, magnetically actuated MARs promise sustainable removal of micro/nanoplastics in water from different sources. Amino-modified polystyrene (PS) microand nanoparticles with diameters of 1.5 μm and 50 nm were used as representative microplastics and nanoplastics (Figure 5a,b). The size distribution of the micro/nanoplastics was also determined by dynamic light scattering, whose results are consistent with the size indicated by SEM analysis, as illustrated in Figure 5c,d. The measured Zeta potential of micro/nanoplastics is shown in Figure 5e. Both types of plastic particles are positively charged because of the amino-modified surface. In particular, the Zeta potential values for nanoplastics and microplastics are +44 and +38 mV, respectively. In comparison, the Zeta potential of MARs is −8 mV, facilitating the electrostatic attraction between MARs and micro/nanoplastics. Based on the information provided by the manufacturer, the micro/nanoplastics exhibit blue fluorescence. Taking advantage of this property, micro/nanoplastics removal was monitored by a spectrofluorometer, which allows for examining the treated solutions. To assess this method, serial dilutions of the as-received micro/nanoplastic suspensions (6 × 109microplastics mL−1and 4 ×1014 nanoplastics mL−1)were performed, and the corresponding fluorescence intensities are Adv. Funct. Mater. 2024,34, 2307477 2307477 (5 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 4. Cell viability of algae in the presence of Fe3O4nanoparticles. a) Growth of C. vulgaris without (control) and with Fe3O4nanoparticles at 0.3 and 0.6 mg∙mL−1concentrations. Every sample was taken daily, dried, and recalculated to dry weight (DW, g∙L−1). Error bars represent the standard deviation, n=3 independent replicates. b) Photographs of C. vulgaris during the experimental testing without (control) and with Fe3O4nanoparticles at 0.3 and 0.6 mg∙mL−1concentrations. Figure 5. Micro/nanoplastics characterization. a,b) SEM images of nanoplastics (the scale bar is 200 nm) and microplastics (the scale bar is 1 μm) in water. c,d) Size distribution of the nanoplastic and microplastic suspensions. The shaded region represents the standard error for n=3 replicates, substituting the discrete error bars. e) Zeta potential of nanoplastics and microplastics. f,g) Fluorescence intensities variation for serial dilutions of nanoplastic and microplastic suspensions. Adv. Funct. Mater. 2024,34, 2307477 2307477 (6 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de shown in Figure 5f,g. It was noted that the fluorescence intensity significantly decreased with the increasing dilution factor, as expected. Considering that 50 nm large nanoparticles cannot be visualized under an optical microscope, only the optical images of microplastic suspensions after serial dilutions were captured and reported in Figure S4 (Supporting Information), evidencing a good agreement with the variation of fluorescence intensities reported in Figure 5g. Additionally, a detection limit of 1.8 ×106 microplastics mL−1and 5 ×1010 nanoplastics mL−1were determined. The micro/nanoplastics capture mechanism is schematically illustrated in Figure 6a. MARs effectively remove micro/nanoplastics due to their powerful motion, induced by a transversal rotating magnetic field (Figure S5, Supporting Information) at 3 mT intensity and 50 Hz frequency, resulting in an average speed of MARs of 7.5 μms −1, promoting electrostatic interactions with targeted plastics. Micro/nanoplastics removal by MARs was evaluated in square-shaped cuvettes under magnetic motion along a predefined trajectory (Figure S6, Supporting Information) or no motion conditions for different treatment periods (0, 5, 10, 20, 30, and 40 min). The fluorescence spectra in Figure S7 (Supporting Information) show the variation of micro/nanoplastics concentrations before and after treatments by MARs without/with applying the rotating magnetic field, respectively, indicating the successful removal of micro/nanoplastics from water. The removal efficiency was calculated based on Equation (1): Removal Efficiency =C0−Ct C0 ×100%(1) where micro/nanoplastics concentrations at time t(Ct)and0min (C0) were determined by measuring the fluorescence intensity at 418 nm, corresponding to the highest peak. Specifically, the removal efficiencies of nanoplastics (Figure 6b) are 92% and 71% for active motion and no motion, respectively, calculated from Figure S7a,b (Supporting Information), demonstrating the contribution of the MARs’ active motion in accelerating nanoplastics removal. Figure 6c reports the microplastic removal efficiencies, which are 70% for active motion and 41% for static condition (Figure S7c,d, Supporting Information). In order to explain the removal efficiency difference between nanoplastics and microplastics, SEM images of MARs after interactions with micro/nanoplastic suspensions for 40 min under the rotating magnetic field were collected and shown in Figure 6d. Nanoplastics exhibit a remarkable affinity to the entire surface of MARs due to the much smaller size compared to microplastics and sufficient electrostatic interactions. In contrast, microplastics, though still minute in size, encounter challenges in occupying the surfaces of MARs due to their similar order of magnitude in size as microalgae cells. Hence, the comparable size between microplastics and algae restricts the microplastics’ ability to effectively cover a significant portion of the MARs surface, limiting their removal efficiency. Consequently, MARs show higher potential in the removal of nanoplastics. Fluorescence images in Figure S8 (Supporting Information) also demonstrate that nanoplastics covered the surfaces of MARs after the treatment. Microplastics removal by the MAR was also observed at an optical microscope (Figure 6e). The time-lapse images captured from Movie S5 (Supporting Information) illustrate the successful capture and transport of microplastics that interacted with the MAR in water, which also elucidates that Fe3O4nanoparticles remained on the surface of the microalgae during the active capture and removal of micro/nanoplastics and barely caused secondary contamination under control of the rotating magnetic field at 3 mT intensity and 50 Hz frequency. On the contrary, thanks to the presence of Fe3O4nanoparticles, MARs empowered with active motion exhibited better performance in removal of micro/nanoplastics than bare algae cells, as illustrated in Figure S9 (Supporting Information). For real world settings it is important to investigate the effect on the removal efficiency of the micro/nanoplastics shape. However, this aspect goes beyond this present study due to limitations in acquiring fluorescent micro/nanoplastics with different shapes at a fixed size. Of note, according to the previous research, different shapes of microplastics show negligible effects on the removal efficiency.[38] The reusable and low-cost characteristics of microrobots are considered the crucial feature that influences their long-term and sustainable applications for environmental remediation.[38] Figure 6f shows that MARs preserved electrostatic attraction capability over five cycles of treatment, guaranteeing ≈80% removal efficiency for nanoplastics and ≈54% for microplastics after the cycles. Through the cycle process, the magnetic response of MARs became weaker due to the vigorous agitation required to release the plastic particles from the robots’ surface, which is ascribed to the loss of Fe3O4nanoparticles from the algae surface, as shown in Figure S10 (Supporting Information). On the other hand, the navigation speed of MARs decreased during the recycling experiments (Figure S11, Supporting Information), which also results in the declined removal efficiencies. Nonetheless, the magnetic properties of MARs can be revived by mixing them with freshly prepared Fe3O4nanoparticles again. The microalgae cells preserved their original shape and green color after recycling experiments, as shown in Figure S12 (Supporting Information), which elucidates their high viability.[61] To prove the practical utilization of MARs, Figure 6g reports the removal efficiencies of nanoplastics by MARs in different aquatic environments, such as tap water, rainwater, and lake water (Figure S13, Supporting Information), confirming the MARs excellent operation not only in DI water samples but also in real contaminated water samples. The removal efficiencies in tap water, rainwater, and lake water were 90%, 86%, and 89%, respectively. MARs performance was also evaluated against the mixture of nanoand microplastics, finding a slight decrease (84%), owing to surface take-up competition between the two classes of plastic particles. These experiments validate the applicability of the proposed strategy in natural settings. 3. Conclusion In conclusion, we have presented eco-friendly and magnetic fielddriven algae-based microrobots for effective capture and removal of micro/nanoplastics from the aquatic environment. The type of algae C. vulgaris, one of the most important microalgae in various biotechnology fields, was chosen as the platform for formulating biohybrid microrobots with a negatively charged surface. After decoration with positively charged Fe3O4nanoparticles, microalgae realized precise actuation and collective manipulation rather Adv. Funct. Mater. 2024,34, 2307477 2307477 (7 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 6. Micro/nanoplastics capture by MARs. a) Schematic diagram of the dynamic removal of micro/nanoplastics by MARs under the rotating magnetic field. b) Nanoplastics and c) microplastics removal efficiencies by MARs as a function of time varying from 0 to 40 min. d) SEM images depicting MARs after being exposed to the suspensions of micro/nanoplastics. Scale bars are 1 μm. e) Time-lapse images showing the transport of microplastics by a MAR. Scale bars are 5 μm. f) Reusability of MARs for nanoplastics (NP) and microplastics (MP) removal from water over five cycles. g) Removal efficiencies of MARs from DI water, tap water, rainwater, lake water, and the mixture of nanoplastics and microplastics. Error bars represent the standard deviation, n=3 independent replicates. than uncontrollable Brownian motion. To investigate the toxicity of Fe3O4nanoparticles for microalgae, biomass experiments were conducted, which demonstrated that the exposure to 0.3 mg∙mL−1Fe3O4nanoparticles resulted in the hormesis stimulation of algae cells growth. The capture of micro/nanoplastics by MARs in water was performed under the control of an external magnetic field to promote the electrostatic attraction of targeted micro/nanoplastics. Taking advantage of their active motion, MARs exhibited considerable efficiencies of 92% for nanoplastics and 70% for microplastics. The reusability of MARs Adv. Funct. Mater. 2024,34, 2307477 2307477 (8 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de was also tested over five cycles, preserving ≈80% removal efficiency for nanoplastics and ≈54% for microplastics. Considering the complexity of real aquatic environments, different water samples (tap water, rainwater, lake water) were collected and examined to validate the applicability of MARs. The introduced biohybrid microrobots, originating from naturally existing microalgae integrated with magnetic Fe3O4nanoparticles, hold considerable promise for addressing environmental challenges associated with micro/nanoplastics in a sustainable and cost-effective manner. 4. Experimental Section Model Organism:C. vulgaris (SAG 211-11b), a strain of green microalgae, was obtained from the Department of Experimental Phycology and Culture Collection of Algae (EPSAG) at Georg-August-Universität Göttingen (Göttingen, Germany). The microalgae were cultivated in Trisacetate-phosphate (TAP) medium, using Erlenmeyer flasks, and shaking at 120 rpm under mixotrophic conditions. The cultures were grown at 23 ±1°C with illumination at 130 μmol m−2s−1in 12-h light and 12-h dark photoperiods. Synthesis of Fe3O4Nanoparticles:Briefly, 0.6 g CaCl2(Sigma Aldrich, ACS reagent, 99%) and 0.6 g FeSO4(Sigma Aldrich, ACS reagent, 99%) were dissolved in 21 mL deionized (DI) water (18 MΩcm), respectively, to form uniform solutions. Then, two solutions were mixed under magnetic stirring for 5 min at room temperature. Subsequently, 1.1 g FeCl3(Alfa Aesar, 97%) was added to the mixture under continuous magnetic stirring for 15 min at 60 °C in an oil bath. Afterward, NaOH solution (Merck, 25%) was added to the mixture slowly until the mixture’s pH value reached ≈11, and the solution turned into a uniform black suspension. After oil bath treatment for another 15 min under magnetic stirring, the resulting product was isolated using a permanent magnet and washed three times with DI water and ethanol, respectively. Finally, the Fe3O4nanoparticles were naturally dried in a fume hood at room temperature. Synthesis of MARs:The green algae were washed using DI water several times to eliminate the TAP medium from the surface. Then, they were suspended in DI water. 0.3 mg∙mL−1(final concentration) Fe3O4nanoparticles solution was sonicated for 15 min, mixed with the algae suspension (VFe3O4:Valgae =1:1), and vortexed for 10 min at 1000 rpm. A permanent magnet was applied to primarily assess the magnetic performance of MARs. Characterization of MARs:To perform SEM characterization, MARs were initially treated with 2.5% glutaraldehyde for an overnight fixation at 4 °C, followed by washing with DI water to remove residues. After overnight drying, MARs were characterized by scanning electron microscopy (SEM, MIRA3-XMU) with an energy-dispersive X-ray (EDX) detector (Oxford Instruments) after coating with 10 nm gold/palladium. X-ray diffraction (XRD) was used to measure the crystal structure (Rigaku SmartLab 3 kW diffractometer, Cu K𝛼radiation). The same methodology was used to treat and examine bare algae. The magnetic hysteresis loop was measured using a Quantum Design VersaLab cryogen-free VSM at 300 K, with an applied magnetic field ranging from −15 to 15 kOe at 10 Oe s−1steps. Micro/nanoplastics and MARs were diluted in DI water, respectively, and then 1 mL solution was transferred into a cuvette. Then, size distribution and Zeta potential measurements were carried out using a Malvern Panalytical Zetasizer Ultra instrument, where the cuvette was placed inside. The measurements were repeated three times to produce the error bars. Motion Analysis:The magnetic movement of MARs was evaluated and recorded using an inverted Nikon ECLIPSE TS2R microscope coupled with a BASLER acA1920-155uc digital camera. No surfactant was utilized during the experiments. A homemade magnetic setup composed of three orthogonal coil pairs positioned on a polylactic acid (PLA) support was employed to regulate the magnetic motion. The setup created a transversal rotating magnetic field. The microrobots were steered under magnetic fields of 3 and 5 mT at various frequencies ranging from 0 to 300 Hz. The captured videos were analyzed using NIS Elements Advanced Research software. The components of the transversal rotating magnetic field, Bx,B y,B z, are expressed by the following equations: Bx=B0cos (𝜔t)sin (𝛼)(2) By=B0cos (𝜔t)cos (𝛼)(3) Bz=B0sin (𝜔t)(4) In these equations, B0denotes the magnetic field’s magnitude, which bears a direct proportionality to the current flowing through the coils. Additionally, 𝜔=2𝜋f,wherefis the frequency in Hertz [Hz], tis the time in seconds [s], and 𝛼is the navigation angle, spanning a comprehensive range from 0 to 360°. It is pertinent to note that by adjusting the value of 𝛼, a remarkable degree of control is achievable to empower the manipulation of the microrobots’ trajectory confined within the XY plane, further ensuring high precision over their navigation. The magnetic field assessments were conducted at the center of the coil holder, wherein a sample holder can be well positioned and the magnetic field was measured by using an A1302 Hall effect sensor. Microalgal Growth Test:Toxicological assays were performed according to OECD guidelines, TG 201, employing 72 h of exposure. In brief, C. vulgaris cells in the exponential growth phase were inoculated into sterile TAP media with/without adding 0.3 and 0.6 mg∙mL−1Fe3O4nanoparticles. The experiments were conducted in 6-well culture plates with 5 mL of medium, and the cultures were maintained under the aforementioned conditions for 72 h. During experimental windows, every sample was taken daily, dried for 24 h, and analyzed gravimetrically. Micro/Nanoplastics Capture Experiments:Fluorescence spectroscopy was used for micro/nanoplastics quantification. Two stocks of micro/nanoplastics were prepared by diluting the commercial suspension, respectively. Specifically, 1.5 mL of MARs and 1.5 mL micro/nanoplastics solutions were transferred in cuvettes. Then, the cuvettes were put in the center of the rotating magnetic setup, set at 3 mT intensity and 50 Hz frequency. Following the treatment, MARs were isolated from the solution by employing a permanent magnet. The MARs and the treated solutions were preserved for subsequent analysis. The experiments were replicated a minimum of three times. To evaluate the reusability of MARs, they were separated by an external magnet from the purified water after the treatment and subjected to vigorous shaking in water for 5 min to dislodge the micro/nanoplastics from their surface. After shaking, MARs were collected again and applied for following cycles. Each cycle was repeated three times to calculate the error bars. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements M.P. acknowledges the financial support of the Grant Agency of the Czech Republic (EXPRO: 19–26896X). X.P. was supported by the China Scholarship Council (CSC No. 202008320382) and acknowledges the Brno Ph.D. Talent scholarship funded by the Brno City Municipality. Czech Nano Lab project LM2023051 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements/sample fabrication at CEITEC Nano Research Infrastructure. Conflict of Interest The authors declare no conflict of interest. Adv. Funct. Mater. 2024,34, 2307477 2307477 (9 of 11) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202307477 by Brno University Of Technology, Wiley Online Library on [10/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License