A Review of Soft Microrobots: Material, Fabrication, and Actuation

Microrobots have shown great potential in many applications, such as non‐invasive surgery, tissue engineering, precision medicine, and environmental remediation. Within the past decade, soft microrobot has become one of the important branches. It is aimed to create soft and deformable microrobots with high bioaffinity, which can perform complex tasks noninvasively in inaccessible small spaces in the body. Herein, the latest research progress of soft microrobots regarding the three cornerstones of this field is reviewed: material, fabrication, and actuation. First, various materials that are used for the fabrication of soft microrobots are summarized, and their characteristics and functions are discussed. Second, various fabrication methods of soft microrobots are introduced, and their applicability to different materials is discussed. Third, the actuation methods of soft microrobots are discussed, as well as their pros, cons, and adaptability. Moreover, the outstanding behaviors of soft microrobots in biomedical and environmental applications are introduced with some typical examples published recently. Finally, current clinical use challenges of soft microrobots are pointed out, and their intelligentization is proposed and discussed for further innovative development.

At present, the study of soft microrobots have been conducted in many aspects.Some of them study the synthesis and selection of proper materials, [38,66] some emphasize the development of suitable fabrication methods, [41,67,68] and others devote themselves to improving motion control. [69,70]In this review, we summarize the progress of soft microrobots regarding the three cornerstones (material, fabrication, and actuation) and point out the valuable achievements and facing challenges of each aspect.The selection of materials is crucial for the application of soft microrobots application prospect and is highlighted in the "Material" section.For the same reason, structural design of soft microrobots is discussed in the "Actuation" section.Besides, some recent applications of soft microrobots with outstanding behaviors in real cases have been introduced.Moreover, the facing challenges and future expectation for the development of soft microrobots have been discussed.This article aims to make a comprehensive and clear summary of the current research status of soft microrobots, providing a beneficial guideline for future research in this field.
[88][89] Based on the special properties of these soft materials, the corresponding soft microrobots are endowed with excellent manoeuvrability and adaptability, resulting in outstanding behaviors in many applications (such as cargo delivery, [34,71] cell therapy, [90][91][92] tissue engineering, etc. [93,94] ).Below, materials used for the fabrication of soft microrobots and the corresponding functions behaved in specific applications are discussed in detail.

Biofriendly Materials for Safe Biomedical Uses
Biofriendly materials can interact harmoniously with biological systems, promoting safe use in various medical scenarios.Soft microrobots made of them can be programmed to perform specific tasks such as drug delivery, [35,39] biosensor, and [95] tissue engineering. [96]Based on detailed properties, biofriendly materials can be further classified into biocompatible and biodegradable materials.

Biocompatible Materials
For safe use in the human body, the biocompatibility of soft microrobots is very important.Thus, biocompatible materials that have the characteristics of histocompatibility and blood compatibility in vivo should be used.So far, biocompatible materials that are commonly used for the fabrication of soft microrobots include poly (ethylene glycol) diacrylate (PEGDA), [37,72,92,[97][98][99] polydimethylsiloxane (PDMS), [73] and Ecoflex silicone rubbers. [79]Soft microrobots constructed with these materials have been used for drug delivery, [77,98,100] cell therapy, [92] and precision medicine. [101][104] Its good photopolymerization characteristics allow microstructures with specific shapes for tissue engineering and regenerative medicine to be easily fabricated. [105]As the elastic modulus of the products increases with the increase of PEGDA's concentration, the mechanical properties of the products are adjustable. [77,105]Recently, Liu et al. prepared soft microhelixes, which exhibit controllable mechanical properties, combining PEGDA with calcium alginate. [106]By adjusting the concentration of PEGDA in the precursor, the elastic moduli of the soft microrobots can be readily adjusted from a few MPa down to tens of KPa.As shown in Figure 2a, due to their excellent self-adaptive deformation capability, the soft microrobots can be navigated through sinuous channels, including orthometric bend capillaries, U-shape bent capillaries, and cross narrow openings with a smaller sectional area than the microrobots.These soft microrobots can be applied in complex environments for biomedical operations.Srivastava et al. realized the fabrication of self-propelled microrobots with PEGDA-based thread-like hydrogel tails by real-time in situ polymerization. [99]he thread-like PEGDA polymeric structure with low mechanical strength is easy to bend and deform, which can realize remote capturing of cells and microparticles.However, some researchers believe that PEGDA is too soft.By combining PEGDA with pentaerythritol triacrylate (PETA), the mechanical strength of soft microrobots would be enhanced to ensure the realization of complex designs without changing their biocompatibility. [37,92]or example, biocompatible burr-like porous sphere microrobots with good mechanical strength have been made with PEGDA and PETA to load and transport therapeutic cells in vascular tissues. [92][111] Soft microrobots made of them exhibited excellent deformability for stretching and shrinking. [73,79,101,112]The deformable multilegged soft microrobot fabricated by Lu et al. using PDMS exhibited the potential to be used for biomedical applications in the stomach. [73]The soft feet, with their remarkable deformability and quick elastic energy release mechanism, can lift the body of the soft microrobot and actuate the whole body to realize superior overobstacle ability.By combining Ecoflex 00-10 polymer with neodymium iron boron (NdFeB) magnetic nanoparticles (MNPs), Ren et al. fabricated a magnetic elastomer, which later on became the core and joints of a jellyfish-like soft microrobot (Figure 2b). [79]The deformability of elastomer enables the bending in the different parts of the jellyfish so that a bionic motion can be achieved through rhythmical beating.

Biodegradable Materials
The biocompatibility of soft materials is not enough to avoid all safety issues for biomedical applications, as the remains of microrobots inside the body could potentially cause side effects.However, it is reasonably hard to retrieve all microrobots from the bloodstream and human body because of their tiny sizes and the complexity of in vivo environment.This issue can be avoided by developing biodegradable soft microrobots that will not produce harmful byproducts during degradation.So far, biodegradable materials commonly used for the fabrication of soft microrobots include nature-derived polymers (such as gelatin, [34,38,46,96,113] alginate, [51,114,115] and chitosan [35,116] ) and other artificial polymers (such as poly (lactic-co-glycolic acid) (PLGA), [117,118] polycaprolactone (PCL) [119,120] ).Their degradation properties are mainly based on enzymatic reaction and hydrolysis.
Gelatin is a nontoxic, harmless, deformable, biocompatible, and biodegradable soft material. [34,96,113,121]It is a natural single-chain polymer derived from natural collagen through moderate hydrolysis and thermal denaturation. [121]When the temperature is higher than 45 °C, gelatin may form a homogeneous aqueous solution with the existence of water.When the temperature is lower than 45 °C, the gelatin solution may transfer into a solid state. [121]Gelatin-based soft microrobots have been extensively used in environmental remediation, [122] drug delivery, [34,64,113] and targeted cell therapy. [46,82]Initially, researchers directly utilized natural gelatin to fabricate soft microrobots.By assembling bovine serum albumin/poly-L-lysine (BSA/PLL) and gelatin hydrogel with gold nanoparticles, Wu et al. fabricated a biodegradable multilayer microrocket that can be used for targeted drug delivery. [46]The photothermal effect of gold nanoparticles under near infrared (NIR) causes a gel-sol phase transformation of the gelatin hydrogel, thus leading to the release of the encapsulated drugs.However, pure gelatin hydrogel can only achieve physical crosslinking at a specific temperature and concentration. [123]This limits the fabrication of complex and functional soft microrobots.Therefore, researchers chemically modified gelatin for more applications.By introducing methacrylamide into gelatin molecular, gelatin methacrylate hydrogel (GelMA) was obtained.Acrylamide bonds in GelMA ensure chemical crosslinking and polymerization of the material so that fine and controllable microstructures can be produced.Although the chemical molecular structure has been changed, GelMA still has deformability and degradability as the same as gelatin. [96,124]eylan et al. proved that GelMA-based soft microrobots can be degraded by matrix metalloproteinase-2 (MMP-2). [34]The diffusion of MMP-2 into the microrobots can lead to uniform swelling of the GelMA hydrogel network, so that effective drug release from the GelMA-based soft microrobots can be realized (Figure 2c).The biodegradable GelMA-based soft microrobots fabricated by Dong et al. have been used for cell transportation. [96]GelMA hydrogel of the soft microrobots provides a reasonable basis for cell growth, ensuring the cells stay alive during transportation (Figure 2d).After a certain time of coculturing with cells, GelMA-based microrobots would be degraded, leading to the release of cells.Gelatin-based gels are undoubtedly popular materials for the fabrication of biodegradable soft microrobots.However, their application in soft microrobots is limited due to their poor mechanical properties and strong adhesion.Therefore, further studies and improvements regarding these properties are urgent.
[116] Researchers have verified that chitosan-based and alginate-based microrobots can be degraded with lysozyme [35,116] and amylase, respectively. [51]Magnetic chitosan microrobots proposed by Go et al. can be used for liver cancer treatment and knee cartilage regeneration by loading and delivering different cells. [116]ere, the enhanced cell adhesion of magnetic particles helps cells adhere to microrobots, while the biocompatible chitosan hydrogel provides the matrix for cell growth.Ding et al. fabricated a sandwich-type micro gripper consisting of a GelMA layer and two oxidized methacrylate alginate (OMA) layers with different amount of methacrylate. [115]ere, OMA with less methacrylate has higher swelling rates and degradation rates.The competitive expansion of the two OMA layers leads to the bending deformation of the Reproduced with permission. [106]Copyright 2021, Elsevier.b) Video snapshots of the Ecoflex-based jellyfish-like soft microrobot capturing a buoyant bead.Reproduced under the terms of the CC-BY license. [79]Copyright 2019, The Authors.Published by Springer Nature.c) Gelatin methacrylate hydrogel (GelMA)-based microrobots release the drug by swelling.Reproduced under the terms of the CC-BY license. [34]Copyright 2019, The Authors.Published by American Chemical Society.d) Cell transportation illustration and experiment of GelMA-based microrobots.Reproduced with permission. [96]Copyright 2020, Wiley-VCH.The scale bar in (a) and (d) represents 500 μm and 100 μm, respectively.microgripper, realizing a variety of shape changes and controlled actuation.
[127] Kim et al. fabricated a biodegradable soft microrobot with PLGA and MNPs for target drug delivery. [128]It has been proved that obvious degradation of these PLGA microrobots in PBS solution at 37 °C can be seen within 6 weeks, and their degradation rate can be modified by adjusting the size and the concentration of MNPs.Pacheco et al. proposed a biodegradable microrobot consisting of PCL, MNPs, and polyethyleneimine micelles for cancer therapy. [120]ipase, which is overexpressed in pancreatic cancer cells, can trigger the hydrolysis of PCL, thereby facilitating drug release to kill cancer cells.By combing PCL and poly (N-isopropyl acrylamide) (PNIPAM), Zhang et al. proposed a biolayer microgripper. [119]This microgripper can bend and recover through NIR stimulation to achieve selective catch and release of objects.

Temperature-Responsive Soft Materials
Temperature-responsive soft materials can reversibly change morphology, responding to the external ambient temperature.][138] The temperature response of PNIPAM is based on its reversible phase transition between the hydrophilic expansion state below its lower critical solution temperature (LCST) and the collapse hydrophobic state above its LCST temperature (32 °C).When the temperature decreases, the hydrogen bond is enhanced, and hydrophilic amide groups are solvated by water molecules, so the hydrogel expands with water absorption.When the temperature increases, the hydrogen bonds are fractured, resulting in the release of water from the structure, and the volume becomes smaller due to shrinkage and dehydration. [139]The thermal response of PNIPAM leads to its volume phase transition.Thus, PNIPAM-based soft microrobots show adjustable deformability, such as shrinkage, expansion, bending, folding, etc. [33,40,[140][141][142] Due to its morphological transformation ability near physiological temperature, PNIPAM has excellent potential for biomedical applications. [33,143]For example, the PNIPAM-based polymer films developed by Magdanz et al. can capture and release sperm at physiological temperatures by folding and expanding. [144]When the temperature of the solution is lower than 38 °C, the microstructure curls to form microtubules so that the sperm can be trapped in the folded cavity.When the temperature reaches 38 °C, the microtubules expand and release the sperm.The combination of PNIPAM and nonthermal responsive polymer can also achieve an excellent self-folding effect. [22,23,69,130,136,144,145]For instance, Ma et al. fabricated a double-layer microrobot through the combination of PNIPAM and fluorescent material perylene bisimide-functionalized hyperbranched polyethyleneimine (PBI-HPEI) (Figure 3a). [136]The self-folding effect of this microrobot enables an "on-off switchable fluorescent color-changing function."At low temperatures, the PNIPAM layer expands to cover the PBI-HPEI layer, so that most of the excitation light would be blocked and no fluorescence can be observed.After the temperature increases, the PNIPAM layer shrinks to expose the PBI-HPEI layer under green excitation light, in which case the fluorescent switch could be triggered.As PBI-HPEI is also pH responsive, the fluorescence intensity could be adjusted by changing the pH value of the surrounding environment.By introducing functional monomers into PNIPAM polymers, a wider temperature response range can be achieved. [146]However, PNIPAM-based soft microrobots still have some common practical problems, such as lacking biodegradability, having low mechanical strength, lacking accurate and repeatable positioning, etc. [147] Therefore, more attempts are needed to optimize and improve temperature-responsive soft microrobots in the future.

Light-Responsive Soft Materials
Among various stimuli-responsive soft materials, lightresponsive ones can meet the needs of high temporal and spatial resolution and therefore, have high applicability and operability. [148]The most typical representative one is LCEs.LCEs are solid soft polymers formed by photoinitiated polymerization of a precursor solution consisting of liquid-crystal monomers, crosslinking agents, and photoinitiators. [149]Such polymers can present in different shapes or structures but still maintain the orderly arrangement of molecules in the liquid crystal phase.Under the stimulation of light or heat, the liquid crystal phase in the polymer changes from anisotropy to isotropy, and the whole system changes from order to disorder.The internal stress caused by the temporary change of molecular order promotes the change of the internal guide of the material and finally leads to the morphology change of LCEs. [133,150]Light simulation allows the LCEs to undergo obvious reversible deformation (shrinkage, expansion, bending, or folding).The sensitive light responsibility of LCEs has been well reflected in the research of soft microrobots. [131,151]In the early stage, fabrication technology and the understanding of LCEs' properties are lacking.Products based on LCEs were mainly developed at the 2D level first and then transferred to 3D with light irradiation.For instance, Haan et al. developed LCEs' circular polymer film. [152]After illumination, the circular sheet became conical, caused by the radial deformation of the liquid crystal sequence during light-triggered heating.Iamsaard et al. proposed to fuse the LCE film with a chiral dopant to form a chiral helix. [63]With the control of cutting angles and light density, the structure produces different shape change modes.With the continuous development of technology, LCEs-based soft microrobots with excellent flexibility and functionality have been realized. [133]In some cases, the deformation of LCEs induced by light could further realize the movability of the soft microrobots, such as walking, rolling, and even flying. [133]Inspired by the kirigami concept, Cheng et al. developed an LCE-based microactuator with a deformable petal shape by laser cutting, which can realize forward and backward rolling movement under the stimulation of light (Figure 3b). [153]Zeng et al. developed a crawler-like soft microrobot based on LCEs, which simulated the caterpillar's movement at the fingertips under visible light stimulation. [151]n addition, intelligent LCEs-based soft microrobots can sense light and adjust themselves according to the changes in light conditions.Inspired by the self-regulation of plants, Wani et al. developed a microflycatcher based on LCEs. [148]As shown in Figure 3c, when the captured object enters the optical fiber field of vision and generates sufficient optical feedback, the microrobot absorbs the energy of reflected light and generates bending to realize the grasping action.The significance of this feedback actuation is that the deformation of the soft microrobot could be adjusted with the change of ambient brightness rather than relying solely on the on/off light trigger.
As light, especially NIR, could be used to generate heat, the deformation of some temperature-responsive soft microrobots, summarized in the previous section, could also be realized under light stimulation. [33,113,138]

pH-Responsive Soft Materials
pH-responsive soft materials can undergo reversible changes based on environmental pH. [154]Their pH-responsiveness is generally based on the protonation/deprotonation of functional groups.With the use of pH-responsive materials, such as Alginate, [155] AA, [132,156] A6ACA, [134] and pHEMA [135] , soft microrobots have been used for applications like responsive actuation [157] and drug delivery. [78,135]lginate, AA, and A6ACA exhibit pH-responsive behavior due to the protonation and deprotonation of their carboxyl groups.In an acidic environment, the carboxyl groups become protonated and positively charged, disrupting electrostatic repulsion and causing a shrinking behavior.In contrast, the carboxyl groups become deprotonated and negatively charged in an alkaline environment, promoting electrostatic repulsion and causing swelling.As a natural polysaccharide derived from algae, alginate exhibits a remarkable response to changes in pH. [71,80,155,158]heng et al. fabricated a sodium alginate-based microactuator that can grasp or release cargo through the shrinkage or expansion of alginate gel under the stimulation of pH or ions. [155]odium alginate can also be used with chitosan as a pair of implicit combinations for fabricating soft microrobots. [71,80]hen et al. fabricated a double-layer micromotor with chitosan and sodium alginate, in which therapeutic drugs and MNPs were encapsulated for intestinal administration. [71]Sodium alginate in this micromotor avoids gastric acid damage to drugs and ensures the smooth release of drugs in the intestine.As another material with carboxyl groups, AA is a weak acid that undergoes reversible swelling and shrinking in response to changes in pH. [132,159,160]in et al. proposed a hollow microball made of hydrogels AA and NIPAM. [159]This microrobot is composed of two layers: an inner layer created with low laser power and an outer layer created with high laser power.With the change in environmental pH, the microball can reversibly transition the shape between a spherical structure and a slender cylinder by swelling and shrinking.In the same work, pH-responsive microrobots with other shapes, such as octagonal prismatic microtubes, microcages, and umbrellas, have also been fabricated.A6ACA is the third pH-responsive material with carboxyl groups introduced here.The terminal carboxyl groups of A6ACA can deionize at a low pH value and form hydrogen bonds with adjacent amide groups to achieve stability polymerization.Jin et al. proposed a pH-responsive self-adhesion hydrogel with A6ACA and NIPAAm to realize the endovascular embolization treatment of aneurysm. [134]Here, after delivering the embolic microrobots to the aneurysm sac by catheter delivery and magnetic field guidance, the self-adhesion behavior can be triggered by the acidic buffer, as A6ACA would deionize to formate hydrogen bonds across the hydrogel interface at an acidic environment, realizing complete embolization (Figure 3d).
][163] Li et al. developed a snowman-shaped hydrogel bilayer microrobot consisting of pHEMA with PEGDA, which can fold itself at about pH 9.58 and unfold at about pH 2.6. [78]This microrobot has been used for pH-triggered drug release.Using the same mechanism, Darmawan et al. developed a bilayer microrobot consisting of a composite resin and pHEMA for pH-triggered drug release.Besides a drug release test in an experiment environment, they also applied the microrobot under an ex vivo environment.Within a pig's stomach, the microrobot moved to the targeted area via rolling motion, and its shape-morphing behavior was realized due to the low-pH-value environment (Figure 3e). [135]he advantage of pH-responsive materials is attributed to their ability to flexibly alter their properties or behavior with the changes in environmental pH.However, these materials usually exhibit relatively low mechanical strength and durability.This weakness may pose challenges in achieving the necessary structural integrity or enduring repeated cycles of shape morphing.

Live Organism Materials
With the unique functional characteristics of high affinity, excellent adaptability, and natural self-actuation capability, live organisms (such as sperm cells, [86] red blood cells (RBCs), [87,164] bacteria, [25] cardiomyocytes, [89] and neutrophil cells (NEs) [165] ) have also been used in a series of attractive research about soft microrobots. [24,43,61]Live organisms have internal sensory units that can dynamically interact with external environments, such as oxygen, chemicals, pH, and temperature gradient. [4,85,166]They also have innate fitness for swimming in biological environments and ability to interact with other cells/tissue. [26]Thus, live organisms have natural advantages in improving cellular drug delivery and bioavailability. [26]Therefore, developing soft microrobots with live organisms is an effective way to realize autonomous biomedical operations. [61]t has been proven that live organisms can encapsulate and transport goods efficiently. [43,86]Alapan et al. obtained a kind of multifunctional biological soft microrobots by adhering RBCs to bioengineering bacteria Escherichia coli (E.coli) (Figure 4a). [43]Here, RBCs were used to load therapeutic drugs and MNPs, and E. coli provided autonomous propulsion ability to turn the RBC into active cargo transport carriers.This microrobot can maintain deformation and attachment stability even after being squeezed.NE-based microrobot proposed by Zhang et al. was constructed by phagocytizing the drug-loaded hydrogel into Nes (Figure 4b). [165]The obtained microrobot maintains the intact membrane structure and nonimmunogenicity of nature Nes.It can squeeze across the blood-brain barrier and deliver drugs to target inflamed tumor area successfully.Magdanz et al. fabricated a biological soft microrobot by coating MNPs on bovine sperm. [86]Under a low Reynolds number, this microrobot realized flexible movement and drug delivery through the spiral propulsion of flagella guided by a magnetic field.Besides, many researchers have coupled biological cells with artificial anchors to fabricate biohybrid soft microrobots. [26,74,167,168]As shown in Figure 4c, Park et al. proposed bacteria-driven microrobots by integrating E. coli with drug-loaded polyelectrolyte multilayer microparticles.With the flagellar propulsion and sensing capabilities of E. coli, the microrobots can target specific cells for drug delivery under the guidance of a magnetic field and chemoattractant gradient. [169]Xu et al. proposed a soft microrobot composed of a sperm and an artificial micromotor, in which the sperm cell is used as a propulsion source and drug carrier, and the micromotor is responsible for guiding and releasing the sperm (Figure 4d). [26]This system can potentially be used for both in situ diagnosis and treatment.
Live organisms have the ability of environmental sensing, autonomous propulsion, and deformation.They can be used as an excellent means of in vivo cargo transportation. [43]owever, most organism-based microrobots can only function for a limited time, varying from a few hours to a few days, and can only be stored and work in biological fluids. [28]esides, there are many uncertainties in the effective combination of living organisms and artificial anchors.As live organisms actuate themselves to move, it is hard to control their motion.Moreover, due to the low contrast between living organisms and body tissues and the current limitations of imaging and tracking technologies, it is also a complex problem to realize the tracking of organism-based soft microrobots with high precision and sensitivity in vivo.

Fabrication of Soft Microrobots
Once the appropriate materials have been identified, it's necessary to carefully select the suitable fabrication methods, as the fabrication methods can significantly impact the final properties and performances of the soft microrobots.Over the past decade, many methods have been studied and applied to fabricate soft microrobots.The most commonly used ones are UV polymerization, [44,97] two-photon lithography, [170,171] and template assisted. [74,172]Besides, there are also other methods used in literature, like soft lithography, [173] electrospinning, [174] 4D printing, [32] and SLF. [49]In addition, there are special fabrication methods for soft microrobots based on live organisms, such as biochemical treatment [75,87] and integration methods. [175,176]n the following part of this section, all these fabrication methods are described in detail.

UV Polymerization
UV polymerization is a commonly used fabrication method for soft microrobots.With materials that have UV active groups, microstructures with specific patterns can be produced under UV light irradiation using corresponding masks.Here, the pattern's shape depends on the mask's design. [69,177]This method has the advantages of mass production and fast reaction speed. [178]The principle of UV polymerization is a chemical reaction initiated by UV light.UV photoinitiators are induced by UV light to form free radicals or ions.These free radicals or ions may cause crosslinking through double bonds in prepolymers or unsaturated monomers, so that polymerization occurs.Specifically, as shown in Figure 5a, the process includes three steps: spin coating photoresist on a substrate, UV exposure with a mask for region-selective photoreaction, and development for the final pattern.During the development step, the nonpolymerized material would be removed by the corresponding developer to get the final pattern.
Kuo et al. fabricated a hydrogel microclip by UV patterning using a photomask. [21]By adjusting the exposure dose, the crosslinking densities can be tuned so that the shrinkage responses of the microclip can be controlled.Go et al. fabricated a thermoelectromagnetically actuated microrobot through UV polymerization. [97]This microrobot combines the electromagnetic actuation layer Fe-PEGDA and the thermal response layer PNIPAM for the targeted delivery of therapeutic drugs.Although UV polymerization technology is difficult to enable 3D structure fabrication, the combination of this technology and shape-deformable or programmable materials can realize the fabrication of microrobots with simple 3D structures.Huang et al. developed a self-folding microrobot using UV polymerization technology by selectively patterning a PEGDA hydrogel layer on a NIPAAm hydrogel layer. [69]With MNPs doped in hydrogels, enhanced NIR heating can be applied to the NIPAAm layer.The orientation of MNP alignment can induce the anisotropic expansion of the microrobot.Therefore, the microrobot can form a 3D structure through stress-induced bending.Using UV polymerization, Nguyen et al. fabricated a magneticguided self-rolling microrobot with a resin material.Self-rolling occurred because of the anisotropic density gradient of the resin along the z-axis generated in the printing process, and the curvature of the self-rolling microrobot can be adjusted by Reproduced with permission. [43]Copyright 2018, American Association for the Advancement of Science.b) Targeted delivery illustration and morphological images of NEs-based microrobot.Reproduced with permission. [165]Copyright 2021, American Association for the Advancement of Science.c) Illustrations of bacteria-driven microrobot for target drug delivery.Reproduced with permission. [169]Copyright 2017, American Chemical Society.d) Biohybrid soft microrobots based on sperm cells for targeted drug delivery and illustration of the corresponding loading and releasing process of the sperm.Reproduced with permission. [26]Copyright 2018, American Chemical Society.
controlling the UV exposure time. [179]Moreover, the innovation of light source devices could also realize 3D fabrication with UV polymerization directly.Zmyślony et al. placed an optical fiber in the liquid monomer to emit UV light so that polymerization was triggered at the tip of the optical fiber to form a conical structure. [149]By connecting two conical structures, a microgripper, which can be used for optical driving operation, was obtained.
UV polymerization has the advantages of high efficiency and time-saving as it can usually fabricate thousands of microrobots simultaneously.However, the structures produced by this method are usually 2D patterns, and the material must be UV curable.For 3D designs, shape-variable materials, special light source devices, or multiple UV photopolymerization steps would be required.

Two-Photon Polymerization (2PP)
Another commonly used fabrication method of soft microrobots is 2PP, known as direct laser writing. [34,38,92,180]2PP is a sensitive and precise fabrication method to ensure stereoselective polymerization with submicrometer resolution.Therefore, it is an ideal technology to fabricate complex microstructures with 3D geometry.Instead of only absorbing one photon at one time (like UV polymerization), in the process of 2PP, photoinitiators would absorb two photons simultaneously from the pulsed infrared laser to produce free radicals or ions so that the polymerization could be initiated. [181,182]As two-photon absorption only occurs at the focus of femtosecond laser, 2PP is highly localized. [183,184]ompared with other fabrication methods, this method can achieve submicrometer resolution without etching or masking. [170,171,185]The fabrication process of 2PP includes three steps.The first step is to drop or spin coat the two-photon light-curable materials on the substrate.Since the fabrication process can be stereoselectively controlled, it is not obligatory to spin coat the materials to obtain a uniform layer.The second step is the polymerization reaction triggered by infrared laser irradiation.For this step, a 3D model should be designed in advance to generate the manufacturing code, according to which the laser will scan inside the materials.The third step is to develop the printed sample in a suitable developer to obtain the target microstructures (Figure 5b).So far, there are already some commercial direct laser writing instruments, such as Nanoscribe [38] and UpNano. [186]Using this technology, researchers have fabricated many complex and tiny high-precision structures for tissue engineering [96] drug delivery. [35,38,64,187]With the method of 2PP, Dong et al. fabricated soft microrobots with GelMA and magnetoelectric nanoparticles (MENPs), and these microrobots can eventually transport nerve cells and stimulate cell differentiation. [96]Using 2PP technology, Lee et al. fabricated size-controllable hydrogel microrollers that can cross narrow channels. [130]Through 2PP fabrication method, Tao et al. obtained programmable stimuli-responsive hydrogel microrobots with submicrometer resolution. [188]Although 2PP technology has a high spatial resolution, it is more time-consuming than UV polymerization as the printing is one by one or layer by layer.In other words, this method is not feasible for fabricating large numbers of structures while maintaining high resolution. [189,190]

Template-Assisted Methods
As simple and controllable fabrication methods, templateassisted methods are suitable for preparing large amounts and highly ordered array microstructures.So far, there are three types of template-assisted methods used for the fabrication of soft microrobots, including micromolding, [191] template-assisted layer-by-layer assembly (LBL), [46] and template-assistedelectrochemical deposition. [189]Many templates, like colloidal monolayers, [192] nanoprinting molds, [193] block copolymers, [194] and anodic aluminum oxide, [1,195] have already been used in these methods.
As a method of mass production of microstructures, micromolding is mainly based on the purpose of replication molding.As shown in Figure 5c, the monomer solution is injected into the micromold hole and undergoes proper treatment for solidification.Then the corresponding microstructures would be achieved after the mold is taken out. [196]Benefitting from the development of various soft materials, monomer solidification can be achieved by photopolymerization, ion-mediated gelation-induced crosslinking, and temperature-induced crosslinking. [196]The mold is usually taken off in two ways, dissolving or splitting. [191,197,198]n Jeon et al.'s work of fabricating a maneuverable soft microrobot using micromolding technology, elastic rubber material and two permanent magnets were combined in a hydrophobictreated PDMS mold. [191]After curing and shaping, the fabricated cylindrical microrobots could be split easily.In the work of Su et al., gelatin and platinum nanoparticles are crosslinked and immobilized in a polycarbonate (PC) mold. [198]After dissolving the mold in chloroform, a soft micromotor was obtained.The achieved micromotor can move independently in hydrogen peroxide (H 2 O 2 ) solution.Micromolding technology is simple and more suitable for mass production.However, like most template-assisted methods, micromolding highly relies on the molds and thus would be limited by the fabrication and use of molds.Besides, for all template-assisted methods, of which the molds need to be dissolved to move at the end, the solvent used to dissolve the mold would be soaked into the soft microrobots, and it is nearly impossible to completely remove the solvent from microstructures.
Like micromolding, template-assisted LBL also relies on a template, which must be removed at the end.However, in this method, multiple films are assembled based on the interaction between molecules and the electrostatic force to ensure the structural stability of the products. [199]Simply, it is a process in which various materials spontaneously associate to form a specific functional microstructure with the help of intermolecular forces through LBL alternate deposition (Figure 5d). [199,200]Using template-assisted LBL technology, Wu et al. fabricated biodegradable PLL/BSA multilayer microtubules for light-triggered drug release. [46]In this work, PC membranes were used as a template, and PLL and BSA were alternately assembled into the inner wall of the template layer by layer.Then, the sample was immersed into a mixture solution of gelatin, gold nanoparticles, and therapeutic drugs to load these materials at the center of the microtubules.Finally, the template was dissolved by dichloromethane to obtain the microtubules.With template-assisted LBL fabrication method, soft microrobots with helical shape can also be fabricated.Using Ca-alginate hydrogel microfiber as the template, soft spiral microrobots have been fabricated by Liu et al. with template-assisted LBL method. [201]As the achieved soft spiral microrobots were made of alginate and chitosan, they have good drug encapsulation ability and ion-responsive drug release ability.The template-assisted LBL technology can alternately assemble various components (such as polymers, biological functional macromolecules, nanoparticles, etc.) into the template without changing their active properties.Therefore, it is an effective strategy for fabricating functional soft microrobots. [46]owever, the fabrication process is often more time-consuming because it consists of multiple alternate depositions and repeated cleaning steps. [200]emplate-assisted electrochemical deposition is mainly based on the electrochemical reaction of discharge particles. [202]The deposited particles enter the lattice template with low energy and rearrange according to a particular law to form a microstructure (Figure 5e).As another effective fabrication method of soft microrobots, this technology has also been used in many studies. [84,203]Hu et al. fabricated templates on silicon chips and then electrodeposited sodium alginate and cobalt-nickel into the templates. [84]The achieved cylindrical microrobots showed good magnetic driving ability.With template-assisted electrochemical deposition, it is easy to control the thickness and length of each functional layer of microstructure.However, this technology often has low throughput and requires special instruments. [204]

Soft Lithography
Soft lithography used for the fabrication of soft microrobots is a soft template-assisted fabrication method that can convert the planar pattern into 3D microstructures. [173,205]The template is usually fabricated from elastomer (e.g., PDMS) by electron beam lithography or molding technology. [206]Since the template is soft, the substrate for fabricating soft microrobots is not limited to a plane.In other words, curved or flexible substrates are also acceptable. [205]When a liquid resist is fulfilled in the template, UV light or heat can be used to polymerize the resist to obtain microstructures with desired shapes (Figure 5f ). [47,205]In Corbaci's work of making a dielectric elastomer actuator using soft lithography technology, a syringe was used to inject the mixture of carbon nanotubes and PDMS into the hollow sealed PDMS mold made in advance.After curing, the soft microactuator was obtained. [47]Soft lithography technology can ensure the fabrication of 2D and 3D structures with low cost and simple operation.No complicated equipment support is needed, and the soft template can be reused.However, because the final effect of soft lithography depends on the elastic template, and the deformation and distortion of the template are not easy to control precisely, the accuracy of this technique is limited.

Electrospinning
Electrospinning is a convenient and straightforward technology for fabricating nano-or micrometer ultrafine fibers from inorganic and organic materials. [174,207]The basic principle of electrospinning is to form a liquid jet by applying a high voltage to the polymer solution and then to convert the microjet into microfibers through solvent evaporation (Figure 5g).The process includes applying a relatively high voltage, electrically spraying the polymer jet, and evaporating the solvent in the jet to produce micro or nanostructures. [208]This method can fabricate large numbers of soft microrobots simultaneously.Using electrospinning, Khalil et al. fabricated soft microrobots with a magnetic head and double tails [209] and also artificial sperm soft microrobots. [210]The motion of these microrobots comes from the flexible tail's periodic change.With the same fabrication method, Shin et al. fabricated a double-layer microactuator by arranging hygroscopic materials in microfibers. [211]The obtained microactuator can bend and deform in response to environmental humidity.Although soft microrobots can be fabricated with electrospinning, the form of them is limited to fiber or silk, and their mechanical strength is often reduced due to the porosity and the increase of the pore size.At the same time, this kind of fiber also has the problem of insufficient anisotropy. [212]Therefore, electrospinning seems to attract less attention to making soft microrobots compared with other fabrication methods mentioned above.

Other Fabrication Methods with Soft Polymer Materials
Other fabrication methods, such as 4D printing and SLF, have also been used to fabricate soft microrobots. [32,40,49,146,213]4D printing technology is claimed by some researchers as a method that uses 3D printing technology and programmable materials to fabricate microstructures that respond to environmental stimuli (Figure 5h). [32,156,214]Using this technique, Jin et al. constructed 3D-to-3D shape-morphing microrobots by programming the gradient distribution of stimulus-responsive hydrogels along arbitrary 3D trajectories. [159]The results indicated that the complex reconfigurable structures (such as microstent and microcage) could achieve rapid and revisable uniaxial or biaxial contraction.Similarly, Hu et al. fabricated a microcapsule by 4D printing. [213]This microcapsule can expand or contract to capture or release goods by adjusting the surrounding pH value.SLF is a method that combines photopolymerization and microfluidic flow control to fabricate complex-shaped microstructures (Figure 5i). [49,146]Wang et al. fabricated a microtemperature sensor with a wide sensing range and sensitive sensing effect using this technique. [146]These methods provide unique insights for the fabrication of soft microrobots and meaningful inspiration for developing other new fabrication methods.

Fabrication of Organism-Based Soft Microrobots
To take advantage of the intrinsic actuation and sensing functionalities of live organisms, researchers developed soft microrobots based on live organisms. [215,216]As shown in Figure 5j, organism-based soft microrobots are usually fabricated with two methods: biochemical treatment for biological soft microrobots [75,87,217,218] and integration method for biohybrid soft microrobots. [74,86,175,176,219]he biochemical treatment mainly refers to the superficial treatment and modification of organisms to endow them certain functionality or tendency. [27,218]Al-Fandi et al. cultured fresh E. coli as live microrobots. [27]Using the natural biological nanosensing system of E. coli and the driving ability of its flagella, the microrobot can effectively identify the overexpressed vascular growth factor VEGF in tumor cells.In addition to the well-known bacterial microrobots, some cells from the body can also be good biological soft microrobots due to their nonimmunogenicity and their ability for cargo transportation. [43,87]Alapan et al. cultured RBCs with streptavidin-conjugated E. coli to obtain biological microrobots. [43]In this work, E. coli was used to provide autonomous propulsion for the microrobots.The external magnetic guidance is realized through the MNPs loaded in the RBCs, transforming the RBCs from passive cargo carriers to active guided cargo carriers.Zhang et al. coated the drug-loaded magnetic hydrogels with the outer membrane of E.coli, and then the E.coli-based drug system was phagocytized by NEs to obtain neutrophil-based microrobots. [165]The E.coli membrane enhances phagocytosis efficiency, and the microrobots inherited the chemotaxis of natural NEs.
The integration method refers to the fabrication method of integrating bacteria/sperm/microalgae with artificial anchors prepared in advance to fabricate biohybrid soft microrobots. [26,61,89,175]At present, biological components can be integrated into microanchors in many ways, such as incorporating live organisms into a microdevice [74,175] or patterning techniques (i.e., attaching bacteria to microspheres using ion etching and surface modification). [29,176,220]Schmidt et al. fabricated biohybrid micromotors by directly coupling sperms with artificial anchors. [175]By mixing sperms and artificial anchors in diluted whole blood, the sperms were trapped in the blood cell pool, and the artificial anchors were coupled with the sperms by applying a magnetic field.Here, the cooperation of sperms and artificial parts enables the micromotors to go against flowing blood and perform the drug delivery function.Behkam et al. used patterning methodology to selectively attach bacteria to the surface of microbeads, achieving biohybrid soft microrobots. [176]Here, one side of the microbeads was treated by ion etching to become hydrophilic, so that bacteria attached to the other side of the microspheres.Park et al. also fabricated biohybrid microrobots using the patterning methodology. [29]Here, microspheres were half immersed in agarose gel, and then the exposed surface of the microspheres was modified with an antibacterial adhesion factor BSA. Finally, Salmonella typhimurium was attached to the unmodified side of the polystyrene microspheres to obtain the biohybrid microrobots that can be propelled along with the movement of the bacteria.
The development of fabrication methods mentioned above enables soft microrobots to take advantage of live organisms' safety, tendency, and deformability.However, due to the vulnerability of organisms and the particularity of their living environment, it is challenging to fabricate organism-based microrobots with complex structures and functions.Further development of suitable fabrication methods for advanced organism-based soft microrobots is challenging and meaningful.

Correlation between Materials and Fabrication Methods
The unique properties of materials play a pivotal role in defining microrobots' functionality and behavior, while the fabrication methods determine how these material properties are preserved, manipulated, and integrated into the final microrobot design.The selection of materials and fabrication methods for soft microrobots are closely intertwined.For certain fabrication methods, specific properties should be possessed by the selected materials.For instance, to use UV polymerization/2PP fabrication methods, the selected materials should be able to crosslink with UV/NIR light exposure.For template-assisted methods and soft lithography, the selected materials should have great moldability and mechanical stability to ensure accurate replication of selected templates with desired shapes.Electrospinning requests materials having solubility, suitable boiling point, and appropriate concentration, so that a liquid jet can be formed under high voltage.While the materials' properties dictate the feasibility of fabrication methods, the fabrication methods limit the final appearance of materials.The final soft microrobots fabricated with UV polymerization fabrication method would normally have a flat pattern structure.2PP allows the production of complex and highprecision 3D microstructures.Template-assisted and soft lithography fabrication methods generally end up with large quantities of consistent microstructures.Electrospinning would produce microfibers.Materials and fabrications are mutually selected and restricted.By flexibly utilizing the properties of soft materials and reasonably selecting the fabrication methods, soft microrobots with superior performances can be developed, being more efficient and adaptable for applications across biomedicine and environmental remediation.Figure 6 summarizes the main materials and fabrication methods of soft microrobots, and their commonly used collocation.

Actuation of Soft Microrobots
The untethered manipulation of soft microrobots is crucial to realize its noninvasive operation, remote control, and precise treatment in vivo. [37]Various nature-inspired soft microrobots have been developed to adapt different actuation methods.Appropriate actuation control methods to ensure the movability of soft microrobots (such as rolling, rotating, jumping, or walking) is an essential step toward practical applications.So far, the commonly used actuation methods of soft microrobots include magnetic actuation, [35,53,92,155,221] light actuation, [62] ultrasound actuation, [222] electric actuation, [59] chemical actuation, [60,223] and biological actuation. [75,175]The principles of them are summarized in Figure 7.
Depending on the actuation source and principle, each actuation method has pros, cons, and adaptability.Choosing actuation methods for a particular soft microrobot will depend on the type of microrobot and the aimed application scenarios.For a concise and visual overview, a summary of each actuation method has been provided in Table 1.In the following part of this chapter, the development status and research progress of these actuation methods are discussed in detail.

Magnetic Actuation
The change of magnetic field in time or space leads to the occurrence of force (and/or torque) on magnetic microrobots so that continuous movement happens (Figure 7a). [191,224,225]Within different magnetic fields, microrobots show different motion modes, such as spiral motion generated within a rotating magnetic field, drag translation generated within a gradient magnetic field, and fluctuation generated within an oscillating magnetic field. [221,226]As a widely used propulsion method for   microrobots, magnetic actuation has also been used for the 3D navigation of soft microrobots. [34,68,97,155,191,227,228]With the actuation and navigation of a magnetic field, the submillimeterscale continuous microrobot fabricated by Kim et al. using programmable ferromagnetic soft materials shows omnidirectional steering ability to realize accurate control in complex and constrained environments. [227]Moreover, magnetically steerable laser delivery was realized by adding an optical fiber to the soft microrobot.Inspired by a magnetotactic bacteria, Xie et al. designed a biomimetic magnetic soft microrobot with speedy motion response and accurate positioning. [228]They embedded MNPs into a nonexpansive gel and assembled them into chains through static magnetic fields.Due to the magnetic anisotropy of the chains, the movement of the microrobot can be accurately controlled by rotating magnetic fields.Under the remote control of a rotating magnetic field, the magnetic chitosan double-helix microrobots developed by Ceylan et al. achieved accurate navigation, steering, positioning, and finally accurately delivered therapeutic drugs to the focus for cancer treatment. [34]esides actuating soft microrobots made of soft polymer materials, the magnetic field has also been used to actuate living organism-based soft microrobots. [86,87,229]As shown in Figure 8a, Wu et al. proposed a magnetic-powered Janus cell microrobot loaded with oncolytic adenovirus (OA) for target therapy. [215]In this work, the asymmetrically coated MNPs endowed the microrobot with effective motion.A rotating magnetic field was used to actuate and position the cell microrobots, and to extend their residence time in the lesion.A soft and resilient magnetic stem cell microrobot developed by Wang et al. can deform to pass through the narrow area and be directionally transported to the target position under the guidance of a magnetic field. [230]By combining endoscopic and ultrasonic imaging for tracking, highly extended working distance and targeting efficiency of this microrobot has been achieved.
The characteristics of high penetration, good biocompatibility, and flexible adjustability endow magnetic fields with the ability to drive soft microrobots in the body efficiently and safely.However, for the actual clinical application of magnetic soft microrobots, it is still essential to develop more advanced magnetic control systems and real-time information feedback systems.

Light Actuation
As one of the most universal physical stimuli, light has a certain penetration and convenient on/off control ability. [55]The movement of light-actuated soft microrobots mainly relies on their body deformation. [231]By adjusting the wavelength, intensity, and polarization direction of the light, the remote intervention of the moving direction and speed of soft microrobots can be realized. [232]Most light-actuated soft microrobots are based on light-sensitive soft materials, which can convert light energy into kinetic energy based on the photothermal effect or photochemical reaction.(Figure 7b) [133] The photothermal effect refers to the phenomenon that soft materials absorb light to achieve local heating, resulting in structural deformation. [56]Photochemical reaction refers to the light-triggered isomerization reactions of photosensitive shape memory polymers, such as LCEs [148,153] and other azobenzene derivatives, [233,234] resulting in deformation of the corresponding macroelastomer. [235,236]oft microrobots actuated by UV light show a good motion effect. [149,237]A microswimmer with a double-layer film structure fabricated by Ma et al. can carry out reversible bending deformation under the control of UV light. [83]With repeated execution of UV-induced bending and stretching, on-demand directional control of this photodriven microswimmer can be realized.Lahikainen et al. proposed a light-triggered microactuator based on LCEs, which can realize reconfigurable actuation behavior under the cooperating control of UV and visible light. [238]As UV light has low penetration ability and obvious biotoxicity, it is not suitable for medical applications.In contrast, NIR light has no apparent damage to cells and tissues and can penetrate deeper than UV light in the body.Therefore, researchers start to explore the biomedical use of NIR-actuated soft microrobots. [239]u et al. fabricated a superhydrophobic microactuator that can be manipulated by NIR light. [240]This microactuator can realize controllable movement on the water surface under the irradiation of NIR, and the motion behavior of this microrobot can be changed by adjusting the illumination direction.A lightpowered microswimmer with a fast NIR response was proposed by He et al. [241] Under the periodic irradiation of NIR light, the microswimmer can move quickly on the water surface (Figure 8b).Wang et al. also proposed a soft microrobot actuated by NIR stimulation. [242]With NIR stimulation at a specific point to induce evaporation of the fuel selectively, this microrobot can move accurately in the corresponding direction.Besides UV and NIR, other light sources could also be used for the actuation of soft microrobots.An LCE-based soft microrobot developed by Palagi et al. can be actuated by a structured monochromatic light field generated by a digital micromirror-device-based optical system to perform complex bionic motion. [235]ike magnetic actuation, light actuation can also realize remote and accurate driving of soft microrobots in different environments.However, due to the relatively low penetration of light, it is usually unrealistic to control soft microrobots in deep tissue with optical actuation.

Ultrasonic Actuation
Ultrasonic actuation is another type of simple, noninvasive, and efficient actuation method that has been used for the propulsion of soft microrobots. [243,244]As shown in Figure 7c, one kind of ultrasonic actuation method is based on the bubble vibration.When the acoustic field frequency reaches the resonance frequency of the bubble, the effective vibration of the bubble will drive the soft microrobot to move forward.For example, Ahmed et al. developed a bubble-based soft microrobot, for which a bubble was trapped in the cavity of a PEG structure (Figure 8c). [245]When the bubble oscillated effectively within an acoustic field, a pair of strong and counter-rotating vortices around the bubble formed so that the microrobot was promoted.However, the bubble size and mechanical response will not remain unchanged under physiological conditions.Thus, the resonance frequency of the microrobot will gradually change, and the soft microrobot's performance will finally be reduced.Once the bubble bursts, the system will lose its driving force. [222]nother kind of ultrasonic actuation methods is based on sharp-edge structures.Under ultrasonic stimulation, vortices are generated around the tip of the sharp-edge structure, and concomitantly tail oscillation occurs, thereby generating driving force.Inspired by flagella, Kaynak et al. designed a microrobot that can achieve linear or rotational motion under ultrasonic actuation by vibrating its flagellum. [246]By changing the design and the number of sharp edge structures in microrobots, the direction and speed of the microrobots can be adjusted. [244]ompared with bubble-based soft microrobots, soft microrobots with sharp-edged structures can achieve more robust acoustically actuated motions, but still have extensive room to improve their moving speed. [244]The third kind of ultrasonic actuation method is based on the pressure gradient.For some specific soft microrobots, a pressure gradient would be generated under ultrasound stimulation, converting acoustic energy into kinetic energy.Wu et al. developed a soft RBC micromotor encapsulated with MNPs that can be propelled by ultrasound. [247]When ultrasound is applied, the movement occurs due to the sound pressure gradient caused by the uneven distribution of MNPs in the micromotor.
As ultrasounds have the same good penetration ability as magnetic fields, ultrasonic-actuated soft microrobots have the potential to be used deeply in the body.However, up to now, there are still some limitations regarding each type of acoustically driven soft microrobots, as mentioned above.Besides, accurate positioning and direction control with ultrasonic actuation are generally hard to achieve.Therefore, many researchers propose to use magnetic fields to assist the direction control of acoustic navigation. [245,247]

Electric Actuation
At present, the electric actuation mechanism suitable for soft microrobots is electro-osmosis.Under the application of an external electric field, local rectification causes the generation of electro-osmotic flow (Figure 7d). [248]The movable cations inside the soft microrobot respond to the electric field moving toward the negative electrode, causing the concentration of cations lower in the microrobots outer side than the inside.The cation concentration gradient leads to solvent diffusion and osmotic pressure.The osmotic pressure balance finally leads to the deformation of the soft microrobots. [58,249]When the signal intensities and angles of the electric field are adjusted, the deformation degree and direction of the soft microrobots change accordingly, leading to on-demand control of soft microrobots.Kwon et al. proposed an electrically actuated soft myriapod aquabot that can walk on the surface of water (Figure 8d). [250]In this work, alternating electric control signals are applied to the microrobot to bend its legs forward and backward based on the electroosmosis mechanism, thereby promoting its forward motion.A human-like soft microrobot proposed by Han et al. can realize a walking motion by electric actuation. [251]Here, before the electric field is applied, the gravity center of the soft microrobot is at its structural center, so that the two legs can stand upright.When the electric field is applied, the gravity center moves backward quickly, and the rear arm acts as its anchoring point.At the same time, the front leg begins to move forward.When the electric field is removed again, the gravity center slowly returns to the structural center, so that the soft microrobot stands again.
The magnitude, phase, and frequency of the electrical signal applied to the soft microrobots can be easily modulated.However, the influence of the electric field on the microrobot rapidly decays as the distance increases.Moreover, the electric field may be incompatible with highly ionic media such as tissue fluid and blood, which limits the biological application of electrically actuated soft microrobots.

Chemical Actuation
Unlike other actuation methods mentioned above, chemicalpropelled soft microrobots must react with fuel to achieve kinetic energy (Figure 7e).This actuation mode requires an anisotropic structure of the soft microrobots, which usually includes catalytic and inert parts. [252]For the chemically powered nanorocket assembled by Wu et al., its platinum layer catalyzes the decomposition of the environmental fuel H 2 O 2 , and the resulting bubbles are ejected from the tube to actuate the motion of the nanorocket. [253]Here, the speed of the nanorocket could be adjusted by changing the fuel concentration.A catalytically propelled soft developed by Lu et al. was used for the pick-up and drop-off of cargoes. [252]The forward thrust is generated by the decomposition of H 2 O 2 aroused by the catalytic layer of the micromotor, and the direction of the micromotor can be guided by magnets.Wilson et al. proposed a bowl-shaped polymer stomatocyte that can be propelled by the decomposition of H 2 O 2 (Figure 8e). [223]H 2 O 2 can freely enter the inner cavity of the cell and decompose, catalyzed by platinum nanoparticles entrapped inside.The oxygen bubble produced during the decomposition would induce the thrust, resulting in the directional motion of this polymer stomatocytes soft microrobot.Chemical-actuated soft microrobots realize self-propulsion by converting the chemical energy into kinetic energy.However, their speed and moving direction are difficult to control, and most fuels are usually not conducive to biological applications.

Biological Actuation
Biological actuation is a particular actuation method that only works for live organism-based soft microrobots (including biological microrobots and biohybrid microrobots).As shown in Figure 7f, this actuation method is based on living organisms' inherent moving ability and taxis behavior in response to diverse environmental factors (e.g., chemotaxis and magnetotaxis). [24,26,75,168,254]Carlsen et al. developed a biohybrid microrobot propelled by multiple bacteria for targeted drug delivery. [255]Comparing the invariance of the instantaneous velocity before and after applying a magnetic field, it was proven that the motion of the microrobot was generated by bacteria propulsion.Combining magnetic steering and biological propulsion, effective motion and direction control of the microrobot actuated by multiple bacteria was realized, which significantly reduced the randomness of bacterial motion.Similarly, a soft biohybrid microrobot developed by Park et al. obtains the moving ability due to the propulsion of Salmonella typhimurium. [29]The experiments showed that microrobots with selective attachment of bacteria showed higher propulsion efficiency than microrobots with full attachment of bacteria.Williams et al. proposed a biohybrid filament microrobot that can be propelled by the contraction of cardiomyocytes. [89]The periodic contraction of cardiomyocytes produced bending waves, so that the microrobot was pushed forward (Figure 8f ).The tropism of the organism to chemical or magnetic factors can guide the soft microrobots swimming toward specific areas [27,165,228,256] .The E. coli.microrobot developed by Al-Fandi et al. can propel itself using its tendency to vascular factors, which are overexpressed on the surface of cancer cells, to realize self-navigation and drug delivery. [27]urthermore, the chemotaxis of natural NEs to inflammatory factors was also used for the in vivo propulsion of neutrophil-based microrobots for delivering cargo to malignant glioma in the work of Zhang et al. [165] As these microrobots have similar biological activity with natural NEs, they could migrate across the blood-brain barrier through the chemotactic motion of NEs along the gradient of inflammatory factors.
As a special actuation technology, biological actuation does not need external energy, so no complex actuation equipment is required.Besides, it is also relatively nontoxic and harmless to the human body.However, biological actuation is difficult to control, and live organism-based microrobots usually have short lifetimes.Therefore, there are still challenges to achieving real applications of biologically actuated soft microrobots.

Actuation Affected by Structural Design
The structural design is a critical determinant for soft microrobots' overall performance and functionality.Soft microrobots with proper structural designs can achieve relatively ideal propulsion performance.Currently, the commonly used structure designs of soft microrobots could be grouped into bionicinspired structures (such as artificial cilia, flagella, etc.) and miniaturization structures (such as microcones, microclips, microgears, kirigami, etc.).Drawing inspiration from nature, soft microrobots can replicate the complex functionality and adaptability of living organisms.For instance, soft microrobots inspired by the movement of cilia or flagella can achieve efficient propulsion, while those mimicking the grasping capabilities of insects can perform delicate manipulation tasks.Soft microbots inspired by organisms with the ability to deform or change shape can navigate through narrow or irregular spaces.For an overview comparison of all the structural designs that soft microrobots used so far, typical design inspirations of soft microrobots are summarized in Table 2.

Application of Soft Microrobots
With the development of materials, fabrication, and actuation, soft microrobots have been developed rapidly in recent years, leading to many innovative applications in disease treatment, [116,257] diagnosis and sensing, [95] and environmental remediation. [122,258]he ability of soft microrobots to adapt to biological environments and to transport cargos (drugs or cells) makes them valuable tools in disease treatment.Zhang et al. reported algae-bioinspired microrobots as a promising cargo delivery platform to treat acute bacterial pneumonia (Figure 9a). [257]These microrobots were introduced into a mouse of acute Pseudomonas aeruginosa pneumonia by intratracheally administered.They can actively move in the lung fluid and permeate throughout the lung tissue within 1 h.Their pulmonary antibiotic delivery efficiency can be improved due to deep tissue penetration and prolonged drug retention.The magnetic stem cell spheroid microrobots proposed by Wang et al. shows an exceptional tissue regeneration ability (Figure 9b). [230]In the demonstration, the soft microrobots were injected in suit into an ulcer area.After 3 days of treatment, the results indicated that the therapeutic effect of soft microrobot was due to the living building block (stem cells) inside, which accelerated the healing of gastric ulcers by seeding, spreading, and growing.Soft microrobots' small size and maneuverability make them ideal for minimally invasive surgery.For example, Go et al. proposed magnetically actuated microrobots with porous structures to load and transport mesenchymal stem cells for knee cartilage regeneration (Figure 9c). [12]Unlike scaffolds that require transplantation through invasive surgery, the microrobots can be navigated to the cartilage defect area by magnetic field after an arthroscope-based minimally invasive surgery.After being delivered to the targeted area, the loaded cells can differentiate into chondrocytes to repair damaged cartilage, and the microrobots can safely degrade after 33 days.
The ability of soft microrobots to detect biomarkers with high sensitivity and specificity makes them a promising platform in sensing and early detection/monitoring of diseases.For real-time determination of the nerve agent neostigmine in field forensic analysis, Prussian Blue (PB)/chitosan soft microrobots functionalized with acetylthiocholinesterase enzyme (ATChE) have been used for on-the-fly biosensing assays. [95]Specifically, neostigmine is a cholinesterase inhibitor drug that inhibits ATChE converse into TCh.Thus, the reaction of TCh with PB is prevented when neostigmine exists.The colorimetric mediator receives the PB inhibition signal to show blue color as the neostigmine concentration increases.Therefore, rapid colorimetric determination of neostigmine can be achieved.For detecting and diagnosing disease with soft microrobots, polydopamine (PDA)-coated spirulina microrobots grafted with fluorescent probes was used for off-on fluorescence diagnosis of pathogenic bacteria.(Figure 9d). [259]Spirulina microrobots offer good biocompatibility and high control accuracy.When fluorescence probes are triggered by the acid environment caused by pathogenic bacterial fermentation, they can be released from the microrobots and labeled on the bacteria, leading to diagnostic fluorescence for the visual tracking of the pathogenic bacteria.As another example of disease diagnosing, Janus soft microrobots were developed for tumor cell capturing and capture-induced ratiometric fluorescence signaling detection (Figure 9e). [260]One side of the soft microrobots is grafted with catalase to catalyze the decomposition of H 2 O 2 for propulsion.The other side is covalently bounded with aptamer, followed by labeling with tetraphenylethene (TPE) derivatives and fluorescein isothiocyanate (FITC).The competitive binding of tumor cells with aptamer causes the release of TPE and FITC from the microrobots.Due to the aggregation-induced emission of TPE and the aggregation-caused quenching of FITC, the release of TPE and FITC would cause fluorescence color change of micromotor.Thus, these soft microrobots can provide real-time and sensitive monitoring of tumor cells.Moreover, soft microrobots have also emerged as potential tools for environmental remediation, as they can navigate through complex environments, detect contaminants, and remove pollutants.As demonstrated in recent studies, iron phthalocyanine (FePc)/gelatin-based soft microrobots were utilized to degrade organic pollutants (Figure 9f ). [122]Under the visible light illumination, these microrobots respond quickly and move toward the light.The autonomous movement of swarm microrobots causes a vigorous solution mixing process and facilitates FePc to degrade the Rhodamine into carbon dioxide and water.

Conclusion and Outlook
With the in-depth study of soft microrobots, people have realized that soft-microrobot is a multidisciplinary and complex research field, integrating material, chemistry, biology, machinery, and motion control.The corresponding study endows soft microrobots with excellent maneuverability and puts practical applications of these small and soft devices in biomedicine, noninvasive surgery, and other fields on the agenda.
Material, fabrication, and actuation are the three cornerstones of soft microrobots.The introduction of various soft materials gives soft microrobots flexibility, changeability, and low invasiveness.Using the special properties of different soft materials, high affinity and various interactions between soft microrobots and the human body could be realized.Biofriendly materials make soft microrobots safe and reliable for medical use.Stimulusresponsive materials enable soft microrobots to perceive external stimuli and conduct responses sensitively.Live organisms can significantly increase the interaction and perception between soft microrobots and the human body.][263] To actualize these excellent soft microrobots, various fabrication technologies have been utilized.UV polymerization can be used to realize low-cost and patterned structure fabrications with photosensitive materials.2PP can be used to meet the needs of having complex 3D structures with submicrometer-level high precision for soft microrobots; template-assisted and electrospinning methods can be applied to the scene of large-mass and efficient fabrication.To promote soft microrobots further toward practical applications, effective actuation methods have been studied.External energy sources used to achieve remote, untethered, and high-precision motion of soft microrobots include magnetic field, ultrasound, electricity, chemical reaction, and biological energy.The three cornerstones of soft microrobots (material, fabrication, and actuation) restrict and interrelate with each other.The future development and breakthrough of soft microrobots would benefit from the continuous renewal of these three aspects.
With further development in the corresponding research fields, soft microrobots will play an essential role in biomedicine, environmental remediation, biosensing, etc., in the foreseeable future.Notably, to utilize these soft microrobots in clinical applications, some challenges and limitations should be addressed, including stability and durability, in vivo imaging and tracking, and in-body adaptation.Stability and durability are crucial factors that determine the efficacy and safety of soft microrobots.Compared with rigid microrobots, soft microrobots are more susceptible to wear and tear due to mechanical stress from movement and deformation.Developing effective imaging and tracking technologies is crucial for the clinical applications of soft microrobots.Although many imaging tracking techniques are already available, such as X-ray, electronic computed tomography (CT), ultrasound imaging, photoacoustic imaging, magnetic resonance imaging (MRI), positron emission-type CT imaging (PET), et al., each technique has its own limitations.For instance, X-rays are harmful to the body and can only provide flat images; CT examination has excessive reconstruction artifacts and is disturbed by respiratory motion; ultrasound imaging images have poor contrast and poor signal-to-noise ratio; photoacoustic imaging is limited by imaging depth; MRI imaging equipment is expensive and has a long scan time; PET scanning requires the use of trace radioisotopes, causing a small radiation dose in the body.Therefore, developing new or combined imaging tracking methods to improve imaging contrast in time and space is still necessary.Moreover, the complex and dynamic physiological fluid environment in vivo poses unique challenges for soft microrobots.The navigation of soft microrobots in vivo is much more formidable due to the unpredictable fluid flows and varying tissue densities compared with the static and controlled environment for laboratory experiments.Furthermore, soft microrobots may trigger immune responses even if they are biocompatible, and the immune response can lead to adverse effects, such as inflammation and tissue damage.
The solving of the above issues will significantly promote the clinical application of soft microrobots.For a more advanced development of this field, further optimization should focus on intelligentization.First, multiple functions, including sense and operation functions, should be integrated for intelligent soft microrobots.For instance, instead of being only able to sense one environmental factor, intelligent soft microrobots should be able to sense different factors, giving different feedback or reactions.At the same time, they should also have multiple operation functions, such as grasp, deformation, speed modulation, etc.Second, real-time communications between intelligent soft microrobots and the controller should be established.Considering all the feedback from intelligent soft microrobots comprehensively, specific tasks should be decided by the controller and assigned to the soft microrobots, for which operation functions of soft microrobots, like cargo grasping/releasing, would be triggered.Similarly, with excellent communication ability, automated navigation within complex in vivo environments should be realized.Based on the dynamic environment feedback from intelligent soft microrobots, motion modulation decisions should be made to avoid obstacles and to improve efficiency and be implemented by the microrobots in time.Finally, collaboration within a swarm of intelligent soft microrobots should be possible.For those tasks that single soft microrobots cannot complete, intelligent soft microrobots should be able to communicate with each other directly or through the controller to collaborate and get the work done optimally.The intelligentization of soft microrobots relies on the further development and application of knowledge in many fields involving micro-/ nanotechnology, physiology, robotics, machine learning, etc.
In summary, soft microrobots represent an exciting and rapidly evolving field.Through continuous innovation, these devices will integrate the abilities of diagnosis, treatment, communication, collaboration, etc., becoming intelligent and efficient tools for minimally invasive medicine.

Figure 2 .
Figure2.Biofriendly soft microrobots.a) PEGDA-based magnetic soft microrobot passing through an orthometric bent capillary and a subuniform capillary.Reproduced with permission.[106]Copyright 2021, Elsevier.b) Video snapshots of the Ecoflex-based jellyfish-like soft microrobot capturing a buoyant bead.Reproduced under the terms of the CC-BY license.[79]Copyright 2019, The Authors.Published by Springer Nature.c) Gelatin methacrylate hydrogel (GelMA)-based microrobots release the drug by swelling.Reproduced under the terms of the CC-BY license.[34]Copyright 2019, The Authors.Published by American Chemical Society.d) Cell transportation illustration and experiment of GelMA-based microrobots.Reproduced with permission.[96]Copyright 2020, Wiley-VCH.The scale bar in (a) and (d) represents 500 μm and 100 μm, respectively.

Figure 4 .
Figure 4. Organism-based soft microrobots.a) Illustration of loading cargoes to an RBC and the NIR-triggered motion control of the RBC-based soft microrobot.Reproduced with permission.[43]Copyright 2018, American Association for the Advancement of Science.b) Targeted delivery illustration and morphological images of NEs-based microrobot.Reproduced with permission.[165]Copyright 2021, American Association for the Advancement of Science.c) Illustrations of bacteria-driven microrobot for target drug delivery.Reproduced with permission.[169]Copyright 2017, American Chemical Society.d) Biohybrid soft microrobots based on sperm cells for targeted drug delivery and illustration of the corresponding loading and releasing process of the sperm.Reproduced with permission.[26]Copyright 2018, American Chemical Society.

Figure 6 .
Figure 6.Commonly used collocation between materials and fabrication methods.

Table 1 .
A summary of different actuation methods.
• Limited actuation lifetimes Biological Self-propulsion organism Biological tissues and fluids • Do not need complex actuation equipment • Natural compatibility with biological systems • Require suitable environment and conditions • Lifetime is short