Progress in Nanorobotics for Advancing Biomedicine

130 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 Progress in Nanorobotics for Advancing Biomedicine Mi Li Member IEEE Ning Xi Fellow IEEE Yuechao Wang and Lianqing Liu Member IEEE AbstractNanorobotics which has long been a fantasy in the realm of science fiction is now a reality due to the considerable developments in diverse fields including chemistry materials physics information and nanotech nology in the past decades Not only different prototypes of nanorobots whose sizes are nanoscale are invented for various biomedical applications but also robotic nanoma nipulators which are able to handle nanoobjects obtain substantial achievements for applications in biomedicine The outstanding achievements in nanorobotics have signif icantly expanded the field of medical robotics and yielded novel insights into the underlying mechanisms guiding life activities remarkably showing an emerging and promis ing way for advancing the diagnosis treatment level in the coming era of personalized precision medicine In this review the recent advances in nanorobotics nanorobots nanorobotic manipulations for biomedical applications are summarized from several facets including molecu lar machines nanomotors DNA nanorobotics and robotic nanomanipulators and the future perspectives are also presented Index TermsNanorobot molecular machine nanomo tor DNA nanorobotics nanomanipulator I INTRODUCTION T HE developments of medical robotics in the past decades have contributed much to the field of clinical medicine Medical robots fundamentally couple information including patientspecificinformationegmedicalimagesandlabtestre sults and general information eg anatomic atlases statistics Manuscript received November 12 2019 revised February 5 2020 and March 17 2020 accepted April 22 2020 Date of publication April 27 2020 date of current version December 21 2020 This work was supported in part by the National Natural Science Foundation of China under Grants 61922081 61873258 91748212 and 61925307 in part by the Key Research Program of Frontier Sciences CAS ZDBSLY JSC043 in part by the Youth Innovation Promotion Association CAS 2017243 and in part by the LiaoNing Revitalization Talents Program XLYC1907072 Corresponding authors Mi Li Lianqing Liu Mi Li and Lianqing Liu are with the State Key Laboratory of Robotics Shenyang Institute of Automation Chinese Academy of Sciences Shenyang 110016 China with the Institutes for Robotics and Intelli gent Manufacturing Chinese Academy of Sciences Shenyang 110169 China and also with the University of Chinese Academy of Sciences Beijing 100049 China email limisiacn lqliusiacn Ning Xi is with the Department of Industrial and Manufacturing Sys tems Engineering The University of Hong Kong Yuechao Wang is with the State Key Laboratory of Robotics Shenyang Institute of Automation Chinese Academy of Sciences with the Institutes for Robotics and Intelligent Manufacturing Chinese Academy of Sciences and also with the University of Chinese Academy of Sciences Digital Object Identifier 101109TBME20202990380 and rules to physical action to significantly enhance humans ability to perform various medical tasks and the medical tasks are often surgical interventions rehabilitation or helping hand icapped people in daily living for macroscale medical robotics 14 The use of surgical robots eg the wellknown da Vinci robot system brings enhanced dexterity greater precision reduced surgeon handtremor intuitive ergonomic interfaces and the ability to access surgical sites remotely with miniatur izedinstrumentationsignificantlybenefitingminimallyinvasive surgery 5 6 Robotic devices have been developed to restore the functionality of patients with movement disorders such as upper limb rehabilitation 7 and lower limb assistance 8 Wireless video capsule endoscopy enables inspection of the digestive system without discomfort or need for sedation and has the potential of encouraging patients to undergo gastrointestinal tract tests 9 which has revolutionized the diagnostic workup in the field of small bowel diseases 10 The emergence of soft robotics which uses soft materials with biocompatibility and biomimicry has opened possibilities for novel biomedical applications in which a soft interaction with a patient is preferred 11 12 Handheld robots which are totally ungrounded and manipulated by surgeons in free space provide specific functions to assist the surgeon in accomplishing tasks that are otherwise challenging with manual manipulation 13 These achievements in macroscale medical robotics undeniably show the impacts of robotics on clinical medicine and healthcare 14 15 It is increasingly evident that making medical robots smaller will have important impacts on the field of biomedicine 16 In fact besides the macroscale medical robotics described above smallscale from several millimeters down to a few nanometers in all dimensions medical robotics has been largely investigated for various biomedical and healthcare applications including singlecell manipulation and biosensing targeted drug delivery minimally invasive surgery medical diagnosis tumor therapy detoxification and so on 1720 So far there is no stan dardized definition of the term microrobot but one common approach defines a microrobot as existing in the size range of hundreds of nm to 1 mm 21 Since this paper focuses on nanorobotics readers are referred to the literatures 2126 for more descriptions about microrobots Thanks to these devel opments the miniatured submarine the imaginary prototype of tiny robots described in the film Fantastic Voyage which is able to autonomously cruise in the blood vessel of patients to cure diseased cells is now becoming true The achievements allow a great leap for the realization of swallowing a surgeon 27 00189294 2020 IEEE Personal use is permitted but republicationredistribution requires IEEE permission See httpswwwieeeorgpublicationsrightsindexhtml for more information Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 131 TABLE I COMPARISON OF DIVERSE NANOROBOTIC SYSTEMS which will probably revolutionize the diagnosis and treatment of clinical diseases As an important type of smallscale medical robotics nanorobotics holds great promise for advancing biomedicine Nanorobotics involves developing robotic devices to perform tasks eg actuation sensing manipulation propulsion signal ing information processing intelligence and swarm behaviors at the nanometer scale 28 Nanorobotic systems may them selves be miniature in size often called nanorobot or they may be designed specifically to interact with nanosized matters of ten called nanomanipulator 29 Nanorobotics is particularly meaningful for biomedicine We are entering the era of personal izedprecisionmedicine30inwhichthereareseveralkeyissues needing to be addressed such as accurate and comprehensive diagnosis of the diseases for individual patients recognizing the potential drugs which possibly benefit the patients assisting physicians to specify therapeutic plans and so on For these issues understanding the pathological situations of individual patients at the singlemolecule level is critical since specific biomolecules on the diseased cells are important indicators for diseases and the interactions between these biomolecules and drug molecules have direct impacts on drug efficacies 31 32 The size of single biomolecules is at the nanoscale 33 and therefore nanorobotics is required for robotic manipulations of biomolecules Consequently the coming personalized precision medicine brings grand opportunities for the development of medical nanorobotics Over the past years a range of nanorobotic devices including nanorobots and nanomanipulators 3436 have been devel oped for diverse biomedical applications significantly demon strating the outstanding capabilities of nanorobotics in address ing biomedical issues with unprecedented spatial resolution and considerably enriching the studies of medical robotics Nanorobots the overall size is in the nanometer scale and nanomanipulators the overall size in the macroscale but their end effectors can perform robotic manipulations on nanosized objects represent two categories of nanorobotic techniques which are complementary with each other in performing dif ferent robotic operations at multiple levels for biomedical appli cations Human diseases such as cancers are extremely intricate and are often associated with multifaceted factors at different levels eg molecule organelle cell tissue organ 3739 requiring nanorobotic devices to probe multidimensional and multiparametric biological signatures for comprehensively char acterizing the pathological situations of diseases Hence sur veying the progress of both nanorobots and nanomanipulators benefits understanding the field of nanorobotics from an overall perspective and may inspire interdisciplinary studies for advanc ing biomedicine In this review we summarize the recent efforts of nanorobots and nanomanipulators for biomedical applications The different types of nanorobotic devices 4046 and their main biomedi cal applications described here are shown in Table I and Fig 1 re spectively mainly including molecular machines nanomotors DNA nanorobotics nanomanipulation systems based on tweez ers atomic force microscopy AFM nanomanipulation system and electron microscopy EM nanomanipulation system In the following sections we will show with illustrations how these nanorobotic systems contribute to addressing biomedical issues at multiple levels from ions and molecules to cells tissues organs and body as shown in Fig 1 These breakthroughs will potentially have significant impacts on both fundamental studies and clinical practice in the coming era of personalized precision medicine II MOLECULAR MACHINES Inspired by the complexity and hierarchical organization of biological machines now researchers can fabricate artificial molecular machines which are capable of performing work or completing sophisticated tasks at the molecular scale 47 48 offering novel possibilities for nanorobotics at the single molecule level Molecular machines can be defined as devices Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 132 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 Fig 1 The main biomedical applications of different types of nanorobotic systems With the use of diverse nanorobotic systems biological samples at multiple scales ranging from single molecules to human body can be probed Molecular machines are able to probe ions and small molecules as well as proteins Tweezersbased nanomanipulators are capable of handling biological samples ranging from a few nanometers such as proteins to tens of micrometers such as cells AFMEM nanomanipulators can manipulate biological samples from singlemolecule level to singlecell level and tissue level After injecting numerous DNA nanorobots into the blood vessel DNA nanorobots carrying drug molecules can move to diseased sites such as tumors in the body and then release drugs to cure the diseased sites for in vivo applications Nanomotors can not only probe single cells but also can pass through the biofluids in the gastrointestinal tract of animal models for precision surgery Notably the labeled scales at the bottom do not correspond to the sizes of nanorobotic systems but correspond to the various biological samples at multiple scales that can produce useful work through the interaction of in dividual molecules whose size is in the range of nanometer 49 and thus molecular machines also fall into the category of nanomachines In living organisms there are many different types of molecular machines which work together to produce store and consume energy so that life can be sustained across a wide range of length scales 50 Rotary motor eg ATP synthase and linear motor eg myosin are the famous natural molecular machines which have been identified and investigated in detail 51 and the dynamic movements of single rotary motor 52 and single linear motor at work 53 have been directly revealed Although natural molecular machines with high com plexity are almost unattainable through synthetic preparations they continuously provide inspiration to scientists to develop artificial systems to mimic the structures and functions of natural molecular machines 54 The effects of random thermal motion heat dissipation solvation momentum inertia gravity and so on differ significantly at the molecular and macroscopic levels meaning that designing nanomachines cannot simply mimic the mechanisms of their macroscopic brethren 55 For detailed descriptions about the design principle of artificial molecular machines readers are referred to the references 5557 The appearance of artificial molecular machines facilitates using molecules to manipulate other molecules in robotic fashion which was proposed by Richard Feynman in his famous speech Theres plenty of room at the bottom 42 In fact researchers have demonstrated the feasibility of molecular manufacturing with the use of programmable molecular machines 58 Molec ular machines also enable the design and synthesis of molecular robotic arm for transporting cargo 59 Hence achievements in the field of molecular machines considerably benefit the studies of nanorobotics at the molecular level In addition evidence has shown that artificial molecular machines are able to open cell membranes and probe intracellular sciences 60 showing the potential of molecular machines in advancing biomedicine Artificial molecular machines having controlled mechani cal motions Fig 2 promote the design and development of nanorobots from the point of bottomup approach for biomedical applications Molecular shuttle I in Fig 2A is an interlocked molecular assembly in which a macrocyclic ring is able to move back and forth between two recognition sites on a linear track terminated by bulky stoppers 61 62 A variety of stimuli have been utilized to trigger molecular shuttles including pH redox light chemical input and microenvironmental changes 63 A recent study II in Fig 2A by Chen et al 64 has shown that a molecular shuttle called rotaxane can operate in lipid bilayers for ion transport The rotaxane is composed of an amphiphilic molecular thread with three binding stations which is interlocked in a macrocycle wheel component that tethers a K carrier The structural characteristics enable the molecular shuttle to be inserted into lipid bilayers to perform passive ion transport through the shuttling motion opening novel possibili ties for developing more effective and selective ion transporters Molecular car has a chassis equipped with some spacer chemical groups to hold it a few angstroms away from the supporting surface and an onboard motor allowing the car to move 65 66 Different types of energy eg thermal 67 electricity 68 light 69 and chemical propulsion 70 have been used as the input to drive the molecular car to move on the metal surface and diverse molecular cars have been developed Fig 2B facilitating using the molecular cars to transport nanocargo for biomedical applications In nature cell membranes can selec tively transport small moleculesions via carrier proteins 71 which plays an important role in regulating cellular behaviors In 2015 Cheng et al 72 developed an artificial dumbbellshaped molecular pump which is able to pump small molecules to create Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 133 Fig 2 Artificial molecular machines for biomedical applications A Molecular shuttle for transporting molecules I Structure of molecular shuttle Molecular shuttle is constructed from a combination of rigid linking struts green and metal nodes brown The axle blue and wheel red of the molecular shuttle are inserted as a crossbar between the struts Reprinted with permission from ref 62 Copyright 2015 Macmillan Publishers Limited II Molecular shuttle for K transport across lipid bilayers Reprinted with permission from ref 64 Copyright 2018 American Chemical Society B Molecular cars with different structural organizations for transporting nanocargo Reprinted with permission from ref 65 Copyright 2017 Macmillan Publishers Limited Reprinted with permission from ref 66 Copyright 2013 American Chemical Society C Molecular pump for pumping molecules The dumbbell and the ring molecule repel each other initially I Reduction favors complexation both thermodynamically and kinetically II and oxidation restores the repulsion between the components III and causes the ring to fall into a kinetic trap during thermal relaxation IV Reprinted with permission from ref 72 Copyright 2015 Macmillan Publishers Limited D Molecular assembly line for peptide synthesis I A ring moves along a linear molecule using its nanorobotic arm to pick up amino acids mounted along the shaft II As the ring moves it links the amino acids into a growing peptide Reprinted with permission from ref 75 Copyright 2015 Macmillan Publishers Limited a gradient in their local concentration Fig 2C The pump uses redox energy and precisely organized noncovalent bonding interactions to pump positively charged rings from solution and ensnare them around an oligomethylene chain as part of a kinet ically trapped entanglement Particularly the artificial pump can cycle repetitively and function against a local concentration gra dient so as to build up potential energy Ribosome is an organelle responsible for protein synthesis 73 In 2013 Lewandowski et al 74 developed an artificial molecular assembly line which is a primitive analog of the ribosome Fig 2D The molec ular assembly line travels along a molecular strand picking up amino acids that block its path to synthesize a peptide in a sequencespecific manner With the use of 1018 molecular machines working in parallel a few milligrams of peptide were produced in 36 hours showing the formation of macroscale quantities of products in the molecular assembly line These miraculous results 62 64 65 72 74 remark ably demonstrate that artificial molecular machines can be de signed to autonomously perform iterative tasks on individual small molecules in robotic fashion for biomedical applications Nevertheless it should be noted that a challenging issue is how to make billions of molecular machines work in concert to produce measurable macroscopic effects 75 Besides the controllability reliability and robustness of the developed artifi cial molecular machines during operations should be improved for example molecular machines must be highly resistant to degradation and break over long time of working 48 III NANOMOTORS Nanomotors are nanoscale devices designed to perform se lected mechanical movements eg rotation rolling shuttling delivery contraction and collective behavior in response to specific stimuli 76 At the micronanoscale the extremely small dimensions and velocities contribute to very low Reynolds numbers ranging from 101 to 105 causing that the motion of micronano objects is dominated by viscous forces rather than inertial forces and Brownian motion becomes significant 77 According to the scallop theorem by Purcell 78 who says that reciprocal motion cannot achieve propulsion in low Reynolds Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 134 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 number environments the movement of nanomotors has to be nonreciprocal to induce propulsion which adds remarkable complication to the design of nanomotors 79 Besides because traditional power supply components and batteries are not pos sible at the nanoscale innovative bioinspired design principles are required to meet the challenging powering and locomotion demands 17 Despite the huge challenges in designing and building nanomotors and propulsion methods various types of nanomotors have been developed details about the fabrication of nanomotors have been reviewed in the reference 80 for diverse applications such as biosensing 25 drug delivery 26 diagnostics 81 environmental microcleaner and sensor 82 targeting and isolation of cancer cells 83 which significantly promote the progress of nanorobotics Nanomotors open the door of autonomous propulsion of nanorobots In 2004 Paxton et al 84 firstly developed cat alytic nanomotors Fig 3A which could move autonomously in hydrogen peroxide solutions The striped platinumgold PtAu nanorods are about 370 nm in diameter and containing platinum and gold segments each 1 µm long The PtAu nanorods could move in the direction of long axis and control experiments show that asymmetric PtAu geometry was necessary for rapid axial movement Further studies show that the selfelectrophoresis mechanism I in Fig 3A plays an important role in the motion of PtAu nanorods in hydrogen peroxide solutions 85 86 According to this mechanism H2O2 is oxidized to generate protons in solution and electrons in the nanorod on the Pt end The protons and electrons are then consumed with the reduction of H2O2 on the Au end This results in an asymmetric distribution of protons near the ends of the nanorods and in turn creates an electric field in the solution near the nanorods which cause the motion of the nanorods in the selfgenerated elec tric field Experiments also show that the bimetallic nanotubes III in Fig 3A move significantly faster than bimetallic solid nanorods II in Fig 3A in H2O2 solution 87 Researchers have also developed nanorockets 88 89 which could propel autonomously and efficiently in extreme acidic environments Nanorockets rely on the bubble propulsion mechanism associ ated with the continuous thrust of hydrogel bubbles generated at the inner of nanorockets Though chemically powered catalytic nanomotors bring breakthroughs in the field of nanorobotics these nanomotors require external fuels such as H2O2 which is usually inaccessible for many important applications such as biomedical applications Therefore developing fuelfree nanomotors is significantly meaningful for the biomedical ap plications of nanomotors In 2015 Li et al 90 developed a magnetoacoustic hybrid fuelfree nanomotor Fig 3B which is composed of a magnetic nanospring Ni coated Pd on one end and a concave Ni coated Au nanorod on the other The nanomo tor can thus be powered by either a magnetic or ultrasound field and change their swimming mode and direction instan taneously upon altering the applied fields Regarding acoustic nanomotors studies have revealed the density and shape effects during the acoustic propulsion of bimetallic nanorod motors 91 For biomedical applications studies have demonstrated the ultrasonic propulsion of rodshaped nanomotors inside living cells 92 which provides a new tool for probing the response of living cells to internal mechanical excitation controllably manipulating intracellular organelles and precisely delivering nanocargos 93 Nanomotors can also be propelled by light and lightdriven nanomotors have the advantages of reversible wireless and remote maneuver on demand with excellent spatial and temporal resolution 94 95 Recently Liang et al 96 reported an innovative mechanism that allowed multifold recon figuration of mechanical rotation of semiconductor nanowires in electric fields by visible light stimulation Fig 3C which have potential impacts on reconfigurable nanorobotic devices for optical sensing communication molecule release detection nanoparticle separation and microfluidic automation The studies of stomatocyte nanomotors based on supramolec ular assembly provide a bottomup approach for designing and developing nanoscale active systems for biomedical ap plications The nanomotors described above Fig 3AC are commonly fabricated by topdown methods and the major downside of these systems is the use of hard metal surfaces that render them unsuitable for biomedical applications due to the lack of biocompatibility and biodegradability as well as feasibility of shape transformation 97 Besides it is quite complex when applying these nanomotors for delivery ap plications In 2012 Wilson et al 98 firstly presented the stomatocyte nanomotors which are bowlshaped structures of nanosized dimensions formed by the controlled deformation of polymer vesicles Fig 3D The design of this nanomotor is a fully bottomup approach based on the selfassembly of biodegradable amphiphilic block copolymers and entrapment of active catalyst platinum nanoparticles within the bowlshaped structures 99 The copolymers can fold inward under osmotic pressure due to the presence of plasticizing organic solvent in both inner and outer compartments 97 The narrow opening of the stomatocyte nanomotor serves as an outlet for the formed oxygen bubbles to facilitate the propulsion of the stomatocyte nanomotors Chemically attaching a stimulusresponsive valve system polymer brushes to the stomatocyte nanomotor allows the control of the motion of the nanomotor without changing the catalyst activity or the shape of the nanomotor 100 The constructed temperatureresponsive nanomotor is able to sense locally the environment temperature and regulate the accessi bility of the fuel and accordingly adjust its speed and behavior showing potentials in controllable cargo transportation The bi layer membrane structure of the stomatocyte nanomotors allows for efficient loading of both hydrophilic and hydrophobic drugs 101 By encapsulating anticancer drugs into the lumen of the stomatocyte nanomotor the nanomotor is able to move to the targeted cancerous cells at a speed of about 39 µms Then the nanomotor can be taken up by cancerous cells and subsequently drugs are released to kill the cancerous cells However it should be noted that the propulsion of stomatocyte nanomotors is based on chemically catalytic reactions and chemical fuels including H2O2 and platinum are required which result in challenges for their biomedical applications Recently in 2019 inspired by the endogenous biochemical reaction in the human body involving conversion of amino acid Larginine to nitric oxide NO by NO synthase NOS or reactive oxygen species ROS Wan et al 102 have reported a nanomotor using Larginine as the fuel Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 135 Fig 3 Typical nanomotors for biomedical applications A Bimetallic catalytic nanomotor I Illustration of the selfelectrophoretic propulsion mechanism for the AuPt catalytic nanomotor II III SEM images of nanorod motors II and nanotube motors III I and III are reprinted with permission from ref 87 Copyright 2013 American Chemical Society II is reprinted with permission from ref 86 Copyright 2006 American Chemical Society B Magnetoacoustic hybrid nanomotor I Schematic of the magnetoacoustic hybrid nanomotor and its dual propulsion modes under the acoustic and magnetic fields II Illustration of the templateassisted fabrication of the bisegment magnetoacoustic hybrid nanomotors Commercial polycarbonate membranes with a pore diameter of 400 nm are first sputtered with a thin Au film as the bottom contact electrode The gold nanorod segment is then electrodeposited within the nanopores step α Codeposition of Pd2 and Cu2 within the nanoscopic pores under an acidic environment yields the helical structures on top of the gold segment step β III SEM image of a magnetoacoustic hybrid nanomotor Reprinted with permission from ref 90 Copyright 2015 American Chemical Society C Lightgated reconfigurable rotary nanomotors I Schematic of silicon nanowire fabrication via nanosphere lithography II Crosssection view of arrays of silicon nanowires III A nanowire IV Schematic of the opticalelectric setup for reconfigurable manipulation Reprinted with permission from ref 96 Copyright 2018 AAAS D Stomatocyte nanomotors fabricated based on bottomup supramolecular assembly I Side view of the stomatocyte nanomotor II Analogy with a miniature propellant III Schematic of the opening of the stomatocyte Reprinted with permission from ref 98 Copyright 2012 Macmillan Publishers Limited Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 136 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 for the production of NO both as driving force and to provide beneficial effects without yielding wastes providing a novel idea for constructing nanomotors for in vivo biomedical applications based on biocompatible chemical reactions IV DNA NANOROBOTICS The appearance of DNA origami technology provides a novel way for the studies of constructing nanorobots DNA is a truly programmable materials and DNA molecules can be as sembled into custom predesigned shapes via hybridization of sequencecomplementary domains 103 Notably nanorobots constructed by DNA techniques belong to nanomotors 76 Due to the importance of DNA techniques for nanorobotics here we describe DNA nanorobotics separately DNA origami which was created in 2006 104 is a DNAbased technology that utilizes programmed combinations of hundreds of short complementary staple oligonucleotides to fold a large single strand of scaffold DNA into precise 2D and 3D shapes stabi lized by thousands of base pairs 105 DNA origami allows the bottomup selfassembly of discrete objects with subnanometer precise features dimensions ranging from the nanometer to the micrometer scale and molecules masses up to the gigadalton scale 106 With the use of DNA origami a wide range of functional static nanostructures and dynamic nanodevices have been constructed more detailed information can be found in the literature 107 paving the way for DNA nanorobotics at the singlemolecule level In 2010 Lund et al 108 showed the robotic motional behaviors such as start follow turn and stop of DNA walkers composed of a streptavidin molecule as the body and three deoxyribozymes as legs on the substrate molecules laid out on a twodimensional DNA origami land scape demonstrating the feasibility of realizing programmed roboticbehaviorsbasedonDNAtechnologyIn2018Kopperger et al 109 developed a selfassembled nanoscale robotic arm by electric fields with the use of DNA origami technology The actuator unit of the system is composed of a 55 nm55 nm DNA origami plate with an integrated 25nmlong arm allowing for electrically driven transport of molecules or nanoparticles over tens of nanometers Experimental results show that the robotic movements achieved are significantly faster than previously reported DNA motor systems and comparable to the adenosine triphosphatasedriven biohybrids Researches have also shown that DNA nanorobots can insert into the 2D DNA crystalline substrate at specific sites 110 and sort pick up transport and drop off cargos 111 These results 108111 signif icantly demonstrate the great potentials of DNA origami tech nology in nanorobotic operations In addition DNA origami has been utilized recently to fabricate DNA devices for generating transmitting and sensing mechanical forces at the nanoscale 112 such as probing molecular forces with DNA origami basednanoscopicforceclamp113andmappingthe3Dorienta tion of integrin traction forces on living cells 114 which facili tatesprobingmolecularforcesinvolvedinmolecularandcellular behaviors for uncovering the underlying mechanisms guiding life activities and shows the potentials of DNA nanorobotics in answering fundamental issues in life sciences DNA nanorobotics in biomedical applications such as drug delivery achieves exciting progress in the past decade DNA nanorobotic structures can be created that contain a cavity for hosting molecular payloads eg antibody fragments that bind to specific antigens and these structures can be switched between open and closed conformations exposing or hiding the cargo depending on the presence of molecular triggers 115 The early therapeutic potential of DNA nanorobotic systems in specifically delivering drugs to tumors while sparing healthy tissues has been experimentally demonstrated 116 117 which has shown great translation significance for smart cancer therapy In 2012 Douglas et al 118 reported an autonomous DNA nanorobot capable of transporting molecular payloads to cells sensing cell surface inputs for triggered activation and reconfiguring its structure for payload delivery Fig 4A The nanorobot in the form of a hexagonal barrel dimensions are 35 nm35 nm45 nm was created based on DNA origami tech nology The barrel consists of two domains that are covalently attached in the rear by scaffold hingers and can be noncovalently fastened in the front by DNA aptamerbased locks The aptamers act as the switch When aptamers recognize the target molecules on the cells the lock dissociates and the nanorobot undergoes a reconfiguration to expose its previously masked surfaces which delivers the drugs encapsulated in the nanorobot to cells The study shows the prototype of nanorobot for delivering drugs to cancer cells in vitro In 2018 Li et al 119 firstly reported the in vivo biomedical applications of DNA nanorobots which are de veloped based on a selfassembled DNA origami nanotube with multiple functional elements Fig 4B Thrombin molecules were loaded inside the nanorobot The nanorobot can protect thrombin until exposure is triggered by the interaction with the tumor vessel marker nucleolin which allows the exclusive delivery of thrombin to tumor sites minimizing the side effects of thrombin in healthy tissues Experiments on mice show that intravenously injected DNA nanorobots deliver thrombin specif ically to tumorassociated blood vessels and induce intravascular thrombosis which results in tumor necrosis and inhibition of tumor growth Besides the nanorobots are shown to be safe and immunologically inert in animal models Also in 2018 the in vivo researches performed by Jiang et al 120 proved that DNA nanorobots can preferentially accumulate in the kidneys of mice with rhabdomyolysisinduced acute kidney injury AKI for repairing the renal tubular epithelial cells Fig 4C showing that DNA nanorobots could become a source of therapeutic agents for the treatment of renal diseases These studies 118120 significantly demonstrate that DNA nanorobotics brings novel possibilities for in vivo drug delivery and disease treatment in biomedicine V NANOROBOTIC MANIPULATIONS BASED ON TWEEZERS Combining robotics with optical tweezers allows precise nanorobotic manipulations of biological samples for life sci ences Optical tweezers are instruments based on a tightly fo cused laser beam that is capable to trap and manipulate a wide range of particles in its focal spot 121 Since the invention in 1970 optical tweezers have advanced significantly in scientific Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 137 Fig 4 DNA nanorobotics for biomedical applications A A DNA nanorobot capable of loading drug molecules I Schematic front orthographic view of closed nanorobot loaded with drug molecules II Open nanorobot after the unlocking of aptamers III Transmission electron microscopy TEM images of nanorobots in closed and open conformations Reprinted with permission from ref 118 Copyright 2012 AAAS B A DNA nanorobot as a cancer therapeutic in vivo α Schematic illustration of the construction of thrombinloaded nanorobot by DNA origami β Schematic representation of the therapeutic mechanism of nanorobot within tumor vessels Reprinted with permission from ref 119 Copyright 2018 Nature America C A DNA nanorobot for treating renal diseases in vivo DNA origami nanostructures DONs can scavenge reactive oxygen species ROS and alleviate oxidative stress locally protecting kidney structures and relaxing kidney diseases Reprinted with permission from ref 120 Copyright 2018 Springer Nature areassuchasatomicphysicsopticsandbiologicalscience122 123 Due to the fact that manual manipulations can induce fatigue of the operator and therefore influence the experimental efficiency and reproducibility 124 automated manipulations of optical tweezers are meaningful for the practical applications of optical tweezers Using the forces exerted by a strongly focused beam of light optical tweezers can function as special robot endeffectors to trap and move objects ranging from tens of nanometers to tens of micrometers in a noncontact manner 125 126 as shown in Fig 5AI With the use of robotaided optical tweezers system robotic operations with high efficiency and high success rate can be performed on cells such as cell transportation 127 and recent studies have demonstrated the capabilities of the optical tweezers robotic system for in vivo manipulations of single cells in the blood flow environments 128 which have potential impacts on biomedicine Besides cells optical tweezers are able to manipulate single molecules such as proteins and nucleic acids 129 which achieves great success in revealing the structural dynamics and force informa tion of molecules by tethered assays 130 In a tethered assay a molecule is attached to an optically trapped bead and the free end of the molecule is attached to a second bead which is held in a second independent optical trap Moving the optically trapped bead results in the stretching or relaxing of the molecule during which the forces and displacements associated with the molecular unfolding or refolding are recorded providing novel insights into diverse molecular and cellular behaviors such as binding of hormone and activation of receptor 131 Fig 5AII molecular mechanisms of molecular motors 132 and plasma membrane tension in cells 133 Notably so far Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 138 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 Fig 5 Nanorobotic manipulations based on tweezers to probe diverse biological samples for biomedical applications A Robotic optical tweezers system for handling single cells and single molecules I Robotassisted optical cell manipulation system The system consists of tree modules for executive sensing and control The executive module contains an XYZ moving stage with a microscope mounted on Z axis and a holographic optical trapping HOT device The sensory module contains an inverted microscope The control module contains a motion controller for the moving stage a phase modulator for the HOT device and a stepping motor controller for the spot moving for the laser scissors Reprinted with permission from ref 126 Copyright 2015 Elsevier Ltd II Manipulating single proteins with optical tweezers Reprinted with permission from ref 131 Copyright 2018 National Academy of Sciences B Robotic magnetic tweezers system for handling intracellular structures I The multipole magnetic tweezers which consist of six magnetic poles with six coils is integrated with a confocal microscope II A magnetic bead denoted by white circle is introduced to the cell by endocytosis III Controlling the bead to move to the target position on cell nucleus The beads trajectory inside the cell is shown as the red line Reprinted with permission from ref 137 Copyright 2018 AAAS C Microfluidic acoustic tweezers automation system for isolating exosomes I Schematic illustration of the system II An optical image of the device III Principle of exosome separation with acoustic tweezers Sizebased separation occurs in each module due to the lateral deflection induced by standing surface acoustic wave SAW field IV Experimental results showing the isolation of exosomes from whole blood with the device Reprinted with permission from ref 143 Copyright 2017 National Academy of Sciences optical tweezersbased singlemolecule assays are dependent on manual labor and developing automated robotic optical tweezers procedures for singlemolecule assays will benefit the molecular studies Nanorobotic manipulations based on magnetic tweezers al low the probing of intracellular structures inside individual living cells Magnetic tweezers use the magnetic particles as endeffectors to manipulate biological samples Magnetic tweezers commonly consist of a pair of permanent magnets placed above the sample holder of an inverted microscope out fitted with a chargecoupled device CCD camera linked to a frame grabber 130 Though optical tweezers have been used to precisely manipulate single cells 125127 utilizing optical tweezers to manipulate intracellular structures inside living cells Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 139 faces challenges since the sample heating and photodamage 134 induced by optical tweezers are harmful to the fragile bi ological molecules inside cells In this case magnetic tweezers which are intangible and safe for biological molecules provide an appropriate tool to sense forces and generate mechanical stimuli inside cells for manipulating cell states 135 Traditional magnetic tweezers are simple force appliers with openloop control for example magnetic forces with desired magnitudes and directions are applied and then the induced motions of the magnetic beads are recorded by the measurement systems and therefore utilizing feedback control facilitates achieving stable positioningandmanipulationofmagneticmicrobeadswithmag netic tweezers 136 In 2018 Wang et al 137 developed a multipole magnetic tweezers system for nanomanipulation involving submicrometer position control and piconewton force control of a submicrometer magnetic bead inside a single cell Fig 5B The bead position was controlled through controlling the driving currents in the coils based on visual feedback from the confocal microscope Magnetic beads were introduced to the cell through endocytosis and the movement of beads was controlled from the initial position to the target location of the cell nucleus When the bead reached the target position on the cell nucleus the system applied a 50pN force in the direction aligned with the major or minor axis By recording the applied magnetic forces and nuclear deformations the Youngs modulus of cell nucleus was obtained showing that the cell nucleus along its major axis is significantly stiffer than along its minor axis Further by controlling beads to apply forces on cell nucleus intermittently the temporal mechanical changes of cell nucleus were monitored The study significantly demonstrates the spatial and temporal intracellular measurement by magnetic tweezers robotic manipulations which will benefit singlecell and singlemolecule analysis The integration of acoustic tweezers and microfluidic automa tion techniques provides a novel way for automatic manipula tions of nanoscopic biological matters with high throughput Acoustic tweezers are a versatile set of tools that use sound wave tomanipulatebioparticlesrangingfromnanometersizedobjects to millimetersized objects 138 With the advantages of non invasiveness labelfree operation and low power consumption acoustic tweezers have been proven to be crucially important for a diverse range of applications particularly for the manipula tions of nanosized biological samples such as exosomes 139 For more descriptions about the advantages and disadvantages of acoustic tweezers and other types of tweezers including optical tweezers and magnetic tweezers readers are referred to the literature 138 Exosomes are small singlemembrane secreted organelles of 30 to 200 nm in diameter that have the same topology as the cell and are enriched in selected proteins lipids nucleic acids and glycoconjugates 140 Exo somes are released by cells and are accessible in biofluids such as saliva urine and plasma 141 Exosomes play an important role in the physiological and pathological processes of cells and living organisms for example during tumor metastasis cancerous cells send signals in advance from the primary tumor via exosomes to make the microenvironment of the distant site suitable for the growth of cancerous cells 142 Hence investigating the structures properties and functions of exo somes is meaningful for revealing the underlying mechanisms guiding life activities The prerequisite of exosomes studies is isolating exosomes from the biofluids Traditional methods for exosome isolation are commonly timeconsuming expensive inefficient and require trained personnel for operation 143 In 2017 Wu et al 143 developed a separation method based on the integration of acoustic tweezers and microfluidics for isolat ing exosomes directly from the whole blood in a labelfree and contactfree manner Fig 5C The isolation chip is composed of two parts including a cellremoval module for removing blood components larger than 1 µm in diameter such as red blood cells RBCs white blood cells WBCs and platelets PLTs and an exosomeisolation module for removing other types of extracellular vesicles such as apoptotic bodies ABs and microvesicles MVs from exosomes The study signifi cantly demonstrated the capabilities of nanorobotic automation techniques based on the integration of microfluidics and acoustic tweezers in isolating nanoscale biological vesicles from native biofluids without pretreatments which expands the capabilities of nanorobotic manipulations for biomedical applications VI NANOROBOTIC MANIPULATIONS BASED ON AFM The development of AFMbased nanorobotic manipulation systems provides a novel way for handling biological samples with unprecedented spatiotemporal resolution AFM is an ex citing powerful tool which is able to probe native biological samples under nearphysiological conditions such as in liquids with nanometer spatial resolution and sub100 millisecond tem poral resolution 144 145 contributing much to molecular and cell biology The commercial AFM is essentially an imaging and measurement instrument Integrating AFM with robotics yields the nanorobotic manipulator which can handle nano objects opening new possibilities of nanorobotics for biomed ical applications Based on AFM with the use of augmented reality and haptic and visual feedback techniques the operator can control the AFM probe as the robotic end effector to manip ulate nanoobjects via joystick yielding the humanintheloop closedloop AFM robotic nanomanipulator 146 as shown in Fig 6AI The exact position of AFM probe tip can be obtained by featurebased localization and planning in nanomanipulations 147 For traditional AFM manipulations with single probe the AFM probe acts as an imaging sensor as well as a manip ulation tool which makes it challenging for threedimensional manipulations The use of dual probes in AFM nanomanipula tions facilitates parallel imagingmanipulation 148 and allows efficient threedimensional manipulations of single cells 149 For individual regular AFM probe mechanical operations eg pushing cutting deforming and touching can be performed on objects with the use of AFM robotic nanomanipulations 150 Combining AFM with microfluidics via microchanneled can tilevers with nanosized apertures enables AFM probe to simul taneouslydeliverdrugstosinglecellsandmanipulatesinglecells 151 as well as electrical measurements and analysis of ions and biomolecules 152 significantly improving the capabilities of AFM nanomanipulations for biomedical applications Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 140 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 Fig 6 AFM nanorobotic manipulations for biomedical applications A AFM nanomanipulator for singlecell operations I Configuration of an AFMbased robotic nanomanipulation system 146 II III The utilization of microfabricated pillar arrays II allows imaging the ultramicrostructures of single living lymphoma cells III by AFM 156 B Probing singlemolecule activities on the surface of single primary cancerous cells by AFM manipulations 162 I Schematic of locating target molecules on cancerous cells with the use of functionalized tip II Bone marrow sample extracted from clinical lymphoma patients III Cancerous cells in the bone marrow sample are recognized by fluorescent staining as denoted by the white arrow IV The nanoscale spatial distribution maps of target molecules on cancerous cells detected by AFM V A typical force curve with a specific molecular unbinding event C Designing hydrogels by AFM for investigating cellmicroenvironment interactions I II III AFM images showing the nanoscale morphological features of diverse biocompatible hydrogels I Natural hydrogel produced by carnivorous plant sundew 175 II Peptideassembled hydrogel 176 III Bioinspired biopolymeric hydrogel 177 IV V The formation of cellular spheroids in culturing cells with the bioinspired hydrogel 177 IV Fluorescent image and V SEM image of cellular spheroid Achievements in the past decade have shown that AFM nanomanipulatorisabletoprobethebehaviorsofsinglecellsand single molecules from the clinical environments For probing cells with AFM the prerequisite is immobilizing cells onto the support The immobilization methods are diverse for different types of cells for example animal adherent cells can be directly probed by AFM without immobilization since these cells can naturally attach to and spread on the substrate microbial cells can be effectively immobilized by porous polymer membrane or polyLlysine electrostatic adsorption and animal suspended cells can be trapped by microfabricated pillar arrays 153 For more descriptions of immobilization methods for AFMbased cellular morphological imaging readers are referred to the refer ences 153155 With the use of micropillar arrays Fig 6AII which are coated by polyLlysine the ultramicrostructures on the surface of single living lymphoma cells Fig 6AIII and the dynamic changes of these fine structures in response to drugs are visualized 156158 Applying AFM tip to indent the cells enables the quantitative measurements of cellular mechanical properties eg cellular elasticity cellular viscoelasticity cellu lar adhesive capability 159 and performing nanodissection on the substructures of cells such as intermediate filaments 160 allows the investigations of cellular activities in response to mechanical disruption providing novel insights into the biome chanics at singlecell level For more descriptions of singlecell mechanical analysis based on AFM readers are referred to the reference 161 AFM nanomanipulator is also able to probe the behaviors of single molecules on the surface of primary cancerous cells prepared from clinical tumor patients 162 Current singlemolecule assays are commonly performed on cell lines cultured in vitro 163 It is widely known that there are significantly structural and functional differences between Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 141 cell lines cultured in vitro and cells grown in vivo 164 and thus results obtained on cell lines cannot fully reflect the real situations taking place in vivo Hence directly probing the pri mary cancerous cells prepared from clinical patients remarkably benefits revealing the underlying mechanisms guiding cellular and molecular processes such as drug actions 165 For this goal an important issue is recognizing and isolating primary cancer cells from the clinical biopsy samples for nanorobotic manipulations With the use of a specific cell surface marker called receptor tyrosine kinaselike orphan receptor 1 ROR1 162 which is exclusively expressed on lymphoma cells but not on other types of cells cancerous cells from the bone marrow Fig 6BII of lymphoma patients can be clearly rec ognized Fig 6BIII Linking antibody molecules onto AFM tip Fig 6BI enables AFM tip to specifically locate the target molecules on cell surface by analyzing the molecular force inter actions Fig 6BV measured by AFM revealing the nanoscale spatial distributions of single target molecules on cell surface Fig 6BIV Integrating the AFM singlemolecule measure ments on primary cancerous cells with the clinical therapeu tic outcomes of lymphoma patients facilitates establishing the relationship between molecular properties of cancerous cells and clinical drug efficacies for individual tumor patients 166 which is notably meaningful for developing drug prediction methods for the coming era of personalized medicine Recent progress has shown the potentials of AFM manipu lator in investigating the regulatory role of microenvironment in cell behaviors Microenvironment plays a crucial role in the physiological and pathological processes of cells and the subse quent changes of tissues and organs 167 168 For example during tumor metastasis the thickening and linearization of collagen fibers in the tumor microenvironment facilitates the migration of cancerous cells in the extracellular matrix 169 After cancerous cells intravasate into the blood vessels platelets and components of the coagulation system support cancerous cell survival by protecting them from cytotoxic immune cell recognition 170 Cells feel and respond to the extracellular matrix such as substrate stiffness which eventually influences the behaviors of cells such as cell differentiation 171 Hence investigating the structures and functions of microenvironment significantly benefit comprehensively understanding cellular be haviors Hydrogels have been a promising material to resemble the extracellular environment of cells for diverse biomedical applications such as stem cell and cancer research cell therapy tissue engineering immunomodulation in vitro diagnostics and so on 172 Particularly adhesive hydrogels have shown great merits in applications in wet surfaces such as tissue adhesives and tissue repair 173 and it is increasingly evident that bioin spiration is now an important way for designing biocompatible hydrogels 174 Carnivorous plant sundew secretes mucilage at the end of leaves for capturing insects AFM imaging of the sundew mucilage clearly shows the nanoparticles Fig 6CI involved in the organizations of sundew mucilage 175 Peptide selfassembly is able to form hydrogels which can be metabo lized by cells AFM imaging of the peptideassemble hydro gels distinctly shows the nanofibrillar structures Fig 6CII of the hydrogels formed by peptide 176 Based on AFM force indentation technique the mechanical properties of individual nanostructures in the hydrogels single nanoparticles or individ ualnanofibrilscanbecharacterized175176whichisuseful for understanding the structures and functions of hydrogels at the nanoscale Inspired by the sundew mucilage and based on AFM characterizations hybrid biopolymeric hydrogels with threedimensional porous scaffolds Fig 6CIII are developed based on the crosslinking reactions of sodium alginate and gum arabic and experiments on cell growth show that the bioinspired hydrogels could remarkably facilitate the formation of cellular spheroids IV and V in Fig 6C 177 which is useful for investigating the cellular behaviors in threedimensional environments All together these studies 175177 show the prominent capabilities of AFM in characterizing the nanoscopic structures and properties of hydrogels which benefits the design anddevelopmentofbiomaterialsforinvestigatingtheunderlying mechanisms of cellmicroenvironment interactions VII NANOROBOTIC MANIPULATIONS BASED ON EM The development of nanomanipulator based on EM provides a novel way for robotic nanomanipulations A nanomanipulation system generally includes nanomanipulators as the positioning device kinds of microscopies as its eyes various end effectors including cantilevers and tweezers among others as its fingers and types of sensors eg force displacement tactile strain to facilitate the manipulation andor to determine the properties of the objects 178 For AFMbased nanomanipulation the slow imaging speed and small scanning area and workspace limit the manipulation throughput 179 Besides the dynamic processes between AFM tip and nanoobjects cannot be directly visualized during manipulations Combining robotics with EM yields a novel type of nanomanipulator 180 which visually fa cilitates precise and rapid nanoassembly and nanomanipulation Nanorobotic manipulation systems constructed based on EM mainly include scanning electron microscopy SEM nanoma nipulator transmission electron microscopy TEM nanoma nipulator and environmental scanning electron microscopy ESEM nanomanipulator 181 The overview of a SEM manip ulatorwith8degreesoffreedomDOFswiththreeunitsforinte gration of the TEM manipulator 182 is shown in Fig 7AI Unit 1 is used for driving the TEM manipulator and immobilizing the samples Unit 2 is used for driving the passive sample stage of the TEM manipulator and Unit 3 is used for the fixation of samples 182 With the use of EM nanomanipulator refined nanoma nipulations can be clearly performed on nanomaterials such as twodimensional and threedimensional handling of single carbon nanotubes assembly of nanodevices 178 destructive fabrication of nanostructures 180 mechanical characteriza tions of nanomaterials 182 and so on significantly promoting the studies of nanorobotics ESEMbased nanomanipulator allows the robotic manipula tions of individual biological cells ESEM which is able to work in gaseous atmosphere allows the morphological imaging of biological samples containing some moisture 183 With the Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 142 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 Fig 7 SEM nanorobotic manipulations for biomedical applications A SEM nanorobotic manipulator for handling single cells I Photo of a SEM nanorobotic manipulator Reprinted with permission from ref 182 Copyright 2006 IEEE II Images of yeast cells recorded under HV mode III Images of yeast cells recorded under ESEM mode Reprinted with permission from ref 184 Copyright 2009 IEEE B Measuring the stiffness of single yeast cells by SEM nanomanipulations I Schematic of the nanoneedle for measuring individual cell stiffness II ESEM image showing the deformation of the nanoneedle beam and cell III Magnified image of the cell indentation Reprinted with permission from ref 185 Copyright 2013 IOP Publishing Ltd C Probing the adhesion features of single yeast cells by SEM nanomanipulations I Schematic of driving AFM cantilever to detach cells with the nanomanipulator Reprinted with permission from ref 186 Copyright 2015 IEEE II III ESEM images showing the dynamic processes of detaching single cells II Before touching II During pushing Reprinted with permission from ref 187 Copyright 2011 IEEE use of ESEM yeast cells can be observed 184 Under the high vacuum HV mode 167C 303103 Pa pressure of SEM yeast cells appeared concave and broken Fig 7AII indicating the damaged morphology of cells Under the ESEM mode 0C 600 Pa pressure yeast cells appeared sphere Fig 7AIII indi cating the intact morphology of cells Based on the observations of yeast cells under ESEM mode nanorobotic manipulations can be performed on single cells to sense the mechanical properties of cells By using a nanoneedle with a buffering beam to press against a single yeast cell the stiffness of yeast cells can be measured 185 as shown in Fig 7B A narrow buffering beam at the end of the nanoneedle was used to sense the applied force avoiding the tip twist or slippage during measurement I in Fig 7B The nanoneedle was fabricated based on the commercial AFM cantilever by the focused ion beam FIB etching technique Under the actuation of the nanomanipulator inside ESEM the nanoneedle touches and deforms single cell II in Fig 7B According to the deformation of the buffering beam of the nanoneedle and the cell obtained from the ESEM images III in Fig 7B the stiffness of cells is calculated The adhesion features of single yeast cells can also be measured based on ESEM nanorobotic manipulations 186 187 as shown in Fig 7C By controlling the AFM cantilever to I in Fig 7C touch the individual yeast cell the pushing of the cell by the AFM cantilever causes the deformation of the cantilever I and II in Fig 7C If the pushing force is larger than the adhesion forces of the cell the cell detaches from the substrate and the detachment force of the cell is obtained from the deformation of the cantilever imaged by ESEM These results 184187 show that ESEM nanorobotic manipulator opens new possibilities for nanorobotic biological manipulations However it should be noted that there are significant gaps between the environment provided by ESEM and the real growth environment of living cells and so far only yeast cells have been shown to be handled in the harsh ESEM environments considerably limiting the biomedical applications of ESEM nanorobotic manipulations Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply LI et al PROGRESS IN NANOROBOTICS FOR ADVANCING BIOMEDICINE 143 VIII DISCUSSION AND PERSPECTIVE The rapid developments of nanorobotics have significantly provided novel possibilities for the medical robotics at the nanoscale Not only different prototypes of nanosized robots emerge for diverse biomedical applications including molecular machines Fig 2 6466 selfpropelled nanomotors Fig 3 8688 and DNA nanorobotics Fig 4 118120 but also nanomanipualatorsappeartobecapableofhandledifferenttypes of biological samples including nanomanipualators based on tweezers Fig 5 126 137 143 nanomanipulators based on AFM Fig 6 177177 and nanomanipualators based on EM Fig 7 184186 remarkably demonstrating the extraor dinary capabilities of nanorobotics in addressing biomedical issuessuchastransportingbiologicalsubstancesegions64 molecules 72 drugs 98 in vivo treating diseases eg can cers 119 renal damage 120 handling different types of bio logical samples eg single cells 126 185 organelles 137 exosomes 143 single molecules on cells 162 characterizing biomaterials for regulating cellular behaviors 175177 and so on Nanosized robots are particularly suited for in vivo appli cations as therapeutic carriers 188 whereas nanomanipulators are mainly suited for detecting the structures and properties of biological samples in vitro These achievements strikingly show the realization of nanorobotics from two different ways nanorobots and nanomanipulator indicating that we are mov ing closer to the scenes of nanorobotics described in science fictionsmovies eg swallowing a surgeon 27 Nanorobotics provides a novel therapeutic approach for dis eases such as tumor Nanoparticles have been widely inves tigated to deliver drug molecules to tumors but so far such nanoparticles have not proved capable of surmounting all of the biological barriers required to achieve this goal 189 and in fact the clinical translation of nanoparticles has been significantly limited 190 DNA nanorobotics allows the construction of nanorobots with tunable threedimensional structures to perform complex robotic tasks for exclusively delivering drug molecules to tumor sites 116 which will benefit the studies of oncology As a novel drug carrier experiments performed on animal mod els have shown the efficacies of nanorobots in treating tumors 119 and the studies directly performed on cancer patients in future are meaningful for evaluating the efficacies of nanorobots in tumor therapy Particularly by attaching different types of biomolecules to nanorobots as the guide nanorobots can target othercellssuchasimmunecellsforactivatingimmuneresponses 191 inspiring the studies of cancer immunotherapy Nanorobotics is a broad scientific field and here we only represent the typical progress with illustrations to show that the nanorobotics is a reality to some extent for biomedical applica tions which will have great impacts in the coming era of per sonalized precision medicine For more related topics regarding the details of nanorobotics readers are referred to the references 192198 such as imaging techniques for nanorobots 192 navigation platforms for nanorobotic agents 193 in vivo appli cations of medical nanorobotics toward clinical uses including toxicology 194 magnetic resonance imaging MRIbased nanorobotics 195 sensing for early diagnosis of cancer by nanorobots 196 selective position and control of nanorobots 197 distribution of nanorobots for in vivo applications 198 and so on It is notable that there are still evident gaps between the current nanorobotics and the requirements of biomedical com munities and there is significantly room for the advancement of nanorobotics for better biomedical applications Current nanorobots for in vivo applications mainly act as drug carri ers based on specific molecular recognition interactions 119 120Theactionsofthesenanorobotsareessentiallypassiveand uncontrollable for example the movements of nanorobots rely on the blood flow and molecular specific binding interactions are closely related to the occasionally adequate collision 199 between the specific molecules coated on nanorobots and the target molecules on tumor cells Besides current nanorobots for in vivo drug delivery to treat tumors are disposable Once the nanorobots bind to the tumors drugs encapsulated inside the nanorobots are released to kill tumor cells and then the nanorobots are degraded and cleared by the immune system 119 120 Hence strictly speaking these nanorobots are more like pharmaceuticals than machines 200 Investigating the reusing of nanorobots will benefit improving the efficiency and intelligence of nanorobots for biomedical applications For this purpose developing novel treatments such as physical treat ments 201 for nanorobots to deplete tumors at the singlecell levels will be meaningful Due to the fact that there are countless nanorobots working in vivo for treating diseases investigating the communication and cooperation of these nanorobots will benefit advancing nanorobotics and its biomedical applications Recently researchers have reported the collective control of group microrobots 202 203 exhibiting reconfigurable mul timode transformation and locomotion behaviors which will inspire the studies of group nanorobots to jointly perform special tasks for biomedical applications Robotic nanomanipulations based on nanomanipulators pro videapromisingwayfordiagnosticsandtreatmentsofcancersat thenanoscaleintheeraofpersonalizedprecisionmedicineDrug response varies between individuals owing to disease hetero geneity 204 and thus the realization of precision medicine re quires the ability to predict the efficacies of different treatments for a given patient 205 Traditional drug prediction methods are mainly based on the detection of cellular biochemical properties 206 while in fact the pathological changes taking place in the cells are often accompanied with the alterations of the physical properties of cells 207 Now it is broadly appreci ated that detecting the physics of cells benefits understanding cell behaviors 208 Consequently applying physical property detection based on nanomanipulators to drug efficacy prediction will undoubtedly promote precision medicine A critical issue needing to be addressed is the isolation and detection of biolog ical samples from clinical patients which should be labelfree and fast to maximally maintain the fidelity 209 of the isolated samples Developing automated hybrid nanorobotic systems which integrate different types nanomanipulators to combine their advantages will benefit handling biological samples with high throughput for example tweezers nanomanipulators are Authorized licensed use limited to UNIVERSIDADE FEDERAL DE SAO JOAO DEL REI Downloaded on September 012023 at 172504 UTC from IEEE Xplore Restrictions apply 144 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING VOL 68 NO 1 JANUARY 2021 suited for rapidly isolating biological samples 138 and ef ficiently manipulating biological samples 126 while AFM nanomanipulators are suited for detecting the structures and properties of samples with high spatial resolution 162 Since cancers are highly complex and many factors are related to the behaviors of cancers we may need to isolate and detect different types of biological samples besides cancerous cells eg cancer associated cells 210 immune cells 211 exosomes 212 which require that the hybrid nanorobotic systems are able to handle different types of biological samples with reliability Taken together the developments of nanorobotic systems and their biomedical applications significantly provide novel possibilities for bridging biomedicine and robotics which will have great impacts on the coming era of the convergence of biomedicine and 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