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Lipid vesicle-based molecular robots
Cite this: DOI: 10.1039/d3lc00860f

Zugui Peng,a Shoji Iwabuchi,a Kayano Izumi,a Sotaro Takiguchi,a Misa Yamaji,aShoko Fujita,a Harune Suzuki,a Fumika Kambara,a Genki Fukasawa,c Aileen Cooney,fLorenzo Di Michele, *efg Yuval Elani, *dg
Tomoaki Matsuura *b and Ryuji Kawano *a
A molecular robot, which is a system comprised of one or more molecular machines and computers, canexecute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology. The
core parts of molecular robots are fairly consistent from system to system and always include (i) a body toencapsulate molecular machines, (ii) sensors to capture signals, (iii) computers to make decisions, and (iv)
actuators to perform tasks. This review aims to provide an overview of approaches and considerations todevelop molecular robots. We first introduce the basic technologies required for constructing the core

Received 10th October 2023,
Accepted 12th December 2023
DOI: 10.1039/d3lc00860f

parts of molecular robots, describe the recent progress towards achieving higher functionality, andsubsequently discuss the current challenges and outlook. We also highlight the applications of molecularrobots in sensing biomarkers, signal communications with living cells, and conversion of energy. Althoughmolecular robots are still in their infancy, they will unquestionably initiate massive change in biomedical

rsc.li/loc

and environmental technology in the not too distant future.

Introduction
Machines equipped to perform specific actions havedisencumbered humans of exhausting labor, helping us
accomplish tasks that would be impossible with only humanhands. The revolution in mechanization began in the second
half of the 18th century and has led to previously unimaginableimprovements in humankind's quality of life. During the past
40 years, this revolution has led to a new generation ofmachines with smaller sizes, pushing the boundaries ofapplications. In the field of organic chemistry, a breakthroughmolecular catenane with two interlocked rings was reported by
Jean-Pierre Sauvage in the early 1980s,1 subsequently inspiringother scientists to develop molecular-scale machines withcomplicated functions such as rotaxanes,2 motors,3 andaDepartment of Biotechnology and Life Science, Tokyo University of Agriculture andTechnology, 2-24-16 Naka-cho, Koganei-shi, Tokyo185-8588, Japan.
E-mail: rjkawano@cc.tuat.ac.jpbEarth-Life Science Institute, Tokyo Institute of Technology, Ookayama 2-12-1,
Meguro-Ku, Tokyo 152-8550, Japanc
School of Life Science and Technology, Tokyo Institute of Technology, Ookayama
2-12-1, Meguro-Ku, Tokyo 152-8550, Japand
Department of Chemical Engineering, Imperial College London, South Kensington,
London SW7 2AZ, UKe
Department of Chemical Engineering and Biotechnology, University of Cambridge,
Cambridge CB3 0AS, UKf
Department of Chemistry, Molecular Sciences Research Hub, Imperial College
London, London W12 0BZ, UK
gFabriCELL, Molecular Sciences Research Hub, Imperial College London, London
W12 0BZ, UK

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nanocars.4 In 2016, three pioneers of molecular machines wereawarded the Nobel Prize in Chemistry, reflecting therecognition of the broad impact of molecular machines.
The concept of molecular machines has motivatedresearch activity in a novel field called “molecular robots”.5
According to the Cambridge dictionary, a robot is “a machine
controlled by a computer that is used to perform jobs
automatically”. By this definition, a molecular robot is asystem composed of molecular-scale machines andcomputers that are used to execute tasks automatically. A
living cell could be considered as one such miraculous robot
produced by nature. With DNA serving as computers toprovide solutions and proteins working as machines toperform specific functions, a living cell performs
sophisticated tasks independent of human control. Taking
inspiration from living cells, the ultimate goal of the field of
molecular robots is to artificially construct an automatedsystem capable of solving problems at the molecular level
using molecular machines and computers.6,7
A molecular robot always includes some or all of thefollowing: a body, sensors, computers, and actuators (Fig. 1).
Pioneers in the field have applied hydrogels as the body ofmolecular robots,5 however, the lack of a barrier between theembeddings and environment can lead to undesired leakage.
Lipid vesicles, which are comprised of lipid membranes
separating an inner lumen from the outer solution, provides
an alternative that could fully meet this problem. The size ofthe lipid vesicles can be tailored from nano- to micro-meterin diameter. For molecular robots, the micro-sized vesicles,

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Lab on a Chip
robots do not have a long history. In 2014, Nishimura et al.
incorporated CFPS into GUVs, and then, in the presence ofamino acids, GFP synthesis was performed as an output.18 In
2017, Sato et al. developed GUVs equipped with actuators
containing DNA clutches and microtubule motors.15 Oncelight irradiation was applied, the clutch was engaged and theshape of GUVs underwent sequential changes. These
pioneering studies provide a clear path towards molecularrobots with increasingly complex functions.
It should be noted that molecular robots sometimes
share related technologies with the field of artificial cells.
The goal of constructing artificial cells is to mimic thefunction of living cells, while molecular robots place greatvalue on developing engineering applications that could
help humans to perform tasks in micro or nanodimensions. There is therefore a drive to engineer
molecular robots with functions exceeding those of living
cells (for details on artificial cells please refer to previous
reviews19,20). This review focuses on the recent progress andchallenges in the core technologies of molecular robots andexplores emerging directions. We also highlight some
applications of molecular robots such as molecular sensing,communications with living cells, and energy conversion.
Finally, we close with a discussion on potential research
trends in this field during the coming decade.

Body of molecular robots
Fig. 1 Conceptual illustration of a molecular robot including: abody to encapsulate molecular machinery, sensors to collectsignals, computers to make decisions, and actuators to performthe tasks.

so-called giant unilamellar vesicles (GUVs), are moredesirable due to the demand for sufficient internal volume tohouse the sensors, computers, and actuators.8,9 Molecularrobots require sensors in order to detect signals in theenvironment. This can be accomplished by ion channels or
nanopores, which punch holes in lipid membranes.
Nanopores can act as a signal filter, selectively transporting
molecular signals based on their size or charge.10,11 Once thesignals are transported, they can be processed and translated
by molecular computing machinery. DNA computing,pioneered by L. Adleman in 1994,12 has evolved in recent
decades into computers applicable in molecular robots, withthe benefit of their capability to perform multiple parallel
computations.12,13 An alternative choice for the computing
machinery is cell-free protein synthesis (CFPS), enabling theoutput of proteins in vitro in response to the input of DNA.14
Actuators for molecular robots, which include DNA
nanostructures,15 peptides,16 and proteins,17 convert signals
to achieve physical movements like deformation or
propulsion.
Although constant progress has been made on theunderlying technology, studies into prototyping molecular

Lab ChipMolecular robots typically require encapsulation within acompartment, which acts as a boundary, separating theinterior from the exterior environment. Various types ofcompartments have been employed to date, including lipid
membranes,21 hydrogels,22 block co-polymers,23 DNA
droplets,24 and coacervates,25 each offering distinct
advantages and limitations. Some researchers have exploredthe formation of hybrid chassis by combining different
compartment types, leveraging the advantages associatedwith each constituent part. For instance, coacervate or DNA/hydrogel systems interfaced with lipid membranes can be
combined to enhance functionality.26–28
Among these compartment types, lipid vesicles are themost commonly used for several reasons. Firstly, they arebiomimetic, closely resembling biological membranes from achemical and morphological perspective. This characteristic
enables facile incorporation of membrane-bound molecularmachinery, including membrane proteins, nanopores, andreceptors, thereby imparting specific functionalities into themembranes.29,30 For example, the controlled flux of cargomolecules in response to stimuli,30,31 which can be used tomediate responses in living cells.32
Furthermore, lipid vesicles are chemically inert and highly
efficient at compartmentalizing large charged molecules fromthe surrounding environment, creating a chemically distinct
internal environment. This feature enables researchers to
exploit the diversity of lipid building blocks, both synthetic
and biological, to create functional membranes with diverse

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Lab on a Chip
behaviors. Examples include membranes capable of selfassembling into tissue-like structures,33 membranes that candisassemble and reassemble in response to physicochemicalcues to reshuffle material between them,34 and membranes
that release cargo triggered by light, temperaturefluctuations, magnetic fields, or biomarkers.31 Suchversatility opens up exciting possibilities for molecularrobotics and targeted drug delivery systems, among otherapplications.
Vesicles can be classified primarily based on their size andlamellarity.35 GUVs have a diameter of approximately 2 μm
and above (making them cell-sized vesicles), comprising asingle lipid membrane (as opposed to multi-layered onionlike structures known as multilamellar vesicles). Other typesof vesicles include small and large unilamellar vesicles,
which fall into the sub-micron size range, as well as multivesicular vesicles (multisomes). Additionally, there have been
intriguing examples of hybrid structures, where vesicles ofdifferent types are assembled into more architecturally
complex arrangements, such as nested36 or layered
geometries.37 A schematic of the different architectures it is
now possible to generate microfluidic techniques andprinciples in biomembrane engineering is shown in Fig. 2.
In molecular robotics, GUVs are often the go-toarchitecture due to their resolvability by optical microscopy.
Moreover, they allow for efficient encapsulation of large andhighly charged building blocks (DNA, proteins, nanoparticles,
and even whole organelles/cells),38,39 which becomes more
problematic when using vesicles in the sub-micron regime.
For instance, to encapsulate the biomolecular components
required for transcription and translation, precise
stoichiometries of plasmids, ribosomes, polymerases, andaround 40 other molecular components must be coencapsulated.40
Achievingsuch
precision
becomesstatistically improbable in lower-size regimes. GUVs thus

Perspective
provide a more suitable environment for accommodatingthese complex biomolecular systems.

Vesicle generation strategies
To generate GUVs, various methods are available, with several
comprehensive reviews published in this area.41–43 Whenevaluating the different methods, it is essential to consider
the relevant Key Performance Indicators (KPIs), which
include:(i) Encapsulation efficiency. The method's efficacy inencapsulating molecules may vary based on the type ofmolecule involved. In molecular robotics applications,achieving a high encapsulation efficiency for large, charged
species (such as DNA, proteins, nanoparticles, etc.) is crucial,
as they contribute to the system's functionality.
(ii) Monodispersity and control over size. The ability to
produce vesicles with well-defined dimensions is ofparamount importance, as size directly influences
downstream applications, and consistent sizes ensure
consistent performance.
(iii) Control of architecture and lipid content. Ideally, themethod should offer precise control over the lipid
composition of the vesicle membrane, ensuring uniformity,
and yielding unilamellar membranes (or membranes of adefined multi-lamellarity).44,45(iv) Generation throughput. For practical applications, it isadvantageous to produce significant quantities of vesicles
within a reasonable time frame to meet industrial demands
efficiently.
(v) Presence of impurities. Some applications, particularly
those related to fundamental biological studies, benefit fromvesicle membranes free of any impurities and oil.
Considering these KPIs when assessing the different GUV
generation methods will aid researchers in selecting the mostFig. 2 Schematic showing examples of different vesicle architectures that can be controllably manufactured using microfluidic techniques.

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Perspective
appropriate technique for their specific applications,ensuring optimal performance and reliable outcomes.
Classical methods of generating GUVs include gentle
hydration, gel-assisted swelling, and electroformation.35
These techniques have played a pivotal role in revolutionizing
our understanding of membrane biophysics phenomena.
They provided crucial insights into the mechanical propertiesof membranes, the influence of lipid composition, thecoupling between mechanics and membrane protein activity,
as well as the principles governing phase behavior andcoexistence of domains (lipid rafts) in cell membranes.46
However, for molecular robotics applications, these methods
have proven largely unsuitable due to their limitations. They
suffer from poor encapsulation efficiency, uncontrolled
production, and yield polydisperse populations concerning
size, architecture, cargo, and lipid composition.
In the past two decades, innovations have led to theemergence of a new class of vesicle fabrication strategies basedon water/oil emulsion technology41,47 (Fig. 3(a)). These methods
rely on the formation of water/oil droplets stabilized by lipid
monolayers. When a second lipid-stabilized water/oil interface
is deposited on top of these droplets (i.e., when they aretransferred from a bulk oil phase to a water phase), it templates
the formation of a second monolayer, resulting in the formationof a lipid membrane. These techniques are sometimes referred
to as emulsion transfer or phase transfer methods.
The simplest version of this method involves the manual
formation of a water/oil emulsion through pipetting and/or

Lab on a Chipagitation, followed by phase transfer under gravity, oftenwith the aid of a centrifuge. This process can be repeatedmultiple times to yield multi-layered arrangements.45 Thismethod is highly adaptable for translation into a
microfluidic format, where devices can be created using
soft-lithography,48,49 glass capillaries,50 or 3D printing.51 Onchip droplet production and transfer across a water/oil
interface can be achieved using microfabricated pillars52 or
flow-based approaches.53 Another approach involves using
bespoke instrumentation to enable the passage ofmonodisperse droplets through a continuously rotating
horizontal column at the water/oil interface, a method
known as cDICE (continuous droplet interface crossing
encapsulation), depicted in54 Fig. 3(b).
An alternative technique employs microfluidics to form alipid-stabilized water/oil/water double emulsion. Upon
evaporation or de-wetting of the intermediate oil phase, thisprocess leads to the formation of GUVs.49,55 The mostcommonly used version of this method employs octanol topromote de-wetting and is therefore referred to as octanolassisted liposome assembly (OLA) (Fig. 3(c)).

Advantages of microfluidics
The use of microfluidics enables high-throughput
manufacturing of monodisperse (<5% C.V.) GUVs withdefined sizes (ca. 5–200 μm diameter) and remarkably highencapsulation efficiency (approaching 100%).41 In the context

Fig. 3 Examples of different techniques used to generate Giant Unilamellar Vesicles (GUVs). a, Emulsion phase transfer, where lipid stabilised w/odroplets are taken through a w/o column through a centrifugal force, resulting in the formation of a second monolayer, and hence theestablishment of a lipid bilayer vesicle.354 b, Continuous droplet interface crossing encapsulation (cDICE) involving instrumentation which houses
concentric layers of fluids with different densities, where aqueous droplets that emerge from a capillary are driven by centrifugal force throughmultiple lipid interfaces.355 c, Octanol-assisted liposome assembly (OLA) method of generating lipid vesicles on-chip. This method involves
generating a lipid stabilised w/o/w double emulsion followed by extraction of the intermediate oil phase, leaving behind a lipid membrane.356
Figures reproduced with permission from: a, ref. 354, copyright 2023, MDPI; b, ref. 355, copyright 2016, the Royal Society of Chemistry; c, ref. 356,
copyright 2016, Nature Publishing Group.

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Lab on a Chipof creating, manipulating, and analysing molecular robotic
systems based on vesicle structures, other lab-on-chip
microfluidic modules come into play. For instance,
microstructure traps have been employed to capture arrays ofhundredsofvesicles,facilitating
high-throughput
experimentation and extended analysis.56 These traps havebeen coupled with valves to control the perfusion ofmaterials for permeation studies. Purification modules havealso been included to remove unencapsulated material anddebris from the surroundings.48
Different chip geometries can be utilized to generate
multi-compartment architectures, such as vesicles-in-vesicles
and arrangements with compartments side-by-side.50,57 By
functionalizing compartments with enzymes, DNA programs,
nanoparticles, and CFPS, various features like signallingcascades, trans-compartment communication, division, andspatial segregation of reactions have been achieved withinthese microrobots.19,20 Moreover, linking up thousands ofvesicles through adhesive membrane patches has led to thecreation of tissue-like structures with self-healing
properties.33 Related technologies include opto-fluidic
methods that employ optical trapping and laser technologies
for the on-demand fusion of compartments. These
techniques trigger biochemical reactions in femtoliter (pL/fL)
reaction vessels, study architectural transformations inducedby lipid mixing,58 and manipulate raft-like domains on thevesicle surface.59

Sensors of molecular robots
In living cells, membrane receptors, transporters, and ionchannels work as sensors to help cells respond to chemicaland physical stimuli. Such functionalities have also been
exploited to implement sensing capabilities in molecularrobots. In particular, nanopores forming stable nanoscale
openings across lipid membranes have been shown tomediate transport of large molecules, enabling theirdetection by the molecular robots.7 The opening and closingof nanopores (gating) can be regulated by environmental
stimuli (e.g., pH, light, temperature, osmotic pressure),
further enhancing the sensing capabilities of the molecularrobots. Various materials have been utilized to assemble
nanopores including proteins,60 peptides,61 DNA,11 andsynthetic materials.62 In the current section, we will mainly
focus on the characteristics, differences, and recent progressof nanopore assembly using different building materials, andwe will also describe some unique approaches that introduce
membrane receptors to GUVs. (For more details on the
introduction of membrane receptors to GUVs, we direct
readers to a review by Tosaka et al.63).
Protein nanoporesProtein nanopores are the most widely-used type ofnanopore, both in planar lipid membrane systems and lipid
vesicle systems. When reconstituted in planar membrane
systems, nanopores are powerful tools for single moleculeThis journal is © The Royal Society of Chemistry 2023

Perspective
detection. The target molecules are detected by recording thechange in ionic current output as the molecules pass throughnanopores under a constant voltage input. Since thedetection sensitivity of nanopore sensing largely depends on
the size and structure of the pores, a wide range of proteins
have been investigated for the detection of specifictargets.60,64 In 1996, α-hemolysin (αHL), with a pore size of
1.4 nm at the restriction, which is compatible with thetranslocation of single-stranded DNA (ssDNA), was initiallyemployedbyKasianowicz
et
al.
for
detectingpolynucleotides.65 In 2010, Mycobacterium smegmatis porin A(MspA) was found to be suitable for discriminating individual
nucleotide bases.66 The above pioneering works finally led to
the release of the first commercial nanopore DNA sequencer
for general use in 2015 by Oxford Nanopore Technologies,
using the E. coli curli transport channel, CsgG.67 Following
the achievement of nanopore DNA sequencing (Fig. 4(a)),
single-molecule analysis of folded proteins68–72 and aminoacid sequences in proteins73–75 are now underway. Advancessuch as the detection of single amino acid mutations in
peptides using aerolysin from Aeromonas hydrophila,76
sequential readout of peptides using MspA,77,78 andidentification of digested protein fragments using
fragaceatoxin C from Actinia fragacea (FraC)79 have beenreported in recent years.
Toward the realization of molecular robots, enormous
efforts have been made to construct a protein nanopore-lipid
vesicle system. Similar to planar lipid membrane systems,
the transport of molecules in the lipid vesicle system ishighly dependent on the size of the protein nanopores. In
addition to conventional studies using protein nanopores ofdifferent sizes to transport fluorophores, there are reports on
the use of protein nanopores to transport molecules into
lipid vesicles, including: the transmission of pH information
by a voltage-dependent anion channel (VDAC, 2.5–3 nm) fromyeast mitochondria,80 the light-/pH-dependent release offluorescent molecules by a mechanosensitive channel of large
conductance (MscL, 3 nm) from E. coli,81,82 the transport ofATP83 and UTP84 by αHL (1.4 nm) from Staphylococcus aureus,
and the translocation of T7 RNA polymerase (98 kDa) bystreptolysin O (SLO, 25–30 nm) from group A, C, and G
streptococci85 (Fig. 4(b)).
Although numerous protein nanopores with pore
diameters ranging from 1.2 to 42 nm have been reported,60
the investigation of novel protein nanopores remains
intriguing due to the need to expand the toolbox for new
target molecules. In 2023, the Maglia group reported thedetection of proteins of different sizes using an α-helicalpore-forming toxin derived from Yersinia enterocolitica,
YaxAB.86 It consists of a dimer of YaxA and YaxB, forming
assemblies of varying sizes, ranging from 8 to 12 dimericsubunits. The characteristic feature of this pore is its huge
conical shape with 15/3.5 nm cis/trans openings (Fig. 4(c)).
Notably, it is the largest protein nanopore ever used for the
analysis of folded proteins in planar membrane systems, withthe ability to capture a wide range of proteins (from 35–125

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Lab on a Chip

Fig. 4 Protein nanopores for transmitting information. a, Illustration of the mechanism of nanopore DNA sequencing.75 b, Transport of T7 RNApolymerase by the SLO nanopore in a lipid vesicle system.85 c, Molecular surface illustration of the YaxAB nanopore.86 d, Protein detection witha monobody containing tFhuA nanopores.94 e, A comparison of the crystal structure (pink) and the designed structure (gray) of a de novodesigned transmembrane eight-strand β-barrel.99 Figures reproduced with permission from: a, ref. 75, copyright 2021, Elsevier; b, ref. 85,
copyright 2021, Wiley-VCH; c, ref. 86, copyright 2023, American Chemical Society; d, ref. 94, copyright 2023, Nature Publishing Group; e, ref.
99, copyright 2021, AAAS.

kDa). YaxAB exhibits a greater electroosmotic flow undervoltage-applied conditions compared to existing nanopores,
which aids the entrapment of proteins within the nanopore.
Using YaxAB, the group successfully discriminated C-reactive
protein (CRP, 125 kDa), hemoglobin (HG, 64 kDa),
streptavidin (SA, 53 kDa), and bovine thrombin (BT, 35 kDa)
from their mixture. Moreover, CRP was detected at clinically
relevant concentrations (>2.5 mg L−1) in 160-fold diluted
depleted human serum.
It is also challenging to detect molecules that are larger
than the pore, or to selectively discriminate molecules of asimilar size and hydrodynamic radius. Binding of targetspecific moieties such as partial antibody domains,87
aptamers (single-stranded nucleic acids with the ability tobind to specific molecules),88,89 biotin (for the avidin-biotin
complex),90 inhibitory peptides,91 protein receptors,92 andgold nanoclusters,93 have been employed to achieve thedetection of large molecules,87,88,90,91 or selective
discrimination.89,92,93 Since the configuration of thedetection system changes depending on the recognition
element used, it has been necessary to engineer a distinct
system for each target protein. Recently, Movileanu andcolleagues reported a general system for the sensitive
detection of target proteins.94 In their study, the tFhuAnanopore, a β-barrel scaffold of ferric hydroxamate uptake
component A (FhuA) from E. coli, is covalently attached to amonobody, a recombinant protein based on the fibronectin
type III (FN3) domain (Fig. 4(d)). The advantages ofmonobodies are that they can be selected from a broad range

Lab Chipof variants against numerous target proteins, and theirrelatively small hydrodynamic radius that does not interferewith pore opening, unlike the larger antibody-derived
proteins. Using the monobody-conjugated tFhuA, the authors
detected human small ubiquitin-related modifier 1(hSUMO1), WD40 repeat protein5 (WDR5). Moreover, thegroup achieved the identification of the 20 nM-epidermal
growth factor receptor (EGFR), which is a 180 kDa prognostic
protein biomarker associated with lung, colon, and breast
cancer, in 5% (v/v) fetal bovine serum (FBS), suggesting thepotential applications of molecular robots in nanomedicine.
In recent years, in addition to using natural membraneproteins, attempts have been made to fabricate nanopores
through de novo design. This approach uses computer-aided
protein design to create proteins with tailored shapes andfunctionalities.95–98 De novo design allows the fabrication of
nanopores with arbitrary structures that do not exist innature, thereby expanding the range of detectable molecules.
Conventionally, de novo design of transmembrane proteins is
considered difficult due to the unclear relationship between
membrane insertion, folding mechanisms and thecorresponding amino acid sequences. In 2021, Vorobieva
et al. achieved the first de novo design of an eight-strand
transmembrane β-barrel99 through a complex optimization
process including: (i) the refinement of the loop structure
and adjustment of hydrophobicity, (ii) backbone design andsequence optimization using Rosetta,100 (iii) additional
design guidelines appropriate for the membrane
environment, and (iv) exhaustive expression trials. The

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Lab on a Chip
expressed structure was confirmed to be highly consistent
with the designed structure (1.1 Å backbone RMSD over all
residues) (Fig. 4(e)). Single-molecule detection by this protein
nanopore has not yet been reported, probably due to thesmaller pore size compared to natural nanopores. However,with the recent popularization of de novo protein design
tools,100–102 the development of protein nanopores suitable
for molecular robotics applications with better molecular
selectivity, transport capacity, stability, and durability are
expected in the future.

Peptide nanopores
As with protein nanopores, peptides that assemble into poresin the membrane are also attractive as sensors in molecularrobotics. Compared with protein nanopores, peptide
nanopores are more tractable, since stable peptide nanopores
can be designed based on naturally occurring sequences orby de novo design, and peptides with the 30–40 residues
required for membrane-spanning can be synthesized in large
quantities by chemical synthesis. Originally, antibacterial
peptides (AMPs) such as gramicidin and alamethicin were
known to assemble to form pores in bacterial membranes,
exhibiting antibacterial activity61,103–106 (Fig. 5(a)). The AMP
nanopores have been reported to enable permeation of small
molecules such as fluorophores,107–111 but it has proven

Perspective
difficult to transport molecules with larger molecularweights.112–114 In recent years, attempts have been made to
design more stable and monodispersely sized peptide
nanopores compared to AMP nanopores.
One approach to obtaining stable peptide nanopores is theredesign of the transmembrane regions of natural membraneproteins.115,116 The advantage of this approach is that no
explicit complex design principles are required, and a certain
degree of stability is expected. Redesigned peptide nanopores
have demonstrated that such transmembrane peptides stably
form pores with a certain number of monomers, opening thefield of peptide nanopores and providing promising nanoporeforming peptides to date. Recently, the Mahendran groupreported that DpPorA, which is a D-amino acid-substituted
pPorA peptide derived from the porin PorACj of
Corynebacterium jeikeium, could form a stable monodisperse
octameric pore116–118 (Fig. 5(b)). Compared to conventional
helix barrels, DpPorA is characterized by very large conductanceand is highly cation-selective, so that cyclodextrins andnonaarginine (R9) can translocate under applied voltage in aplanar membrane system, while there is negligible interaction
with anionic cyclodextrins and oligopeptides. Notably, whilst
their L counterparts are readily degraded by proteinase K,
DpPorA is highly resistant to protease K treatment, indicatingpromising potential for use as a stable sensor in molecularrobots in biological environments.

Fig. 5 Peptide nanopores for transmitting information. a, Formation of nanopores in lipid membranes by alamethicin.357 b, DpPorA peptide
sequence and CD spectra for DpPorA (blue) and LpPorA (red).118 c, X-ray crystal structure of the nanopore formed by the de novo-designed
α-helix peptide.358 d, SVG28 sequence and simulated pore structure.122 e, Utilizing CFPS to synthesize variants of SVG28.123 Figures reproducedwith permission from: a, ref. 357, copyright 2014, Hindawi Publishing Corporation; b, ref. 118, copyright 2022, Nature Publishing Group; c, ref.
358, copyright 2022, American Chemical Society; d, ref. 122, copyright 2023, Nature Publishing Group; e, ref. 123, copyright 2023, American
Chemical Society.

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Similar to protein nanopores, another approach to obtain
peptide nanopores is via de novo peptide design. Several denovo-designed α-helix peptides have been reported in recent
years,119,120 as α-helices allow for the evaluation of inter-helix
stability and the precise control of packing interfaces. In
2021, Woolfson and colleagues reported the de novo design ofan α-helix peptide that forms stable, monodispersely sizedpores.120 They began by designing a water-soluble α-helicalbarrel by introducing polar residues on the lumen of the
α-helical bundles of pentameric or higher order (Fig. 5(c)), to
support the helical interface and the passage of watermolecules, resulting in a parallel hexamer water-soluble pore.
By incorporating hydrophobic leucine and membraneinterface-preferring tryptophan residues into the outersurface of the water-soluble pore, peptides that form
monodisperse pores in the membrane were achieved. To
further improve the inter-helix packing, computationalscoring was used to fabricate possibly hexameric nanoporeswith a longer lifetime and fewer open–close states (gating).
Although single-molecule detection has not been attempted,
probably due to the small pore size, the group has
demonstrated the fabrication of water-soluble helical barrels
from 5 to 9-mers by changing the residue size at specificpositions on the helical wheel,121 leading to the possibility ofpore size expansion and greater molecular transport ability.
In 2022, Shimizu et al. reported the de novo design of
β-hairpin peptides SV28 and SVG28, which form
transmembrane β-barrel nanopores following four simple
design principles: (i) alternating hydrophilic and hydrophobictransmembrane residues, (ii) introduction of membraneinterface-anchoring residues, (iii) introduction of charged
residues for membrane insertion by electrophoresis, and (iv)
introduction of a glycine kink to reduce the strain of the
β-barrel (only for SVG28)122 (Fig. 5(d)). SVG28 forms almost
monodisperse heptameric pores under optimized conditions
and can discriminate between L-PLL (molecular weight 30 000–
70 000) and S-PLL (molecular weight 10 000). In addition, SVG28
detects cationic peptides more efficiently than the commonlyused αHL nanopore due to its terminal charge. Moreover, Fujita
et al. have found that a hydrophilic variant of SVG28 can besynthesized using CFPS, which retained the pore-forming
ability123 (Fig. 5(e)), promising easy access to peptide nanoporesfor molecular robotics. Furthermore, we showed that SV28, anantecedent of SVG28, can form a wide range of pore sizes from
1.7 to 6.3 nm and can translocate the G4 structure of DNA when
the pore size is sufficiently large. Therefore, we believe that it ispossible to find SVG28 variant sequences that form nanoporeswith a suitable size for molecular transport. Future efforts are
required not only to design assembled pore structures but also
to add vestibule regions124 and lining structures125 that stabilize
the pore structure.

DNA nanopores and receptors
DNA nanotechnology presents a unique tool for mimicking
the function of biological nanopores as sensors for molecular

Lab ChipLab on a Chip
robots, due to its advantages in design flexibility andprogrammability. Nanopores formed by DNA nanotechnology,
so-called DNA nanopores, can be realized by DNA origami
strategies which use a long single-stranded scaffold DNA andhundreds of staple DNA strands,126,127 as well as DNA
nanostructures strategies which use few short DNA
strands.128,129 To embed the DNA nanopores into lipid
membranes, the DNA nanopores carry hydrophobic
molecules such as cholesterol and tocopherol so that they
can interact with the hydrophobic membrane core. From thefirst two groundbreaking DNA nanopores that transported
ions through planar lipid membranes,130,131 in 2016,
Krishnan et al. then reported the successful transport ofsmall dye molecules through lipid membranes of GUVs usingsquare-shaped DNA nanopores with a 4.2 nm-wide channel
lumen132 (Fig. 6(a)). Since then, several DNA nanopores thatfunction on GUV membranes have been reported.133–136
The current strategies to improve DNA nanoporesconstructed by DNA origami mainly focus on two areas:
expanding the pore diameter,133–137 and adding uniquefunctions for selective transportation.133,134,137 A larger pore
not only allows larger molecules to be transported, but also
improves the efficiency of transport. In 2021, a 35 nm-wide
DNA nanopore was reported with the capability oftransporting dextran with a molecular weight of up to 250
kDa135 (Fig. 6(b)). Furthermore, in 2022, Xing et al. developed
DNA nanopores with different polygonal shapes and sizes
that permit the transport of antibodies.138 Meanwhile, adding
functions such as control over gating can provide the ability
to selectively transport molecules. To date, various methods
for controlling the gating have been demonstrated, including:modifying the pore lumen with PEG molecules,133 changing
the number of lid-like DNA strands on top of the pore134(Fig. 6(c)), and reversible opening and closing with key DNA
strands137 (Fig. 6(d)).
While DNA origami is advantageous in constructing poreswith large diameters, their versatility and stability is hinderedby the complicated design procedure. In contrast, DNA
nanopores based on DNA nanostructures have generated
great interest due to their simpler design strategy. To date,DNA-nanostructure-based nanopores with six-helix11,139–141(Fig. 6(e)) or eight-helix142 bundle structures, and wireframesof triangular143 and square144 shapes have been reported tofunction on GUV membranes. The relatively small lumen size
provides the merit of charge-selective molecular transport,
since negatively-charged DNA molecules have strong
electrostatic interactions with charged cargo molecules11,139(Fig. 6(f)). Moreover, similar approaches have been described
to control the gating of DNA nanopores with inputs including
specific
molecules,145
temperature,146
light,147
and148
mechanical stimuli.
Receptor-mimicking DNA nanostructures, which do not
puncture a pore but simply span the lipid membrane, arealso potential sensors for molecular robots. Unlike DNA
nanopores, receptor-mimicking DNA nanostructures do not
suffer from leakage issues, and hence can achieve signal

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Fig. 6 DNA nanopores and receptors for transmitting information. a, First reported DNA origami nanopore that transported dye molecules acrossthe lipid membranes of GUVs.132 b, DNA origami nanopore with the largest inner diameter.135 c, DNA origami nanopore for size-selective transport,
controlled by changing the number of lid-like DNA strands on top of the pore.134 d, DNA origami nanopore that reversibly opens and closes upon
addition of key DNA strands.137 e, DNA nanopore composed of six DNA strands.359 f, Charge selective transport of dye molecules by a six-helix
DNA nanopore.139 g, Receptor-mimicking DNA nanostructures capable of detecting ATP and lysozyme.149 h, Receptor-mimicking DNA
nanostructures capable of detecting DNA strands.150 Figures reproduced with permission from: a, ref. 132, copyright 2016, Nature PublishingGroup; b, ref. 135, copyright 2021, American Chemical Society; c, ref. 134, copyright 2021, Royal Chemical Society; d, ref. 137, copyright 2023,
Nature Publishing Group; e, ref. 359, copyright 2023, Wiley-VCH; f, ref. 139, copyright 2021, American Chemical Society; g, ref. 149, copyright2021, Royal Chemical Society; h, ref. 150, copyright 2021, American Chemical Society.

transduction with high accuracy. Typical receptor-mimicking
DNA nanostructures include two transmembrane DNA with arecognition domain, an anchor domain, and an amplification
domain. When the signal inputs arrive at the GUV outermembrane, the two transmembrane DNA hybridize with eachother and hence enter close spatial proximity. The
amplification domain consequently forms a G-quadplex
structure or complement strands, resulting in the generationof further chemical reactions inside the GUVs. To date,
receptor-mimicking DNA nanostructures capable of detecting
ATP,149 lysozyme149 (Fig. 6(g)), and DNA messengers150(Fig. 6(h)) have been reported.
All the above reports have been achieved during the past
decade. Thus, the construction of DNA nanopores andreceptor-mimicking DNA nanostructures with more
complicated functions is expected to accelerate in the next
decade. For DNA nanopores, current studies perform
molecular transport using simple diffusion, whereas active

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transport of molecules is highly desired. For receptormimicking DNA nanostructures, systems that can detect
multiple molecules and generate multiple reactions
simultaneously are yet to be realized.

Synthetic channels
Synthetic supramolecular complexes have emerged as a typeof pore capable of functioning in lipid membranes.151 These
pores are always smaller than 1 nm in diameter and thus
only transport ions, similar to ion channels in living cells.
Previous studies have referred to these pores as “nanopores”
or “nanochannels”, but without clear criteria. In a distinction
from the nanopores that can transport both ions and larger
molecules, in this review, we define pores based on synthetic
supramolecular complexes as “synthetic channels”. Thanks
to the wide chemical repertoire, synthetic channels withdifferent functions have been generated to date. A

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comprehensive introduction to this field is beyond the scopeof this review, so we will focus on the recent advances in thisfield and discuss how they can impact the field of molecularrobots (for a full survey of synthetic channels, we direct thereaders to previous reviews31,152–154).
Selective ion transport is the most well-establishedfunction for synthetic channels and is a highly desired
function for molecular robots because it affords theopportunity for strict regulation of information input. Similar
to natural ion channels,155 the selectivity of synthetic
channels is highly dependent on both the pore lumen size
and the type of functional groups in the pore lumen. Startingfrom several ground-breaking works in 1990s,156–158 synthetic
channels that can selectively transport K+,159 Cl−,160 I−,161 andH2O162 have been reported. Currently, efforts are focusing on
constructing channels that can selectively transport smaller
cations.163–165 In 2023, Zhang et al. reported a Li+-selective
synthetic channel using self-assembled aromatic molecules

Lab on a Chipwith well-adjusted lumen size and coordination numbers163(Fig. 7(a)). The group found that the channel, with a 1.8
Å-diameter lumen and four ion coordination sites inside thecavity, exhibits higher transport selectivity for Li+ over Na+,
achieving Li+/Na+ selectivity ratios ranging in value from 15.1
to 23.0. Meanwhile, activities in developing new types ofanion-selective channels continue unabated.161,166,167 In
2020, Roy et al. reported a fully hydrogen-bonded, helicallyfolded aromatic foldamer-based synthetic channel, with acentral lumen rendered by the many methyl groups161(Fig. 7(b)). The methyl groups provide a positive electrostatic
potential, resulting in a selectivity towards anions overcations. Moreover, I− was observed to be more favourablytransported than Cl− due to having fewer hydrogen bonds
than Cl− (I−/Cl− selectivity ratio was around 11).
Responsivity to external stimuli is another essentialfunction for synthetic channels and would provide the ability
for remote-control in molecular robots. Light is one of theFig. 7 Synthetic channels for transmitting information. a, Chemical structures of aromatic molecules (left). Single crystal structure of aromatic
molecules and its linearly self-assembling channel structure (central). Illustration of selective transmembrane transport of Li+ (right).163 b, Chemical
structure of foldamer (left). Side and top view of the foldamer-based synthetic channel. Illustration of selective transmembrane transport of I−(right).161 c, Schematic showing light-driven transport of metal ions and an enlarged view of the molecular motor.170 d, Chemical structure ofamphiphilic cyclophanes with perfluorinated aromatic units (left) and its response to mechanical stress with selective transport of K+.172 e,
Illustration of benzoimidazole (Bzim)-modified 5hmC-containing DNA2 translocation through a SWCNT.181 f, Illustration and single-crystal X-ray
structure of the tetrahedral MOCs (left). Transport process of amino acids through the tetrahedral MOCs (right).187 Figures reproduced withpermission from: a, ref. 163, copyright 2023, Wiley-VCH; b, ref. 161, copyright 2020, Wiley-VCH; c, ref. 170, copyright 2021, American Chemical
Society; d, ref. 172, copyright 2022, American Chemical Society; e, ref. 181, copyright 2013, Nature Publishing Group; f, ref. 187, copyright 2021,
American Chemical Society.

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most widely used stimuli due to having high biocompatibility
and ease of spatio-temporal control. So far, light-responsive
synthetic channels which are irreversibly168 and reversibly169
photo-controlled have been developed, and recent studies
focus predominantly on reversible photo-control. In 2021,
Wang et al. reported a light-driven synthetic channel that can
actively transport alkali ions170 (Fig. 7(c)). To achieve active
transport, the group included a light-driven rotary motor
around the channel. Under light irradiation, the motor
provides thermal energy to the channel to help overcome theactivation barrier necessary to translocate ions between
macrocycles, leading to an increase in Na+ transport activity(up to 400%). In living cells, mechanical stress is a major
stimulus involved in the regulation of structure and function.
In the cytoplasmic membrane, ion channels called mechanosensitive channels (MSCs) sense mechanical stress andenhance substance influx/efflux as an output. Synthetic
channels that mimic the function of MSCs to respond tomechanical stress are now attracting great attention. The firstmechano-sensitive synthetic channel was reported by
Muraoka et al. in 2017.171 Using a channel consisting ofrepeating oligo-(ethylene glycol) (OEG) and aromatic units,with subunits that destabilize the pore structure, the iontransport activity was observed to decrease with expanding
membrane tension. In 2022, the group further extended theconcept to develop a synthetic channel with potassium
selectivity by using amphiphilic cyclophanes withperfluorinated aromatic units172 (Fig. 7(d)), opening up the
possibility to process multiple signals with synthetic
channels. Other synthetic channels responding to stimuli
including voltage173,174 and the presence of ligands175,176
have also been reported in recent years.
Over the past decades, many efforts have been made toconstructsynthetic channels
employingmacrocyclic
molecules,177 foldamers,178 and π-stack architectures.171
Nevertheless, the synthesis of conventional synthetic channels
varies case-by-case, making it challenging to prepare channels
with different functions using a general protocol, and hence
restricting their use in molecular robots. Carbon nanotubes(CNTs), which are all-carbon hollow nanobarrels with adiameter in the range of 0.8–2 nm,62 are considered as aversatile synthetic channel that can be easily prepared.
Benefiting from a larger size than conventional synthetic
channels, previous reports have shown that CNTs can transport
not only ions and water molecules,179 but also larger molecules
such as amino acids180 and ssDNAs.181 Importantly, Liu et al.,utilized CNTs to selectively detect benzoimidazole (Bzim)modified 5-hydroxymethylcytosine (5hmC) in ssDNA181(Fig. 7(e)), opening the door to the use of CNT-based molecularrobots for the diagnosis of gene mutations. Moreover, a recent
study by Marcotte et al. has shown that CNTs enablemechanically-activatedionictransport
thatdepends
quadratically on the applied pressure,182 making CNTs apotential candidate as mechanical stress sensor.
Metal–organic polyhedra (MOPs), also known as metal–organic cages (MOCs), have emerged as another popularThis journal is © The Royal Society of Chemistry 2023

Perspective
option in the synthetic channel family.183 MOPs/MOCs are
nano-sized cages constructed by linking transition metalclusters with organic ligands, hence benefiting from theability to control functionality by choosing appropriate metalclusters and ligands from a wide variety of options.184 The
first report that studied the interaction between MOPs/MOCs
with lipid membranes was by Jung et al. in 2008, and theirresults suggested that MOPs/MOCs synthesized from
5-dodecoxybenzene-1,3-dicarboxylicacid(5-OC12H25mBDCH2) and Cu(CH3CO2)2·H2O have transport selectivity
dependent on cation size.185 In 2017, Kawano et al.
synthesized rhodium metal–organic polyhedrals (RhMOPs)
and demonstrated their multiple conductance states, which
are proposed to result from reversible geometry switching of
the RhMOPs, between square and triangular geometry of theapertures exposed to the aqueous phase in the lipid
membranes.186 In 2021, Li et al. developed tetrahedral MOCswith hydrogen bonding units inside the cage cavity using six
chiral BINOL-derived ligands and four n-Bu3-Cp3Zr3 clusters,
and demonstrated their ability to transport amino acids187(Fig. 7(f)). Importantly, the chiral cavity enables
enantiospecific recognition of amino acids, which is a rarelyreported function for conventional synthetic channels. The
endless potential combinations of metal clusters and ligands
make MOPs/MOCs sensors for molecular robots an excitingprospect with limitless possibilities.

Computers of molecular robots
Molecular robots go hand-in-hand with biological computers,
where biological computers take available inputs andtranslate them into appropriate outputs. Thanks to advancesin synthetic chemistry and biology, biologically derived
molecules such as DNA and proteins have become readily
available, leading to the development of biological computerssuch as DNA computing systems and CFPS.188,189 In thissection, we will briefly explain the characteristics and reveal
the research trends in DNA computing and CFPS.
DNA computing
Due to the remarkable programmability of DNA molecularbehavior based on sequence-dependent hybridization,
enzymatic reactions, and strand displacement reactions, DNA
computing has emerged as a promising candidate for thecomputational machinery of molecular robots. The genesis of
DNA computing can be traced back to Adleman's pioneering
work: massively parallel computation using artificiallysequence-designed DNA.12 He encoded the nodes and pathsin the Hamiltonian path problem onto different ssDNA to
execute hybridization-based parallel exploration of the correct
Hamiltonian path. By exploiting DNA self-assembly, thismethodology allowed large-scale computations to be
performed with low energy consumption. Besides thesubsequent implementation of mathematical computations(satisfiability problem,190,191 maximal clique problem,191
etc.), Benenson et al. constructed DNA-based finite automata

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Fig. 8 DNA computing for signal processing. a, Suggested mechanism of operation of the automaton by DNA-based state transition using the
restriction nuclease FokI and ligase (right).193 b, “Seesawing”, “thresholding”, and “reporting” DNA reactions.360 c, The decision architecture (left)of the neural network. Fluorescence levels of α, β and γ, measured in approximately 25 000 droplets (right).361 d, Illustration of the AND gate
operation using DNA droplets (left). CLSM images for droplet phase separation corresponding to four input patterns (right). Scale bars: 10 μm.212
Figures reproduced with permission from: a, ref. 193, copyright 2003, National Academy of Sciences; b, ref. 360, copyright 2012, Elsevier; c, ref.
361, copyright 2023, Royal Chemical Society; d, ref. 212, copyright 2022, Wiley-VCH.

with two states using programmed DNA sticky/blunt ends,
restriction nuclease, and ligase192 (Fig. 8(a)). The two statesin the automata run at a rate of 109 transitions per second in
an input-responsive manner, being the prototype of thesmallest biocomputer193 as certified by Guinness World
Records. Originating from the above single-information
processing systems, the development of DNA computing has
recently steered towards multiplex information processing.
As a multi-input responsive module, logic gate operations
were adopted due to the inherent simplicity in harnessing
binary information represented by ‘0’ and ‘1’. Stojanovic
et al. first demonstrated a DNA-based logic gate using
deoxyribozymes (DNAzymes).194 In their pioneering approach,
two ssDNA were defined as inputs and a DNAzyme was
employed as a computational module to catalytically cleave
DNA in a logically controlled manner, producing a distinct
output that was detected via fluorescence readout. Based on
this principle, they successfully implemented NOT, AND, and
XOR gates. This was followed by other groups that reported
the construction of OR, NOR, and NAND gates using
DNAzyme/ribozyme-based methodology.195,196
Having developed basic logic gates, the next phase of theresearch effort focussed on cascading the individual gates
towards the construction of DNA circuits. DNA strand
displacement has emerged as a pivotal technology for
implementing cascades, providing a more permissive
operating framework than the use of DNAzymes/ribozymes
that require strict experimental conditions.197 In strand

Lab Chipdisplacement reactions, an input ssDNA reacts with a doublestranded DNA (dsDNA), where it selectively binds to an
exposed single-stranded region. This binding event leads tothe displacement and release of a pre-existing hybridized
ssDNA component. In 2006, Seelig et al. proposed a way tocascade DNA logic gates using strand displacement
reactions.198 In their approach, the output ssDNA released by
the strand displacement reaction in the first layer gate was
exploited as a subsequent input for the next layer gates. By
programming short oligonucleotides to bind/release andincorporating fluorophore/quencher modification, AND, OR,
and NOT gates were cascaded without any enzymatic
reactions. The same group then extensively applied thisstrand displacement principle to construct a larger-scale
circuit consisting of AND and OR gates using 130 DNA
strands, with a combination of “seesawing”, “thresholding”,
and “reporting” DNA reactions199 (Fig. 8(b)). Importantly, thegroup demonstrated the circuit's ability to compute the floorof the square root of a four-bit binary number, enabling
digital logic networks to be compiled into DNA-based
implementations. Moreover, circuits with unique functions,for example, DNA circuits with enhanced reaction speed,
responsiveness, and robustness using the cationic polymer,
poly(L-lysine)-graft-dextran,200 in addition to temporal DNA
circuits that can respond to both the presence and history ofa molecular environment,201 and pH-responsive switchable
DNA circuits202 have all been reported. Through suchcascading, scaling, and additional functionalization, DNA

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circuits can potentially attain the capabilities of reliable
computation, precise error correction, and automated circuitcompilers, ultimately achieving a computational behaviour
reminiscent of electronic computers in wet biologicalenvironments.
Besides the binary information processing as describedabove, what types and level of complex information can DNA
computing handle? One intriguing topic is the constructionof neural networks, mathematical models that mimic theinterconnected neurons of the human brain, using DNA
molecules. Qian et al. demonstrated the translation ofarbitrary linear threshold circuits (neural network model)
into strand displacement cascades as a Hopfield network
consisting of four interconnected artificial DNA-basedneurons.203 By setting the proper weights and thresholds foreach neuron, the network was able to ‘remember’ a series ofbinary patterns, opening up possibilities for embedding‘learning’ into DNA computations. Following this remarkabledemonstration, several types of neural networks with training
processes were constructed such as winner-take-all neuralnetworks that recognized 9 patterns from 100-bit inputs,204
convolutional neural networks that recognized 32 categories
from 144-bit inputs,205 and oscillation-based reservoir
computing as a type of recurrent neural network.206 Focusing
on the practical applications of DNA-based neural networks,
decision-making functionality is useful for clinical diagnosis
in healthcare. Okumura et al. recently proposed nonlinear
decision-making using DNA-based enzymatic neurons withadjustable weights and biases. Combining a hidden layer
with two linear classifiers and a logical layer with a NOR gate,
the network correctly partitioned the concentration space ofthe two cancerous miRNAs (X1 and X2) as inputs into three
nonlinearly separable regions using distinct fluorescence (α(green), β (red) γ (blue))207 (Fig. 8(c)). As represented in thisreport, oligonucleotide biomarkers, such as miRNA, can beused as inputs in DNA computation. Diagnostic applications
are therefore an emerging trend in the development of
medical DNA computation,208,209 holding the promise of
substantial impacts on the advancement of medical
molecular robotics. Furthermore, the above DNA-basedneural network architectures offer the fascinating prospect ofendowing molecular robots with ‘intelligence’, including therealisation of intricate human-brain-like functionalities fromperception to memory and consciousness, using DNA.
In addition to the liquid-phase DNA computations
described so far, other computational methods using DNA
droplets/condensates have also attracted attention. For
instance, the Takinoue group developed DNA droplets basedon liquid–liquid phase separation of Y-motif DNA consistingof three ssDNA, whose sticky ends enabled selective andexclusive fusion of the droplets.210,211 Based on this DNA
droplettechnology,
the
group
furtherdeveloped
computational DNA droplets with AND gate functionality,
where the mixed droplet separated into two distinct droplets
only in the presence of two input DNA (= input (1, 1))212(Fig. 8(d)). Moreover, using cholesterol modification as anThis journal is © The Royal Society of Chemistry 2023

Perspective
alternative to DNA sticky ends, the Di Michele group
established DNA condensates with a responsive core-shellstructure213 and with internal domains induced by reaction–diffusion waves.214 These technologies can be integrated with
GUVs-based molecular robots as components with inputresponsive computational capabilities.

Cell-free protein synthesis (CFPS)
The current developments in the field of molecular robots
have focused on using DNA as the output for thecomputational machinery. On the other hand, the diverse
building blocks of proteins make them an attractive
alternative output, capable of increased information relay
compared with DNA. To this end, another promising tool bywhich molecular robots respond to external stimuli andconvert these into signals or outputs is CFPS. CFPS is capableof synthesizing proteins in a test tube via transcription andtranslation from DNA, without the use of living cells.215,216
CFPS can be divided into two types: a reconstituted system
and a cell-extract-based system. A reconstituted system is a
CFPS where components essential for protein synthesis arehighly purified individually and reconstituted in a test tube
to carry out the reactions, while the cell-extract-based systemis essentially a crude cell extract that contains not only theprotein translation machinery but also additional molecules
involved in other reactions. A reconstituted system has theadvantage of less contamination by factors unrelated toprotein synthesis and the possibility of tailoring thecomposition of the reaction on demand. For these reasons,
not all but many of the molecular robots consisting of CFPS
reported to date use a reconstituted system, the PURE
system,14 with its high adjustability.
Input signals for controlling the fate of molecular robots
consisting of CFPS can be divided into two major categories:
1) chemical and 2) physical signals. In response to the inputof various chemical signals, intravesicular CFPS has beenshown to synthesize proteins, most often reporter proteins.
Kamat's group has developed a sensor that expresses
luciferase in response to nitrate.217 In the bacterial twocomponent sensing system, NarX–NarL, the binding of
nitrate to the sensor domain of NarX induces
phosphorylation of NarL by the kinase domain of NarX. The
phosphorylated NarL acts as a transcriptional activator anddownstream reporter proteins are expressed (Fig. 9(a)). The
authors reconstituted the NarX–NarL system inside GUVs andshowed not only the sensing of nitrate but also that of nickel,
iron, and vancomycin is possible, by only modifying thesensor module. In addition, the same group has recently
shown the use of a fluoride-responsive riboswitch that allows
detection of the presence of fluoride in the environment bycoupling it to the expression of reporter proteins.218 Whilethe examples above use previously reported or natural
response elements, Dwidar et al. started by creating a novel
riboswitch. They created a histamine-responsive riboswitch
using SELEX. The riboswitch was encoded upstream of a

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Fig. 9 Cell-free protein synthesis system (CFPS) DNA for signal processing. a, Using a two-component sensing system (NarX–NarL) to sense
various substances.217 b, Controlling the fate of GUVs with a histamine-responsive riboswitch.219 c, Synthesis of a reporter protein using lightirradiation of ATP synthase and bacteriorhodopsin-containing GUVs.223 d, Deforming GUVs from elliptical to spherical shape by degrading CFPS
synthesized BtubA/B formed microtubes into monomers using light irradiation. Scale bars: 5 μm.224 e, Expression of a bacterial cytoskeletal proteinwith GUVs loaded with CFPS and the mechanosensitive protein MscL by exposing the GUVs to hypo-osmotic solution.225 f, Activating the CFPSsynthesized ion channel Pkd2 with osmotic stress on the membrane to enhance the influx of calcium ions. Scale bars: 10 μm.226 Figures
reproduced with permission from: a, ref. 217, copyright 2023, National Academy of Sciences; b, ref. 219, copyright 2019, American Chemical
Society; c, ref. 223, copyright 2020, Nature Publishing Group; d, ref. 224, copyright 2021, American Chemical Society; e, ref. 225, copyright 2019,
American Chemical Society; f, ref. 226, copyright 2022, American Society for Cell Biology.

reporter protein, pore-forming protein, or phospholipase,which allowed the GUVs to show fluorescence, release small
chemicals, or self-destruct, respectively219 (Fig. 9(b)). Other
examples of chemical signalling control over molecularrobots include membrane deformation of GUVs by external
supply of FtsZ synthesized by CFPS,220 a fusion of two
different vesicles by calcium and polyethylene glycol toinitiate protein synthesis inside GUVs,221 and driving an inchip and lipid-compartmentalized protein synthesis using
CFPS by supplying an energetic substance to the outside ofthe lipid membrane.222
In addition to chemical signalling, light and osmotic stresshave been used as physical input signals. Kuruma's groupsynthesized F0F1 ATP synthase and bacteriorhodopsin using
CFPS and incorporated both into small lipid vesicles to prepareenergy-generating proteoliposome (PL). Upon light irradiation,
bacteriorhodopsin created a proton gradient across the

Lab Chipmembrane, which then was used by ATP synthase for thephosphorylation of ADP to ATP. The PL encapsulated inside
the GUVs was used as an ATP regeneration system and using
the generated ATP, GFP synthesis was performed with CFPS223(Fig. 9(c)). Danelon's group showed the synthesis of BtubA/B
microtubule formation inside of GUVs, which ultimately
deformed GUVs from spherical to elliptical shapes. Light
irradiation cleaved the microtubes into monomers and restored
the spherical shape of GUVs224 (Fig. 9(d)). When MscL, amechanosensitive protein, was synthesized using CFPS inside
GUVs and osmotic stress was applied, MscL changed its
conformation and opened a pore, resulting in the influx of an
inducer which then triggered the expression of a bacterial
cytoskeletal protein225 (Fig. 9(e)). Another example synthesized
Pkd2, an ion channel, inside GUVs and upon an increase inosmotic stress, an influx of Ca2+ ions was observed via thebinding of the ion with its indicator G-GECO226 (Fig. 9(f)).

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As described above, molecular robots, in response tochemical and physical inputs, have exhibited various outputs
using CFPS as a computer. For chemical input signals, thepermeability of the chemicals plays an important role.
Membrane-permeable chemicals can directly affect the CFPS
inside the GUVs, whilst membrane-impermeable chemicals
require receptors or transporters to transmit the chemicalsignal to the intravesicular CFPS. Several examples have beenreported,217,227 however, since many input signalling
molecules are charged and have little membrane
permeability, the development of a sensing domain using
receptors or transporters is a future challenge. For physicalinputs, temperature shift, pH change, redox state, andelectron transfer are yet to be realized. As for the outputs,
many studies are limited to the expression of reporter
proteins. These are sufficient as proof-of-concept experiments
or sensor developments. However, more sophisticated output
including motility and cooperative interaction withextravehicular environments are yet to be realized.

Actuators of molecular robots
Molecular robots that achieve complicated tasks are desired to
exhibit specific physical movements such as deformation andpropulsion. So far, we have demonstrated the power of DNA
and proteins as molecular tools to construct sensors andcomputers for molecular robots. In a similar vein, DNA andproteins also provide a unique path forward in building
actuators for molecular robots.228,229 This section aims to givea brief overview of the strategies to actuate molecular robots.
DNA nanotechnology
DNA nanotechnology enables exquisite control over the
structure of self-assembled macromolecular and nanoscale
motifs.230 DNA nanodevices are however far from static andhave been engineered to reconfigure, change shape, andmove in response to a wide array of stimuli.230,231 From theground-breaking examples of DNA tweezers197 andwalkers,232 to the origami crank-sliders and joints
demonstrated by the Castro group,233 to the bioinspired
rotors built by the Dietz and Simmel groups,234,235 DNA
nanomachines and nano-actuators have demonstrated an
unparalleled ability to control different types of motion at thenanoscale. Furthermore, these nanodevices can be actuatedthrough a variety of different stimuli, from strand
displacement236 to the species and concentration of
cations,237,238 to changes in pH,237 light exposure,239 andenzymatic action.240
As discussed in the section on DNA nanopores, DNAnanostructures can be mechanically coupled to lipid
membranes using lipophilic anchors, typically cholesterol or
tocopherol.241,242 This coupling unlocks vast opportunities toengineer both the morphology and dynamic responses of
GUV-based microrobots and artificial cells throughmembrane-anchored DNA devices that imitate the functionsof membrane proteins.

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Perspective
Membrane adhesion is among the most basic functions
mediated by cell-surface receptors, underpinning a plethoraof biological processes, including motility, tissue formation,mechanosensing, and endocytosis. Exploiting the selectivityof base-pairing interactions, synthetic, membrane-anchored
DNA linkers have been used to induce and program adhesion
between lipid membranes242 (Fig. 10(a)), starting with theseminal works of Höök and coworkers,243,244 Boxer andcoworkers245,246 and Beales and Vanderlick247,248 (Fig. 10(b)).
Parolini et al. have then demonstrated the DNA-mediatedassembly of thermoresponsive synthetic tissues249 and,leveraging toehold-exchange reactions, established control
over the kinetics of tissue formation.250
Membrane fusion is another ubiquitous mechanism thatliving cells use to manipulate the structure and compositionof their lipid membranes, manifesting, for instance, inneurotransmitter release from synaptic vesicles and in theinvasion by enveloped viruses. Dynamic DNA nanostructures
have been designed to replicate the response of extant
fusogenic protein machinery, notably SNARE proteins.251
Höök and co-workers first demonstrated that zipper-like DNA
constructs anchored to membranes can induce fusion bybringing the membranes into molecular proximity,252 asshown in Fig. 10(c). The efficiency of fusion has been
observed to depend on nanostructure design, for instance thepresence of non-binding DNA spacers between the lipophilicanchors and the zipping domains, as well as on the number
and chemistry of the hydrophobic anchors and the surface
density of the DNA-zippers.253,254 Later designs have included
dsDNA “tendrils”, where the fusogenic zipping action ismediated by four-way branch migration, which were found to
improve fusion efficiency and facilitate its modulation withsoluble DNA strands255 (Fig. 10(d)). Membrane composition
can also significantly influence fusion efficiency,252,253 which
increases substantially in the presence of conical lipid
species(e.g.
DOPE)255
andforphase-separated
256
membranes.
Fusogenic DNA constructs have been applied
to engineer complex behaviors in artificial cell systems, forinstance triggering CFPS256 (Fig. 10(e)) and mediating
intricate fusion pathways that mimic cell differentiation.257
Such nanodevices have also been shown to facilitate fusion
between synthetic and biological cells for the purpose of
intra-cellular delivery258 or to generate “hybrid” cells.259
Similar to smaller nanostructures, DNA origami can belinked to membranes via multiple hydrophobic moieties.260
Owing to their larger size, stiffness, and precisely
programmable 3D geometry, membrane-bound origami canbe used to influence the morphology of synthetic lipid
membranes, mimicking membrane-sculpting proteins.
Czogalla et al. first demonstrated that, for sufficiently highcoverage, polymerizable, brick-like DNA origami can induce
large-scale deformation in GUVs, which were found to lose
their equilibrium spherical shape.260 Franquelim et al. later
showed that GUVs decorated with convex origami imitating
natural bar-domain proteins, acquired shallow invaginations
upon osmotic deflation.261 In turn, functionalization withLab ChipView Article Online

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Perspective

Lab on a Chip

Fig. 10 DNA nanostructures for actuating lipid membranes. a, Schematic of DNA linkers anchored to membranes and mediating adhesion
between lipid vesicles. Linkers may feature a double-stranded DNA spacer (top) or be fully single-stranded (bottom).242 b, Confocal micrograph of
GUVs adhering due the action of single-stranded DNA linkers (right), adapted from ref. 247. c, DNA “zipper” constructs mediating lipid vesicle
fusion.252 d, Comparison of fusion efficiency (right) from various DNA zipper and “tendril” constructs (left).255 e, Cascades of lipid vesicle fusion
reactions mediated by DNA zippers trigger CFPS.256 f, Curved DNA origami (top) influence the morphology of GUVs as determined with confocal
microscopy (bottom).261 g, Clathrin-like DNA origami triskelia of controllable curvature (top) and their TEM images (bottom).262 h, DNA origami
lineactants (left) accumulate at the interface between liquid ordered and liquid disordered domains in phase-separated GUVs (confocal projection,right) thanks to the phase-selectivity of double cholesterol (dC) and single tocopherol (sT) anchors. Scale bar: 10 μm.271 i, Virus-like DNA particle
obtained by templating the formation of a lipid vesicle around a spherical DNA origami decorated with lipids (left) and TEM images of a bare (topright) and lipid-enveloped (bottom right) origami. Scale bar: 50 nm.272 j, Lipid vesicles of controlled size templated by lipid-modified DNA-origami
rings. Images are TEM micrographs of the constructs shown on the immediate left. Scale bars: 50 nm.362 k, Liposomes captured by dynamic DNA
origami arrays. Scale bars: 100 nm.363 Figures reproduced with permission from: a, ref. 242, copyright 2019, IOP Publishing; b, ref. 247, copyright
2007, American Chemical Society; c, ref. 252, copyright 2008, American Chemical Society; d, ref. 255, copyright 2022, Royal Chemical Society; e,ref. 256, copyright 2019, Wiley-VCH; f, ref. 261, copyright 2018, Nature Publishing Group; g, ref. 262, copyright 2019, American Chemical Society;h, ref. 271, copyright 2023, American Chemical Society; i, ref. 272, copyright 2014, American Chemical Society; j, ref. 362, copyright 2017, Elsevier;k, ref. 363, copyright 2020, Wiley-VCH.

concave origami induced tubulation, which became more
prominent for increasing origami coverage261 (Fig. 10(f)).
Following a similar principle, Journot et al. designed convex
DNA-origami “triskelia” resembling clathrin proteins. The
origami triskelia could be polymerized to form a twodimensional network, producing membrane deformation
reminiscent of clathrin-coated invaginations262 (Fig. 10(g)).
Membrane-adhesion and the polymerization state of DNA
nanostructures can be externally controlled through a variety
of different mechanisms, including strand displacement
reactions,263,264
pH-responsive
non-canonicalDNA
constructs,265 light-responsive azobenzene-modified DNA
linkers,266 and by modulating ionic composition andmembrane phase.267 These tools open up vast opportunities
for controlling dynamic reconfiguration in lipid-based
microrobots and engineering biomimetic pathways forenvironmental adaptation.
The differences in affinity that different lipophilic anchors
display for co-existing lipid phases also offer a useful

Lab Chipmechanism for programming dynamic responses in DNAfunctionalised synthetic membranes.268,269 Rubio-Sánchez
et al. considered phase-separated GUVs with liquid-ordered(Lo) and liquid disordered (Ld) domains and decorated themwith DNA nanostructures using both double-cholesterol (dC)and single-tocopherol (sT) anchors, which display apreference for Lo and Ld, respectively.270 The authors
demonstrated that connecting and disconnecting selectedanchors via strand displacement could induce the redistribution of the DNA nanostructures on the surface of the
GUVs – a simple example of directed cargo transport.
Specifically, linking dC and removing sT would cause thedevices to accumulate in the Lo phase, while removing thedC and bringing back the sT would trigger migration to
Ld.270 In a subsequent contribution, Rubio-Sánchez et al.
noted that DNA origami plates functionalized with both dC
and sT, arranged at opposite ends of the plate, showed thetendency to accumulate at the line-interface between Lo and
Ld (Fig. 10(h)). These DNA-origami line-actants (DOLAs) wereThis journal is © The Royal Society of Chemistry 2023

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Lab on a Chipshown to reversibly stabilize small lipid domains against
coalescence, establishing a route to control the surface
patterning of GUVs. DOLAs were also shown to provide
control over a biomimetic membrane-fission pathway,
whereby budding-off of DOLA-stabilised domains could be
induced upon de-activating the line-actants under
hyperosmolar conditions271 (Fig. 10(h)).
When the overall size of the lipid vesicles one seeks toengineer approaches the nanoscale, DNA origami can beused to directly template the formation of vesicles with
precisely controlled size and shape. This concept was first
proposed by Perrault and Shih, who used spherical, lipidmodified DNA-origami to template the formation ofmonodisperse lipid vesicles (diameter ∼76 nm)272 (Fig. 10(i)).
The lipid vesicles would form around the origami following
lipid addition and removal of an initially present surfactant.
The lipid envelope was found to protect the DNA
nanostructure from enzymatic degradation – a useful featurefor envisaged in vivo deployment.272 A similar strategy has
been pursued by Yang et al. using DNA-origami rings ofvarious diameters that, thanks to a lipid-modified inner edge,

Perspective
could template the growth of size-controlled lipid vesicles in
a Saturn-ring-like geometry273 (Fig. 10(j)). Zhang et al. later
demonstrated that similar DNA-origami exoskeletons could
be polymerized, generating arrays of lipid vesicles with
controlled inter-membrane distance, or tubular vesicles274(Fig. 10(k)). The devices could also be actuated to induce
lipid vesicle fusion into and bending of the lipid tubes.274 In
addition to providing control over lipid architectures, lipid
vesicle-templating DNA origami has also been applied toprecisely position molecules onto membranes and study
reaction kinetics with absolute control over stoichiometry.275

Protein and peptides
Owing to their high affinity to lipid membranes, proteins andpeptides act as promising actuators in molecular robots, inthe same way that they deform or propel living cells.
Deformation could allow molecular robots to overcome
complex terrains. Various proteins/peptides have beenreported to deform GUVs.17,276–283 Membrane deformation
can be classified into two broad categories: outward

Fig. 11 Proteins and peptides for actuating lipid membranes. a, GUV deformation triggered by phase separation of FUS LC adhered on the lipid
membrane. Scale bars: 10 μm.279 b, GUV deformation triggered by de novo peptides.285 c, Propulsion of ATPase-coated GUVs.286 d, Light-induced
propulsion of a phase-separated GUVs driven by local peptide nanofibre growth (top) and the photocleavage reaction of a DNA-peptide conjugate(bottom).289 e, Illustration of the biochemical process inside OLVs. Vesicle fusion is triggered by the interaction between peptide K and peptide
E.364 Figures reproduced with permission from: a, ref. 279, copyright 2021, National Academy of Sciences; b, ref. 285, copyright 2018, American
Chemical Society; c, ref. 286, copyright 2019, American Chemical Society; d, ref. 289, copyright 2018, Nature Publishing Group; e, ref. 364,
copyright 2021, Royal Chemical Society.

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Perspective
deformation and inward deformation. Inward deformation isconsidered more energetically unfavorable because it leads tomembrane expansion.284 As a consequence, most of theprevious studies focus on outward deformation. However,inward deformation is also an attractive route towards
molecular robots with increased functionality, such as
endocytosis-like membrane fission. In 2021, Yuan et al.
reported inward bending of the membrane of phaseseparated GUVs of membrane-bondable N-terminal lowcomplexity domain of fused in sarcoma (FUS LC) proteins279(Fig. 11(a)). Meanwhile, Yu et al. designed a de novo
amphiphilic bola-type peptide composed of lysine andleucine that regulates inward budding of lipid membranes285(Fig. 11(b)).
Propulsion could help molecular robots to reach theirvarious destinations. One strategy to perform propulsion is tomake use of the diffusive movement of enzymes. In 2019,
Ghosh et al. showed that GUVs with ATPase-tagged lipid
membranes exhibited enhanced mobility in the presence of
ATP.286 In 2023, Jin et al. demonstrated that horseradish
peroxidase (HRP)-decorated Janus GUVs undergo directionalmotion in the presence of H2O2, while such enhanced
mobility is absent from homogeneously enzyme-decorated
GUVs287 (Fig. 11(c)). Another attempt reported by Kurakazu
et al. involved GUV propulsion using motor protein flagellaisolated from Chlamydomonas.288 Interestingly, the mobility
was further enhanced when the GUVs were coated withannexin. On the other hand, there are no reports of peptidebased GUV propulsion, with the need for peptides to convert
chemical energy to kinetic energy.
Morphological change with a combination of proteins/
peptides and DNA is also of great interest owing to the highprogrammability and versatility of DNA. A groundbreaking
work by Sato et al. reported a system using kinesin-DNA
complexes to deform GUVs. The group showed that kinesin
can be attached to lipid membranes using a light-triggered
DNA displacement reaction, leading to a continuous shapechange of the GUVs driven by microtubule sliding on themembrane.15 Meanwhile, Inaba et al. have developed asystem in which propulsion is achieved by partial growth of
peptide nanofibers on a phase-separated membrane.289 In
their system, photocleavable peptide-DNA complexes are
specifically conjugated to the lipid-disordered membrane.
Upon exposure to light, the peptides were released from themembrane and locally self-assembled into nanofibers,
resulting in autonomous GUVs propulsion (Fig. 11(d)).
Unique applications have been realised when combining
protein/peptide
actuators
withmulti-compartment
architecture, such as vesicles-in-vesicles. Chen et al.
engineered ‘artificial beta cells (AβCs)’ that sense glucose insolution and release insulin by membrane fusion.290 They
first prepared insulin-encapsulated inner small vesicles(ISVs) with a membrane composed of lipids, peptide(peptide K), and pH-sensitive polyethylene glycol (PEG)
conjugates which detach from the membrane at a low pH
environment. A mixture of ISVs, glucose oxidase, andLab ChipLab on a Chip
catalase are then encapsulated in outer large vesicles (OLVs)
containingalipid
membranewithpeptideE(complementary to peptide K) and glucose transporters.
When glucose was transported from the outside to theinside of the OLVs, a low pH environment was generated
due to the glucose oxidase and catalase reaction. The pH
decrease triggered PEG detachment from the membrane,
and consequently, the unshielded peptide K formed coiled
coils with peptide E, promoting the fusion of two vesicles
and the release of insulin (Fig. 11(e)). We anticipate future
research to develop in the use of proteins/peptides to form
complex structures, fine-tuning of morphology changes, andcooperative interaction with each other.

Applications of molecular robots
Robots that can extend the reach of human engineering
capability in the microscale are in high demand. Compared
to micro-sized robots using alloys,291,292 oxide minerals,293,294
and ceramics,295 molecular robots are biocompatible andbiodegradable due to their use of biological materials. This
also affords versatility in biological environments. At thesame time, the large toolbox of sensors, computers, andactuators, as mentioned above, enables them to execute
multiple and complicated tasks which are challenging foralternatives such as hydrogel-based microswimmers.296,297
Additionally, the leakage issues for such microswimmers areresolved in molecular robots owing to the lipid membrane
functionality as a semipermeable barrier. Accordingly,
molecular robots have exhibited great potential in various
applications as a promising alternative to conventional
microrobots. In this section, we will provide some
representative examples of emerging state-of-the-art
applications of molecular robots.
Detection of molecules
Small toxic molecules (≤1000 daltons), for example, heavy
metals and organochlorine, are well-known pollutants inwater. Though spectroscopic methods such as inductively
coupled plasma atomic emission spectroscopy and mass
spectrometry are powerful for molecular detection, therequirement of real-time monitoring of the local molecularconcentrations cannot be met with such methods inprinciple.298 On the other hand, molecular robots withsensors and computers may be able to fill this gap. Previous
studies have reported that GUVs with nanopores are usefulfor the detection of K+,56 Cu2+,299 and Cl−.300 Recently, aunique strategy using GUVs with a DNA nanopore and circuit
offered by Peng et al. suggested switchable detection of Sr2+
(ref. 141) (Fig. 12(a)). In the presence of ATP, the DNA
nanopore switches from closed to open, which enhances theinflux of environmental Sr2+. The Sr2+ sequentially triggers a
set of confined downstream cascade reactions to output botha fluorescence signal and switching of the nanopores back to
the closed state. Another pioneering work that uses CFPS
inside GUVs to detect fluoride was demonstrated by Boyd

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Lab on a Chip

Perspective

Fig. 12 Molecular robots for detection of molecules. a, Detection of Sr2+ by GUVs with a DNA nanopore and circuit (left), as observed by theincrease of 488 fluorescent signals from SG-I in the presence of ATP and Sr2+ (right).141 b, Illustration of the GUVs designed to detect sodium
fluoride (top) and its absorbance over time in response to in vitro samples (bottom left) and real-world samples (bottom right).218 c, Using
nanopores and DNA computing to detect five types of miRNAs (left) from histograms of the unzipping time of each miRNA pattern (right).304 d,
Illustration of DiffusiOptoPhysiology (DOP) method (left). Comparison of the signal of Fluo-8 with/without dsDNA in the solution (right).310 Figures
reproduced with permission from: a, ref. 141, copyright 2020, Nature Publishing Group; b, ref. 218, copyright 2023, AAAS; c, ref. 304, copyright2022, American Chemical Society; d, ref. 310, copyright 2019, AAAS.

et al.218 In their system, the membrane-permeable sodium
fluoride (NaF) initializes the riboswitch-mediated synthesis ofcatechol (2,3)-dioxygenase (C23DO), which subsequently
catalyzes the conversion of its colorless substrate, catechol, totheyellow-colored2-hydroxymuconate
semialdehyde,
constituting a colorimetric response (Fig. 12(b)). Importantly,
they successfully showed the possibility of detecting fluoride
in real-world samples from Lake Michigan and the Evanston,IL municipal tap water supply. Though the sensitivity ofmolecular robots remains inferior to that of spectroscopic
methods, they provide the opportunity to probe in detail theconcentration of small molecules in a biologicalenvironment. We infer that research into other novel
applications, for example, real-time monitoring of thepollutants in aquatic life, is also underway.301
Biomarkers such as DNA, microRNAs, and peptides areanother essential target for molecular robots. So far, we havereported that nanopores have a strong synergy with DNA
computing to allow for highly sensitive detection.13,302–304 In
our most recent study, we proposed a system for patternrecognition of five types of miRNAs overexpressed in bile

This journal is © The Royal Society of Chemistry 2023

duct cancer (BDC) with an αHL nanopore, diagnostic DNA,and a planar lipid membrane304 (Fig. 12(c)). With our system,
we succeeded in the label-free detection of miRNA expression
patterns from the plasma of BDC patients at sub-femtomolar
concentrations. On the other hand, detecting peptides using
nanopores is more challenging due to peptides' relatively
small size and heterogeneous charge compared withpolynucleotides. During the last decades, academic efforts
have been invested into designing mutant nanopores305,306 or
optimizing solution conditions307,308 to address the above
problems. However, there remains a long way before therealisation of peptide detection with amino acid resolution.
Meanwhile, biomarker detection with GUV-based molecularrobots also likely faces an arduous path to development. Liuet al. reported GUVs that detect environmental DNA
messengers,150 while Barba-Bon et al. have used GUVs tomonitor the transport of cell-penetrating peptides.309
Nevertheless, the sensitivity and amount of information
conferred using fluorescence-based detection is several times
lower compared with electrical recording technology.
Recently, the Huang group has reported an electrode-free

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nanoporesensingsystembyaso-called
DiffusiOptoPhysiology (DOP) method, which opticallymonitors fluorescence emission resulting from diffusive
binding of Ca2+ with its indicator dye Fluo-8 through ananopore on a planar lipid membrane by total internalreflection
fluorescence,anddetected
dsDNA310,311(Fig. 12(d)). Accordingly, the combination of GUV-based
molecular robots with new technology (for example, singlemolecule localization microscopy312 and stimulated emission
depletion microscopy313) is worth exploring to facilitate
higher levels of detection sensitivity.

Communication with living cells
Engineering communication between molecular robots andliving cells would provide novel strategies to address issues
in fields such as drug development or bioremediation.
Communication is a process of signal production andtransmission. Attempts have been made towards both DNA
computing256,314,315 and CFPS316,317 for signal processing.

Lab on a Chip
The use of such systems can greatly improve the complexityand programmability of signalling, endowing molecularrobots with the potential of performing complicated tasks.
For instance, Adamala et al. constructed two populations oflipid vesicles that contain different genetic circuits called“sensor liposomes” and “reporter liposomes”.317 When themembrane-permeable arabinose (Ara) was applied to the bulk
solution, αHL expression started in the sensor liposomes,
consequently enabling the release of the non-membranepermeable activator β-D-1-thiogalactopyranoside (IPTG). The
IPTG was then recognized by the reporter liposomes,
triggering the output of luciferase (Fig. 13(a)).
Signal transmission of molecular robots can beaccomplishedbynanopores
including
αHL,317–320
321,322316
melittin,and perfringolysin O (PFO).
Importantly,
the nanopores not only facilitate the transmission of signals
but can also cause leakage problems of other encapsulated
molecules. For this reason, heptakis(2,3,6-tri-O-methyl)-betacyclodextrin (TRIMEB), which is noncovalently bound into
the lumen of αHL, is sometimes introduced to prevent theFig. 13 Molecular robots for communication with living cells. a, Modular design of genetic circuit interactions within and between GUVs.365 b,
Controlling the distance between GUVs with light-oxygen-voltage 2 protein. Scale bar = 25 μm.326 c, Schematic representation of communication
between GUVs with bacteria (top). GFP is expressed only when the GUVs are exposed to UV light (bottom). Scale bar = 200 μm. Scale bar (bottom
right) = 20 μm.329 d, Schematic representation of communication between GUVs with neural stem cells (top). GUVs function to enhance stem celldifferentiation, as revealed by the increase in the percentage of bIII-tubulin-overexpressing neurons (bottom). Scale bar = 50 μm.316 Figures
reproduced with permission from: a, ref. 365, copyright 2020, Royal Chemical Society; b, ref. 326, copyright 2019, Wiley-VCH; c, ref. 329,
copyright 2023, Nature Publishing Group; d, ref. 316, copyright 2020, AAAS.

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Lab on a Chipleakageofencapsulated
content.319,320
Meanwhile,
considering the approximation diffusion equation t = L 2/2D
(here, t is the elapsed time since signal transmission began,
L is the distance from the GUVs to the target, and D is the
diffusion coefficient), the distance between the GUVs also
plays an important role in signal transmission. To this end,
significant advances have been made in recent years toengineer membrane adhesion with DNA nanostructures323
and proteins.319,324,325 Notably, the Wegner group showedthat membrane adhesion could be controlled with the use of
an improved light-induced dimer protein based on the lightoxygen-voltage 2 (LOV2) domain from Avena sativa, opening
up a new horizon for designing efficient communication
networks319,325,326 (Fig. 13(b)).
The above progress has paved the way for the realizationof communication between molecular robots and living cells.
Quorum sensing, which is the communication behavior ofcells to detect and respond to cell population density, hasbeen extended to molecular robots and bacterial
populations.327–329 Remarkably, Smith et al. reported lightactivated GUVs that could perform quorum-sensing-based
communication with bacteria329 (Fig. 13(c)). The system isachieved by installing the T7 promoters of DNA templateswith photocleavable biotinylated (PCB) and monovalent
streptavidin (mSA) that could impede T7 RNA polymerase
from binding to the T7 promoter. When exposed to UV light,
the mSA was liberated, resulting in the expression of the BjaI
enzyme followed by the production of N-isovaleryl-Lhomoserine lactone (IV-HSL) in GUVs. As the bacterial
populations contained a gene circuit highly sensitive to IVHSL, they responded to the IV-HSL signal with resultant GFP
expression. This work sets the stage for the development of aremote-controlled molecular robot for communication withliving cells. Meanwhile, communication between molecularrobots and mammalian cells provides another area ofinterest. A remarkable work by Kaneda et al. reported thatcalcien dye could be transferred from Connexin43 (Cx43)expressing GUVs to cultured Cx43-expressing U2OS cells
through the gap junction.330 Recently, Toparlak et al.
demonstrated GUVs that can drive mammal neuraldifferentiation.316 The GUVs contained transcriptiontranslation machinery and DNA templates that coded forbrain-derived neurotrophic factor (BDNF), LuxR, and PFO. In
the presence of N-3-oxohexanoyl homoserine lactone (3OC6HSL)–responsive transcriptional repressor LuxR, PFO was
expressed and assembled into nanopores in lipid
membranes, enabling the release of BDNF. After 19-day
coincubation with mouse embryonic stem cell-derived neuralstem (mNS) cells, the group successfully observed thecapability of GUVs to enhance the neural differentiation andmaturation, with an increase in pan-neuronal and matureneuronal markers βIII-tubulin and microtubule-associatedprotein 2 (MAP2) (Fig. 13(d)). We anticipate that future efforts
will be geared toward adapting multiple molecular robots
and living cell populations to build communication networks
with higher complexity.

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Perspective
Conversion of energy
The operation of sensors, computers, and actuators inmolecular robots requires energy sources, hence ATP issometimes encapsulated in GUVs or added to the external
solution.141,316 Moreover, such molecular robots may also
provide solutions to global energy issues. In a livingorganism, energy production is powered by a transmembrane
proton gradient, meaning that the membrane-spanning ATP
synthase turns ADP into ATP only when there is a proton
gradient.331 In thylakoids, this proton gradient is caused by alight energy-driven proton pump. Accordingly, it has attracted
the interest of scientists to incorporate ATP synthase andproton pumps into GUVs to build a self-fuelling system. A
groundbreaking work in this field was reported by Lee et al.
in 2018.332 The group built an artificial organelle containing
ATP synthases and two light-driven proton pumps,
photosystem II (PSII) and proteorhodopsin (PR), andencapsulated it into GUVs. The PSII is a proton pump thatcan be activated by red light, whereas PR proton pumping is
mainly initiated by green light. Moreover, due to the pHdependent bidirectional proton-pumping ability of PR, PR
and PSII work in conjunction to increase the proton gradientat low pH, but counteract the action of PSII at high pH. This
makes it possible to facilitate or impede ATP synthesis with
exposure to different colors of light. The ATP-dependent
polymerization of cytoskeletal proteins was used to confirm
ATP synthesis. As expected, the red light facilitated and thegreen light impeded the growth of cytoskeletal proteins,
responding to the operation of PR and PSII. Moreover, thepolymerized cytoskeletal proteins showed the ability to
deform GUVs with an adjusted lipid composition (Fig. 14(a)).
A similar idea was demonstrated by Berhanu et al.223 Insteadof cytoskeletal protein reaction, successful ATP synthesis wasmonitored by GFP expression in a CFPS.
In eukaryotic cells, energy conversion mainly occurs in the
mitochondria. Li et al. encapsulated mitochondria extracted
from C6 glioma cells into GUVs and showed ATP synthesis
with the addition of pyruvate which is a trigger molecule tostimulate mitochondria to produce ATP333 (Fig. 14(b)).
Though this work paves the way to develop mitochondriabased self-fuelling systems in GUVs, it remains difficult toconstruct an artificial mitochondrion from scratch, due tothe complexity of the citric acid cycle and the electrontransport chain. Another fascinating work demonstrated by
Hicks et al. inserted carbon nanotubes in GUVs to mimic
voltage-dependent anion-selective channels, which were able
to transfer electrons via redox upon a change in potential.334
The group showed that a 1.5 V voltage stimulation triggers
the reduction of gold chloride to solid gold deposits on the
carbon nanotube terminus (Fig. 14(c)). This work opens up
the potential for nanopore transistors, able to convert
electrical energy to chemical energy in molecular robots.
Subsequent attempts in this field focus on improving theenergy conversion efficiency to accelerate the development ofmolecular robots towards practical applications.

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Perspective

Lab on a Chip

Fig. 14 Molecular robots for conversion of energy. a, Facilitating or impeding the ATP synthesis with different light color exposure. (left) Opticalstimulation couples ATP synthesis with ATP-dependent actin polymerization and morphological change of the GUVs (top right). Confirmation of
the formed cytoskeletal proteins and their impact on membrane deformation with microscopy (bottom right). Scale bar = 20 μm.366,367 b,
Mitochondrion-containing GUVs' response to pyruvate addition (top). Time-dependent process of cytoskeletal proteins polymerization triggeredby the addition of pyruvate (bottom). Scale bar = 10 μm.333 c, Schematic showing polarization of carbon nanotubes within lipid membranes,
triggering the reduction of gold chloride to solid gold deposits (left). Observed deposition of gold on GUVs (right). Scale bar = 20 μm.334 a, ref.
366, copyright 2023, Frontiers Media and ref. 367, copyright 2020, Wiley-VCH; b, ref. 333, copyright 2022, Wiley-VCH; c, ref. 334, copyright 2022,
Wiley-VCH.

Conclusion
The multi-functionality, programmability, and controllabilityof building materials for molecular robots engendermolecular robots with superior potential to other types of
microrobots. However, constructing molecular robots is notan easily achieved task, as it requires not only parts with highquality but also great synergy between each part. In thisreview, we have introduced several recent advances in thecore parts of molecular robots. Looking to the future, whatshould be the direction of development for these core parts?
The lifetime of a GUV is reported to be up to several
months,335 in contrast to the 10 to 20 years' average lifetimeof industrial robots,336 reflecting a great hurdle in the
successful implementation of molecular robots. Adding asecond component to the lipid membranes, for example,
polymers337,338 or surfactants,339 could make a giant leap indeveloping GUVs with higher stability. However, these
impurities would also change the permeability of lipid
membranes and impair the functionality of the sensors,
computers, and actuators. A comprehensive list of suchcomponents with their advantages and drawbacks will haveto be obtained using both experimental and in silico

Lab Chipapproaches. In addition, while the water/oil emulsion
technology-based strategy is the leading contender for GUV
fabrication, adding polymers and surfactants may change theinterfacial tension between water and oil, making GUVs
fabrication difficult. Consequently, new GUVs fabrication
methods must be continuously explored.
For nanopores, future improvements should be directed
towards increasing their homogeneity and imparting
selectivity. So far, we have shown that each of the building
materials for nanopores provides unique benefits: proteins
are highly compatible with lipid membranes, peptides areeasy to synthesize, DNA offers high programmability, andsynthetic materials offers strong operability. Accordingly,
combining these materials has gained great interest as aunique strategy to achieve homogeneity and selectivity.
Recent reports have demonstrated methods to tune thediameter of protein340,341 and peptide342,343 nanopores using
DNA nanostructures/origami as a scaffold. Such DNA
nanostructures could also impart selectivity to the protein/
peptide nanopores, whilst the incorporation of synthetic
materials is also worth exploring.
Building a molecular computer that can perform tasks in
an intelligent way like a human being is the ultimate goal for

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Lab on a Chip
molecular robotics. The first step towards intelligence is theability to learn. Neural networks are the backbone of deeplearning algorithms, and a neural network capable of 144-bit
pattern recognition with a two-layer implementation function
has already been achieved using DNA computing.205
However, research into DNA computers with the capability tolearn remains in the early phase due to the challenges inscalability (this is discussed in detail in a survey by Nagipogu
et al.344). Consequently, new methods to address thescalability problems are highly desired. On the other hand,
computers based on CFPS are still in their infancy. Whilstarithmetic operation in digital computers is based on a
combination of logic gates, the use of protein logic gates israrely reported. One difficult issue is the limited pool ofbuilding blocks due to the complicated interaction between
proteins. Recently, Chen et al. reported a de novo design ofthree-input protein logic gates, giving careful consideration
to the interaction between each building block.345 Rapid
advances in de novo design will bring out the full potential of
CFPS as computers for molecular robots, and the great
diversity in the types of functional outputs of CFPS may leadto new breakthroughs in computational intelligence.
Despite the significant progress in actuating lipid
membranes with DNA nanostructures/origamis and proteins/
peptides, most of the reported methods rely on
uncontrollable thermal fluctuation-dependent molecular
processes, hence restricting their operability. Applying acontrollable external field may help meet this challenge, as
achievements in the actuation of DNA nanostructures/
origamis with both electric346 and magnetic fields347 haveprogressed in recent years. The incorporation ofelectrochemically, magnetically, or acoustically sensitive
chemical groups would allow development of increasingly
superior actuators for molecular robots.
Improving the cooperation between sensors, computers,
and actuators is the key to realizing the potential ofmolecular robots. Previous molecular robots mainly utilize
open-loop control systems, meaning that the output
generated by computers or actuators does not affect the input
from sensors. Though the simplicity of such an open-loop
control system offers many benefits, its poor accuracy limits
the reliability of molecular robots. In conventional
mechanical engineering, closed-loop control systems are
known to improve accuracy, and we argue that the same ruleof-thumb should be valid for molecular robots. Only alimited number of molecular robots utilizing closed-loopsystems have been reported. In one example, Peng et al.
developed a molecular robot that could detect Sr2+ with a
DNA computer while regulating the Sr2+ input from DNA
nanopores141 (this is also discussed in the section on
Applications of Molecular Robots). Usage of sensors,
computers, and actuators that can precisely control thefunction of each other is expected to become mainstream in
molecular robots' development.
Another challenge that needs to be addressed is how tomaintain molecular robots' sustainability. The working

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Perspective
principles of current molecular robots follow theminimization of free energy, limiting the lifetime of theirfunctionalities. Looking ahead toward a versatile molecularrobot, the ability to sustain non-equilibrium conditions
where energy and materials are continuously supplied ishighly desirable. As we discussed earlier, the introduction of
a self-fuelling system may solve the energy supply issue(readers are referred to the section on Applications ofMolecular Robots for details). On the other hand, therequirement to supply new material can be addressed byutilizing membrane fusion. Zhao et al. have demonstrated
that CFPS in GUVs can be triggered by the fusion of GUVs
and large unilamellar vesicles encapsulating DNA
plasmids.348 Enabling sustained out-of-equilibrium operation
represents a critical step towards the deployment ofmolecular robots for practical application.
Looking at the success of currently reported applicationsof molecular robots, how can these applications be further
expanded in combination with other technologies? Wearable
sensors, which are electronic devices that can be comfortably
worn, provide promising approaches to monitor physiologicalinformation in real-time.349 To provide sensing selectivity
and specificity, biological systems such as livening
engineered bacteria350 and CFPS351 have been incorporated
into wearable sensors. Compared with previous systems,
molecular robots can combine both the functionality of livingorganisms and the durability of CFPS, making them a strong
candidate for new wearable sensors. Another likely trend will
be using molecular robots in the atmosphere to performtasks such as monitoring atmospheric pollution or delivery of
specific substances. While this may seem to run contrary to
the principle of molecular robots, due to the absence ofaqueous media, we consider the soap bubbles as a potential
solution to address this issue because they are stable in theatmosphere and can trap molecules in their wall. Inspired bysome interesting studies that used soap bubbles to detect
methamphetamine in aerosols352 or to pollinate flowers,353
we expect that such soap bubble-based molecular robots
incorporating sensors, computers, and actuators can berealized in the near future.
In conclusion, the molecular robot is the fruit ofnumerous
technologicalprogressesinbiophysics,
biochemistry, and systems engineering. With an increasing
number of reports on new findings in these areas, we believe
the development of molecular robots will be further
accelerated, opening the door to a new era in robotics.

Author contributions
Z. P. and R. K. conceptualized the article. Z. P. prepared theinitial draft of section: Introduction, Applications ofMolecular Robots, and Conclusions. Y. E. prepared the draftof section Body of Molecular Robots. Z. P., S. I., M. Y., and S.
F. prepared the draft of section: Sensors of Molecular Robots.
T. M., S. T., G. F, and F. K. prepared the draft of section:
Computers of Molecular Robots. L. D. M., Z. P. and K. I.

Lab ChipView Article Online

Perspective
prepared the draft of section: Actuators of Molecular Robots.
Z. P., H. S. and A. C. edited the manuscript with expert
guidance, suggested edits, and supervision from R. K.

Conflicts of interest

Open Access Article. Published on 19 January 2024. Downloaded on 1/19/2024 8:46:10 AM.
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There are no conflicts to declare.

Acknowledgements
R. K. acknowledges support from JSPS (19H00901, 21H00390,
21K19786,
and21H05229)
andfrom
JST-CREST(JPMJCR21B2). T. M. acknowledges support from JSPS
(22K21344 and 21H05228) and from Human Frontier Science
Program (RGP003/2023). Y. E. acknowledges support from a
UKRI Future Leaders Fellowship (MR/S031537/1) as well as
BBSRC grant BB/W00125X/1. L. D. M. acknowledges support
from a Royal Society University Research Fellowship(UF160152, URF\R\221009) and from the European ResearchCouncil (ERC) under the Horizon 2020 Research andInnovation Programme (ERCSTG No 851667 NANOCELL).

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