Acrylic acid (AA), trimethylolpropane ethoxylate triacrylate (Mn = 428 g mol⁻¹, PEG₄₂₈-triacrylate), polyvinylpyrrolidone (Mn = 360,000 g mol⁻¹, PVP), 2,2-dimethoxy-2-phenylacetophenone (DMPA), dimethylformamide (DMF), isopropyl alcohol (IPA), sodium hydroxide (NaOH), ferric chloride hexahydrate (FeCl₃·6H₂O), ferrous chloride tetrahydrate (FeCl₂·4H₂O), ammonium hydroxide (NH₃·H₂O), and silver nitrate solution were purchased from Aladdin.

The material composition of the initial pre-polymer mixture used to create the HMs consisted of 15 wt% AA, 15 wt% PEG₄₂₈-triacrylate, 10 wt% PVP, 3 wt% DMPA, and 57 wt% DMF.

The HMs were prepared by UV-initiated free-radical polymerization of acrylate in the pre-polymer mixture. The HMs were fabricated using UVL, as shown in Figure 1. First, a uniform layer of the pre-polymer mixture was formed by spinning 3 mL of the mixture onto a silicon wafer at 1,000 rpm for 60 s. The wafer was then prebaked at 95°C for 3 min to evaporate the organic solvent DMF completely, which resulted in a homogeneous, hard film on the wafer. The condensed long PVP chain acts as a scaffold to prevent PEG₄₂₈-triacrylate from flowing freely. After cooling to room temperature, the hard film was exposed to UV light (Mask Aligner, SUSS MA6, Germany) for 0.2 s at 8.9 mW cm⁻² through a chrome photomask. The photoinitiator DMPA initiates PEG₄₂₈-triacrylate cross-linking polymerization under UV light, and PVP and PEG polymer networks were formed with permanent polymerization structures. After exposure, the HMs were developed by immersing the wafer in IPA to wash away the unpolymerized region to reveal the polymerized HMs. The HMs were washed three times with DI water and visually examined under an inverted optical microscope (MJ33, Mingmei, China).

In situ coprecipitation was previously used to grow iron oxide nanoparticles inside the microparticles (Suh et al., 2012). Here, a similar method was used to grow iron oxide nanoparticles inside the HMs. To fully deprotonate the COOH groups of AA to COO⁻ groups, the HMs were dispersed in 0.5 M NaOH solution for 15 min and washed three times with DI water to remove excess NaOH solution. Then, 0.2 M FeCl₃ and 1 M FeCl₂ solutions were mixed in a ratio of 1:15 with the HMs for 20 min. After the iron ions diffused into the HMs and fully formed a chelate with COO⁻ groups, the excess solution was removed by using an absorbent paper. NH₃·H₂O and DI water were mixed in a ratio of 1:2 with the HMs at 65°C for 15 mins, resulting in iron oxide nanoparticles nucleated and growing inside the HMs, which yielded the MHMs. The MHMs were washed three times with DI water. To further increase the magnetic content, the in situ coprecipitation process was repeated five times.

A layer of silver nanoparticles was grown on the surface of the MHMs through the silver mirror reaction to enable SERS detection capability (Wang, 2020). First, 1 g of PVP was dissolved in 10 mL of ethanol. Then, 200 μL of silver nitrate solution and 100 uL of the MHM solution were added to the mixture and shaken for 5 min. Finally, the mixture was transferred to a reaction kettle and heated at 160°C for 4 h. After transfer to room temperature, the MHM probes were collected by centrifugation (5,000 r/min for 3 min) and washed three times with DI water.

The size and surface morphology of the HMs, MHMs, and MHM probes were characterized using scanning electron microscopy (SEM, Merlin, ZEISS, Germany) at 2 keV. The elemental composition of the MHM probes was characterized using energy-dispersive spectrometry (EDS, Octane Pro, United States) at 5 keV. The magnetic hysteresis loop of the MHMs was obtained using a vibrating sample magnetometer (VSM, 7404, Lake Shore, United States) at room temperature.

The MHMs were actuated using a control system composed of an imaging system (microscope and camera) and a three-dimensional Helmholtz coil system. For motion control test, 300 μL of the solution containing the MHMs was dropped onto a sealed PDMS chamber. A 10-mT RMF produced by a magnetic coil system was used to manipulate the MHMs. By adjusting the rotation frequency and direction of the RMF, the propulsive velocity of the MHMs can be adjusted. The imaging system recorded their movement at 30 fps, and a MATLAB tracking algorithm was used to analyze the propulsive velocity and trajectory of the MHMs.

As shown in Figure 5, 50 μL of crystal violet (CV) ethanol solution with a gradient concentration of 10^–5 ∼ 10^–8 M was first dropped into a PDMS chamber. Then, 50 μL of ethanol containing the MHM probes was then transferred to the CV solution for molecular adsorption. After 60 min of molecular enrichment, excess CV solution was removed, and the MHM probes were dried for Raman measurements. The Raman spectra were collected using a confocal Raman spectrometer (LabRAM HR Evolution, Horiba, Japan) with a 532 nm laser and an integration time of 30 s. As shown in Figure 6B, the active MHM probes were actuated by a 15-Hz RMF for active molecular adsorption. As shown in Figure 6D, the active MHM probes were actuated by a 2-Hz, 5-Hz, and 10-Hz RMF.

The MHM probes were carefully examined at the three major stages of fabrication: HMs after UVL, MHMs after in situ coprecipitation, and MHM probes after the silver mirror reaction. Their morphology and elemental composition were characterized using SEM/EDS. The SEM images of HMs, MHMs, and MHM probes are shown in Figure 2A–D, respectively. EDS mapping showed the uniform distribution of C\N\Fe in the MHM after the in situ coprecipitation process (Figures 2E–G) and the uniform distribution of silver nanoparticles on the MHM probes (Figure 2H). The uniform coating of silver nanoparticles enables the function of SERS detection. To verify the consistency of the UVL fabrication of the HMs, the size distributions of the HMs were measured, which included the distribution of short arms, long arms, and body lengths (Figure 2I). As shown in Figures 2J–L, the size distributions of the short arm, long arm, and body lengths were 20.2 ± 0.7 μm, 39.9 ± 0.8 μm, and 69.0 ± 1.2 μm, respectively. On a single substrate, 280,000 HMs can be obtained from 3 mL of the pre-polymer mixture, which indicates the high manufacturability of this method. These experimental results demonstrated that the UVL process can create MHM probes with high uniformity, efficiency, and consistency.

The in situ coprecipitation method was used to grow iron oxide nanoparticles inside the MHMs. Several in situ coprecipitation cycles were performed to increase iron oxide nanoparticle content; the morphology and magnetic properties of MHMs were tested after several cycles. The optical images of HMs before in situ coprecipitation, MHMs after the first cycle, and MHMs after the fifth cycle are shown in Figures 3A(a–c). The results showed that the MHMs gradually darkened with the increase in the number of cycles, which suggests that more iron oxide nanoparticles were grown. For quantitative analysis of the magnetic property of the MHMs after in situ coprecipitation, the hysteresis loops of the MHMs were obtained using a VSM (Figure 3B). After the first, third, and fifth cycles, the saturation magnetizations of the MHMs reached 0.7, 3.3, and 12.0 emu/g, respectively, which indicates that multiple cycles can enhance the magnetism of the MHMs. In addition, Figure 3C shows that the magnetization values of the MHMs are close to zero when no magnetic field is applied, which indicates that the synthesized iron oxide nanoparticles are superparamagnetic. To verify the biocompatibility of the MHMs loaded with iron oxide nanoparticles, cell survival experiments were carried out, and the results showed that MHMs had very low toxicity to cells (Supplementary Figure S1).

MHMs are capable of precise movement guided by an external RMF generated using a magnetic coil system (Figure 4A) (Cheang et al., 2017; Sachs et al., 2018). The motion schematic of a representative MHM is shown in Figure 4B. The RMF actuated the MHMs by generating a magnetic torque that drove the MHMs to rotate synchronously with the RMF; this allows the MHMs to convert rotational motion into translational motion. Since the MHMs were close to the bottom of the PDMS chamber, their total velocity consisted of two components: swimming velocity, V _s , and drifting velocity, V _d (Figure 4B). The drifting motion was due to the interaction with the substrate (Peters et al., 2014).

The relationship between the velocity of MHMs and the RMF frequency is shown in Figure 4C (also see Supplementary Video S1). As the frequency of the RMF increased, the resultant velocity ( V r = V s 2 + V d 2 ) and V _d increased significantly. The V _r of MHMs reached the maximum value of 214 μm/s (∼3.1 body length/s) at 10 Hz. After 10 Hz, V _r decreased slowly but the MHMs maintained a steady rotation. Interestingly, this is different from the direct loss of motion ability after step-out for rigid microswimmers. We speculated the reason to be subtle deformations of their soft bodies (unobservable from Supplementary Video S1), leading to this behavior after max velocity. The relationship between V _s of the MHMs and the RMF frequency is shown in Figure 4D. The average V _s profile is highly linear from 1 to 11 Hz, and the maximum V _s reached was 74.8 μm/s at 11 Hz. The maximum average swimming efficiency was 0.1 (swimming efficiency = V _{s, max} /(Lf), V _{s, max} = 74.8 μm/s, body length L = 69.0 µm, and frequency f = 11 Hz). Supplementary Table S1 summarizes the parameters of the magnetically actuated hydrogel-based microswimmers reported in the last 5 years. Supplementary Table S1 shows that the MHMs have higher step-out frequency than many of the previously reported magnetically actuated hydrogel-based microswimmers that used physical mixing or chemical conjugation to incorporate magnetic material (Ceylan et al., 2019; Park et al., 2019; Lee et al., 2021; Park et al., 2021). Microswimmers that utilized surface coating of magnetic nanoparticles have higher step-out frequencies (Wang et al., 2018; Dong et al., 2020); however, their surfaces are completely covered which may hinder further functionalization. Furthermore, the MHMs have a swimming efficiency on par with the other types of microswimmers (Wang et al., 2018; Ceylan et al., 2019; Park et al., 2019; Dong et al., 2020; Lee et al., 2021; Park et al., 2021). The high step-out frequency of the MHMs was due to the increase in iron oxide content from the in situ coprecipitation process that allowed them to have a larger magnetic moment. This comparison highlighted that in situ synthesis offers a way to increase the magnetic moment of the hydrogel microswimmers without modifying the surfaces of the microswimmers. Furthermore, we also analyzed the drift angle [θ = cos⁻¹(V _s /V _r )], which represents the influence of the surface on the swimming state of the MHMs. As shown in Figure 4E, the drift angle decreased linearly as the frequency increased. This indicates that the MHMs are less affected by the interaction with the substrate at higher frequencies. In addition, the synchronized motion of two MHMs under an RMF (10 mT, 6 Hz) is shown in Figure 4F (also see Supplementary Video S2). The red and yellow curves represent the trajectories of MHM1 and MHM2, respectively, as shown in Figure 4F(a). As shown in Figures 4F(b–e), the two MHMs can move along their respective square trajectories under the precise control of the RMF.

To demonstrate that the in situ coprecipitation can yield MHMs with surfaces that are free to be functionalized for different applications, silver nanoparticles were grown on the surfaces of the MHMs to enable SERS detection capability. Silver nanoparticles are a common, highly active, and biocompatible SERS substrate (Liu et al., 2009). They can enhance the Raman spectrum signal of analyte molecules and improve the detection sensitivity of the analyte, which is very suitable for biological analysis. To test the SERS sensing ability of the MHM probes, CV was selected as the analyte. CV is a commonly used analyte, and its chemical structure is shown in Figure 5A. The main Raman peaks of CV are observed at 912, 1,172, 1,372, 1,587, and 1,618 cm⁻¹ (Raman peaks and corresponding vibrational functional groups are shown in Supplementary Table S2) (Liu et al., 2009). First, the MHM probes were immersed in CV ethanol solution at different concentrations (10^–5 ∼ 10^–8 M) for 60 min. Then, the Raman spectrum of CV was collected by laser Raman spectroscopy to analyze the relationship between concentration and Raman intensity, as shown in Figure 5B. The peak position of the Raman spectrum was almost consistent with the peak position corresponding to vibration functional groups in Supplementary Table S2, verifying the SERS capability of the MHM probes (Figure 5C). The variation in Raman intensity at different CV concentrations at the same peak position is shown in Figure 5D. As the CV concentration gradually decreased (10^–5 ∼ 10^–8 M), the intensity of all peaks gradually decreased, indicating that the detection ability of CV by MHM probes is positively correlated with CV concentration.

Traditional SERS probes lacked propulsion and could only rely on passive diffusion to collect analyte molecules, so the detection time was limited by the diffusion rate of analyte molecules in the solution. MHM probes could accelerate the diffusion between silver nanoparticles and analyte molecules through active motion, improve the collection efficiency of analyte molecules, and thus reduce the collection time of analyte molecules and enhance SERS intensity. Here, the detection performances of the MHM probes on CV analytes under active and inactive states were compared. As illustrated in Figure 6A, the active state refers to the MHMs being in motion under an RMF, and the inactive state refers to the MHMs being at rest. CV molecules were collected by active and inactive MHM probes at the concentration of 1 × 10⁻⁵ M CV for 30 min. The Raman spectra of the two groups of probes are shown in Figure 6B. The red curve and the black curve represent the active and inactive MHM probes, respectively. The results show that the Raman intensity of the active MHM probes is approximately 170% that of the inactive probes (Figure 6C), which indicates that the active MHM probes can collect more CV molecules within the same time duration and has higher collection efficiency. Furthermore, Raman measurements were performed in different active states with the frequencies of 2, 5, and 10 Hz. The CV concentration for these tests was 1 × 10⁻⁵ M. As shown in Figures 6D,E, as the rotation speed of the MHM probes increased, the SERS intensity increased accordingly, indicating that the CV collection efficiency of the MHM probes in the active state was positively correlated with the rotational frequency of the MHM probes.