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Cite this: Mater. Adv., 2021,
2, 6428

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Catalase-driven protein microtube motors with
diﬀerent exterior surfaces as ultrasmall biotools†
Mizuki Umebara, Natsuho Sugai, Kohei Murayama, Tomonao Sugawara,
Yushi Akashi, Yoshitsugu Morita,
Ryo Katoand Teruyuki Komatsu *This report describes synthesis of catalase-driven protein microtube (MT) motors with diﬀerent exterior
surfaces, highlighting their abilities of bacteria capture, reaction enhancement by self-stirring, andvelocity control with light irradiation. Common precursor MTs with an internal wall of avidin (Avi MTs)
were prepared using template synthesis with layer-by-layer (LbL) assembly in a track-etchedpolycarbonate (PC) membrane. Subsequently, (i) avidin–biotin complexation immobilized biotinylated
catalase in the liberated MTs, and (ii) the outside wall was covered with lectins, enzymes, or Au nanoparticles (AuNPs). The designed MTs of four kinds (1.2 mm outer diameter, 24 mm length) were selfpropelled in aqueous H2O2 solution powered by O2 bubble ejection from the terminal opening. The
swimming (concanavalin A)-wrapped MTs captured Escherichia coli bacteria. The self-stirring motion of(a-glucosidase)-coated MTs promoted the hydrolysis reaction of a-glucoside linkage. The similar(horseradish peroxidase)-covered MT motors used H2O2 as both a propulsion fuel and an oxidizing

Received 14th July 2021,
Accepted 8th September 2021

agent. Furthermore, the velocity of the swimming AuNP-bound MTs can be modulated by visible light

DOI: 10.1039/d1ma00610j

irradiation. Photothermal heat generation by AuNPs increased the Cat enzyme activity. Notably,proteases digested the four MTs, demonstrating sufficient biodegradability. These results showed that

rsc.li/materials-advances

the catalase-driven MT motors with different outer surfaces act as ultrasmall moving biotools.

Department of Applied Chemistry, Faculty of Science and Engineering,
Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.
E-mail: komatsu@kc.chuo-u.ac.jp; Fax: +81 3 3817 1910; Tel: +81 3 3817 1910
† Electronic supplementary information (ESI) available: Experimental details,
Fig. S1–S5 and Videos S1–S4. See DOI: 10.1039/d1ma00610j

a powerful enzyme capable of H2O2 dismutation, is an attractive
alternative to Pt catalyst. Several pioneering achievements of
Cat-powered MTs have been reported.22–26 Sanchez et al. fabricatedAu-based MT motors in which Cat was immobilized by covalentbond.22 Wang et al. prepared polymer/Au-Cat MT motors that
respond to various organic and inorganic toxins.23 Nonetheless,they still show poor biocompatibility. He et al. synthesized conicalprotein microrockets containing gelatin hydrogel with Cat.24 Free
diffusion of H2O2 into the gel produced O2 bubbles to swim. The
rockets, however, showed a limited lifetime for bubble generation.
Furthermore, light-guided and driven MT motors have attracted
considerable attention due to their potential in a variety of
applications.5,27–31
Template-assisted synthesis using alternate layer-by-layer(LbL) assembly in a track-etched polycarbonate (PC) membrane
is a beneficial procedure to create smart MTs comprising soft
materials.32–41 Aqueous solutions of biopolymers such as
polypeptides, DNAs, and proteins are filtered through the
PC membrane and are deposited onto the pore walls.
Subsequently, dissolution of the template in organic solvent
provides uniform hollow cylinders. An important benefit of thisstrategy is that one can readily manipulate the interior wall bychanging the final filtration material. Over the last decade,we demonstrated that various protein-based MTs and nanotubes (NTs) show unique functionalities such as molecular

6428 | Mater. Adv., 2021, 2, 6428–6438© 2021 The Author(s). Published by the Royal Society of Chemistry

Introduction
Swimming microtube (MT) motors with autonomous
propulsion have been of particular scientific interest during
the last decade1–12 because of their potential utility as pollutant
removers,13,14 separation devices,15–18 and analytical sensors.19–21
Most synthetic MT motors possess an inner wall of platinum (Pt)thin film, where disproportionation reaction of H2O2 (2H2O2 2H2O + O2) takes place. They are then powered by continuous
jettingofO2
microbubbles
fromtheopen-end
terminus.1,2,8,12,13,15–17,19–21 Such noble metal pipes were generally
preparedusingarolling-up
procedurewithphotolithography1,13,15 or electrochemical deposition in a porous
membrane.2,8,16,17,19–21 An ultimate challenge for the MT motors
is their implementation in life science applications such as ondemand drug carriers and as diagnostic microchip elements.
However, the stiff MTs involving Pt walls are not biodegradable,
which is the most important feature for biological and biomedical
applications. Catalase (Cat.; Mw, 240 kDa; isoelectric point (pI), 5.5),

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Papertransport,37 virus trapping,38,39 bacteria killing,40 and molecularconversion.41 Furthermore, creation of a Cat wall in the capillary
allowed the protein MTs to swim in aqueous H2O2 by jetting O2
bubbles from the terminal opening.26 The obtained hollow
cylinders are digested properly by proteases, demonstrating agood biofriendly nature. To confer another task to the swimming
protein MTs, the outer surface wall of the tubule must be used. It
is nonetheless limited to change of the exterior surface in theinitial step of template synthesis. Therefore, we first fabricatedprecursor MTs using LbL assembly in the porous PC membrane.
Thereafter (i) the interior surface of the released tube was coatedwith Cat using avidin (Avi)–biotin complexation in aqueous
medium, and (ii) the exterior surface was wrapped with desired
materials using electrostatic interaction.
As described herein, we present the synthesis of protein MT
motors having a Cat engine layer as an internal wall and diﬀerent
exterior surfaces (1.2 mm outer diameter (O.D.), 24 mm tube length(T.L.)) (Fig. 1). Moreover, we emphasize their diverse functionalities
with autonomous propulsion powered by H2O2 fuel. The swimming
tubes covered with lectin bioreceptor can capture and release
Escherichia coli (E. coli) bacteria. The enzyme-wrapped MT motors
facilitated the catalytic reaction by their self-stirring motion. The
velocity of the swimming MTs coated with gold nanoparticles(AuNPs) was modulated by light irradiation. The stratiform tubular
walls were decomposed by proteases. Our findings constitute a newchemistry of soft protein MT motors that can become a platform of
ultrasmall biotools for future biomedical applications.

Results and discussion
Synthesis of protein MTs with Cat interior surface (Cat MTs)
For our template synthesis using electrostatic LbL assembly ina microporous PC membrane, the first layer adhered on the

Materials Advances
negatively charged pore wall must be positively charged
material.7,33,37 However, the lectin, enzymes, and AuNP that
we would deploy on the exterior surface of the MT possess total
charges of negative. In addition, the Cat layer deposited at the
inner surface of the tube was denatured during the PC
membrane dissolution in DMF.26 Given the diﬃculty of
preparing the designed MTs directly by template-assisted synthesis,
we used a two-step procedure combining (i) electrostatic LbL
deposition in the PC membrane and (ii) LbL coating in aqueous
solution (Fig. 1 and 2). First, the human serum albumin (HSA)based precursor MTs containing an Avi (Mw, 68 kDa; pI, 10.0–10.5)
internal surface, PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi MTs (AviMTs), were prepared in the PC membrane (Fig. 2A) [HSA as the
major component, Avi as an innermost layer, magnetite NP (MNP)
as the magnetic active layer, poly(L-arginine) (PLA) as a positively
charged glue, and poly(L-glutamic acid) (PLG) as a negatively
charged glue]. After etching the PC template in DMF solution,
the released Avi MTs were freeze-dried, affording a lyophilized
pale-yellow powder. Scanning electron microscopic (SEM) images
showed the formation of uniform hollow cylinders with 1.14 
0.04 mm O.D. and 138  8 nm wall thickness (W.T.) (Fig. 2B).
The maximum T.L. (approximately 24 mm) coincided well with thepore depth of the PC membrane used (Fig. 2C). We proposed a
16-layer model. Under an assumption that the sizes of PLA, PLG,
HSA, and Avi are 8.7, 11.0, 8.0, and 6.0 nm, respectively, from ourearlier reported studies,26,37 the W.T. of the tube is estimated as
136 nm. This value is close to the observed width of the dried MT(Fig. 2B).
Second, specific immobilization of Cat onto the internal Avi
layer was conducted using Avi–biotin bonding in aqueous
medium (Fig. 2D). Avi can bind four biotins with tremendously
high affinity (K 4 1015 M1).42 The lyophilized powder of Avi MTs
was suspended in phosphate-buffered saline (PBS) solution (pH7.4, +150 mM NaCl) by ultrasonication. Thereafter, biotinylated
Cat [bCat; ten biotin arms were introduced on the surface lysine
residues of Cat]26 was added and the mixture was incubated for
3 h. Before the experiments, we confirmed that the enzyme activityof Cat was unaltered by the introduction of biotin groups.26
To prevent nonspecific adhesion of bCat to the positively charged
exterior surface of the tube, the NaCl concentration was increased
to total 287 mM. Removing the unbound bCat by centrifugation
conferred the PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs(Cat MTs).
ConA-covered Cat MTs (ConA/Cat MTs) and capture of E. coli

Fig. 1 Protein MT motors with a Cat engine layer as an internal wall and diﬀerent
exterior surfaces (concanavalin A, ConA; a-glucosidase, aGD; horseradish peroxidase, HRP; and Au nanoparticle, AuNP). PLA/HSA/MNP (PLA/HSA)5PLA/PLG/Avi/
bCat MT, Cat MT; (PLA/HSA)7PLA/PLG/Avi/bCat MT, Cat2 MT (poly(L-arginine),
PLA; magnetite nanoparticle, MNP; and poly(L-glutamic acid, PLG).

First, we explored to capture bacteria using the bubblepropelled MT motors. Gram-negative bacteria such as
Escherichia coli (E. coli) possess lipopolysaccharides (LPS) on
their outer membranes. Concanavalin A (ConA; Mw, 104 kDa;
pI, 4.5–5.5), a Ca2+/Mn2+ dependent lectin extracted from
Canavalia ensiformis (Jack-bean), binds certain structures ofvarious sugars and glycolipids, mainly terminal a-D-mannosyl
and a-D-glucosyl residues.43 This lectin is known to recognize
the specific terminal carbohydrates of LPS on E. coli surface andto form multiple valent binding.44 Wang et al. reported bacteria
isolation using polymer-Pt MT engines.16 The ConA was© 2021 The Author(s). Published by the Royal Society of Chemistry

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Fig. 2 (A) Synthetic scheme of a precursor PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi MT (Avi MT) using LbL deposition in microporous PC membrane.
(B and C) SEM images of Avi MTs. (D) Synthetic scheme of Cat MT by LbL coating of Avi MT with Cat in aqueous solution.

immobilized on the tube surface by gold–thiol chemistry.
Quantitative evaluation, however, has never been done. For
this study, we designed ConA-wrapped Cat MT motors that cancapture E. coli while swimming in aqueous medium.
The Cat MTs were redispersed in 4-(2-hydroxyethyl)piprazineethanesulfonic acid (HEPES) solution (pH 6.8, 10 mM,
including Ca2+ and Mn2+). Then, the negatively charged ConA wasadded to cover the exterior surface of the tubes. After incubationfor 30 min, the unbound ConA was removed by centrifugation,yielding ConA/PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs(ConA/Cat MTs) (Fig. 1). Transmission electron microscopic(TEM) image of the air-dried sample revealed that the MTs swelledconsiderably in water. The W.T. became 283 nm (Fig. 3A). Because
the dimensions of the proteins and MNP are constant, thepolypeptide (PLA or PLG) layer thickness would be 25 nm, whichis greater than that observed in the freeze-dried sample of Avi MT(Fig. 2B). We ascertained the swelling ratio of the polypeptide
layer as 2.5. This value was found in the range of normal
polyelectrolytes (1.2–4.0).45,46
To ascertain the ConA coverage rate of the tube surface, weused fluorescent Cyanine 5.5 (Cy5.5)-labeled ConA (CyConA).
The mixture of Cat MTs and CyConA was centrifuged to spin
down the tubes. The fluorescence intensity of the supernatant
was 39% of that observed for the identically treated CyConA
solution without the tubes. This result implied that the numberof adhered CyConA molecules per tube was approximately 2.3 
108 and that the coverage rate of the MT surface with CyConA
was 55%. Moreover, similar tubules were prepared using
fluorescein-labeled bCat (FCat) as an internal wall. Confocal
laser scanning microscopy (CLSM) images of the CyConA/FCat
MTs exhibited dual fluorescence emission (Fig. 3B). We
inferred that (i) the homogeneous immobilization of bCat on
the interior wall was achieved through strong avidin–biotin

interaction and (ii) the CyConA molecules are adhered on theexterior wall of the tubules.
The ConA/Cat MTs were self-propelled in aqueous H2O2
solution by jetting O2 bubbles from the open end terminus
with average velocity of 60  13 mm s1 (2.5 body-lengths per s)(pH 6.8, 2 wt% H2O2, 0.1 wt% Triton X-100) (Fig. 3C, D andVideo S1, ESI†). The disproportionation of H2O2 was catalyzed
by the innermost bCat engine layer. The continuous O2 bubble
expulsion from one end is responsible for autonomous
propulsion. After the first bubble exits one of the terminations,a one-way liquid flow of H2O2 fuel is generated in the onedimensional (1-D) channel. The swimming trajectories of theMTs mostly show a turning motion, which is attributed to slight
diﬀerences of the tube’s body symmetry and mouth shape. The
structure was suﬃciently robust to endure the continuous
expulsion of O2 bubbles. Our MT motors retain their bubble
ejection capability for 6 h at 2 wt% H2O2. This stability
contrasts with the fact that the conical protein microrockets
with Cat lose their bubble propulsion ability within 10 min.24
One possible explanation is that the enzyme inhibitors such ashydroxyl radical are washed out from the channel of the tubes
with water flow. We reasoned that the hollow cylinder structure
is more preferred for biocatalytic O2 bubble propulsion for long
period. The tubes including MNP layer can be collected easily
by the magnet-field assistance. By bringing a neodymium (Nd)magnet (468 mT) close to the vessel, the swimming ConA/Cat
MT was attracted by the magnet.
For bacteria capture experiments, we exploited a geneticallyengineered E. coli in which green fluorescent protein (GFP) is
secreted (F-E. coli) (Fig. S1, ESI†). Before the experiment, we
ensured that the majority of F-E. coli can survive (remain viable
and culturable) after exposure to the working solution containing 2 wt% H2O2 fuel and 0.1 wt% Triton X-100 used in our

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Materials AdvancesFig. 3 (A) TEM images of ConA/Cat MT. (B) CLSM images of CyConA/FCat MT (left – lex, 633 nm for CyConA; right – lex, 488 nm for FCat). (C and D)
Snapshots taken from Video S1 (ESI†) of microscopic observations of self-propelled ConA/Cat MT by jetting O2 bubbles in HEPES solution (pH 6.8, 2 wt%
H2O2, 0.1 wt% Triton X-100) at 25 1C under bright field mode. (E) Residual rate of E. coli after incubation with self-propelled ConA/Cat MTs. Results wereascertained using the fluorescence spectral intensity of GFP in the remaining F-E. coli. (F) Schematic illustration of catch and release of E. coli on theexterior surface of a bubble-propelled ConA/Cat MT.

experiments for 15 min (ESI†). The Con A–LPS binding allows
the tube to capture F-E. coli regardless of their bacterial viability
or calturability. To the HEPES dispersion of self-propelled
ConA/Cat MTs (pH 6.8, 2 wt% H2O2, 0.1 wt% Triton X-100),
the F-E. coli suspension was added. The ConA/Cat MTs were
swimming in the bacteria suspension by spouting out of O2
bubbles. The mixture was incubated for 15 min under dark
conditions at 25 1C. Later, the tubules were collected using a
Nd-magnet. The supernatant was centrifuged to precipitate theunbound F-E. coli, which was redispersed in HEPES solution.
The fluorescence intensity of the dispersion was only 20% of thatobserved for the identically treated F-E. coli without the tubes(Fig. 3E). The 80% removal of F-E. coli from the sample solution
was achieved by single treatment with the swimming MTs (Fig. 3F).
The average number of F-E. coli adhered on the exterior surface wascalculated as 3.9 per tube. Additional experiments were conductedwith control MTs. Incubation with the non-swimming ConA/PLG
MTs, which were always sinking at the bottom, showed 94% fluorescence intensity relative to the control value (Fig. 3E). Furthermore,
the HSA/Cat MTs, which have no ConA wall, exhibited 90% of thecontrol. Our results demonstrated that self-propelled ConA/MT
motors capture F-E. coli efficiently and showed that the swimming
motion plays an important role in accelerating the collision
frequency to bind the bacteria.

© 2021 The Author(s). Published by the Royal Society of Chemistry

Once F-E. coli attached to the swimming ConA/Cat MT, it
became tightly bound onto the cylinder and never was shaken
oﬀ. To release the captured F-E. coli, mannose was added. It isnoteworthy that eﬃcient unloading of bacteria occurred byaddition of D-(+)-mannose to the tube dispersion. Dissociationof F-E. coli–ConA complex caused the elevation of fluorescenceintensity to 78% of that for the total captured F-E. coli.
We concluded that (i) the specific binding between the ConA
surface of the tube and LPS surface of F-E. coli is solely responsible
for the capture. Also, we concluded that (ii) autonomous
propulsion is crucially important to increase the molecular
contact frequency and to promote bacteria catching. The selfpropelled ConA/Cat MTs can act as ultrasmall removers andseparators for E. coli.
Enzyme-covered Cat MTs and self-stirring biocatalyst
The next challenge in using bubble-propelled MT is tocreate self-stirring catalysts. Immobilization of an enzyme onto
the outer surface would allow the MT motors to havebiocatalytic activity. The self-propulsive motion is anticipatedto facilitate the reaction. The a-glucosidase from Saccharomyces
cerevisiae (aGD; Mw, 68.5 kDa; pI, 4.0–5.5) is a typical exo-type
carbohydrase to cleave the a(1 - 4) bond at the non-reducing
terminal end of the substrate to release an a-D-glucose

Mater. Adv., 2021, 2, 6428–6438 | 6431

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Materials AdvancesPaper(eqn (1)).47
aGD

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RO-a-D-glucose þ H2 O ! ROH þ a-D-glucose(1)

The aGD was added to the PB dispersion (pH 7.0) of Cat
MTs and the mixture was incubated for 30 min. The MTs
were collected using a Nd-magnet and the supernatant was
discarded, yielding aGD/PLA/HSA/MNP(PLA/HSA)5PLA/PLG/
Avi/bCat MTs (aGD/Cat MTs) (Fig. 1). TEM image of the
air-dried sample revealed that the MTs swelled in water.
The W.T. of the MT was 283 nm (Fig. 4A), which is identicalto that of ConA/Cat MT. The aGD coverage rate of the tube
surface was determined using fluorescein-labeled aGD (FaGD).
By attaching a Nd-magnet to the vessel, the FaGD/Cat MTs
were attracted at the bottom. The supernatant fluorescence
spectrum was measured to ascertain the concentration ofunbound FaGD. The fluorescence intensity declined to 30%of that for the identically treated FaGD solution without the
tubes. This result signifies that the number of adhered FaGD
molecules per tube was approximately 1.9  106 and that thecoverage rate of the MT surface with FaGD was 30%. A CLSM
image of the FaGD/Cat MTs showed wrapping of the outside
wall of the cylinder by FaGD (Fig. 4B). The aGD/Cat MTs werealso self-propelled in aqueous H2O2 solution by jetting O2
bubbles from the open end with average velocity of 68 
15 mm s1 (2.8 body-lengths per s) (pH 7.0, 2 wt% H2O2,
0.1 wt% Triton X-100) (Fig. 4C, D and Video S2, ESI†). The
swimming behavior with a turning motion resembled that of
ConA/Cat MT.
As a fluorogenic substrate, we used 4-methylumbelliferyl-aD-glucopyranoside (MUGlc) (Fig. 4E). The reaction product,
4-methylumbelliferone (MU), is a coumarin compound showing
fluorescence at 447 nm. Upon addition of MUGlc to the PB
solution (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100) of free aGD,
fluorescence (lem, 447 nm) and UV absorption (l, 370 nm)
increased gradually, demonstrating the generation of MU(Fig. 4F). The MU concentration reached 50 mM after 15 min.
Later, identical experiments were conducted using aGD/Cat MT
motors. After adding MUGlc to the PB dispersion of swimming
tubes (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100), we observed afluorescence increase at 447 nm, from which the MU concentration was calculated. It reached maximum 30 mM after 50 min(Fig. 4F), representing that the enzyme activity of the swimming
aGD/Cat MTs was lower than that of the free aGD. This restrictedactivity might be attributable to the sparse distribution of theaGD molecules on the tube surface with statistic geometries. We
presumed a four-part model of aGD in which an active site
locates in one of the four parts (Fig. S2, ESI†). The probability of
the active site facing the aqueous solution is half (50%) or
three-fourths (75%), with an average of 63%. This presumably
engenders lower enzyme activity. The result is consistent
with our earlier reported data for the protein MTs and NTs with
an aGD surface.12,48 The same reaction using non-swimming
aGD-covered aGD/PLA/HSA/MNP(PLA/HSA)5PLA/PLG MTs (aGD/
PLG MTs) showed considerably low activity (‘‘Non-stirring’’ inFig. 4F). This low activity was expected, but surprisingly, stirringof the reaction mixture of MUGlc and aGD/PLG MTs using amagnetic stirrer (300 rpm) did not promote the reaction(‘‘Stirring’’ in Fig. 4F). It is noteworthy that the product MU
concentration was equivalent to that with the non-stirring aGD/
PLG MTs after 50 min. Two causes can be reasonably inferred.
The aGD/PLG MTs including MNP layer were absorbed tightly
onto the magnetic stirrer bar. Thereby, (i) collision between aGD
and substrate occurred less frequently, and (ii) aGD immobilized

6432 | Mater. Adv., 2021, 2, 6428–6438© 2021 The Author(s). Published by the Royal Society of ChemistryFig. 4 (A) TEM image of aGD/Cat MT. (B) CLSM images of FaGD/Cat MT(lex, 488 nm). (C and D) Snapshots taken from Video S2 (ESI†) of microscopic observations of self-propelled aGD/Cat MT by jetting O2 bubbles in
PB solution (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100) at 25 1C underbright field mode. (E) Hydrolysis reaction of fluorogenic substrate MUGlc
to generate MU. (F) Time course of MU concentration in the PB solution(pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100) of MUGlc with swimming aGD/
Cat MTs or non-swimming aGD/PLG MTs at 25 1C.

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Materials Advanceson the tube was denatured momentarily by rubbing with highspeed mechanical rotation.
Next, horseradish peroxidase (HRP; Mw, 40.2 kDa; pI, 3.0–
9.0) was used as a surface enzyme of the MT motor. Actually,
HRP, a heme-containing enzyme, catalyzes the oxidation of
widely various organic substrates using H2O2.49 The high turnover rate of this protein allows generation of a strong signal in ashort time. The characteristic color of the products can beobserved readily using simple spectrophotometry. For thatreason, HRP is ideal for many applications and is commonlyused in enzyme-linked immunosorbent assay (ELISA) andimmunohistochemistry. Because our working solution of the
Cat MT motors contains 2 wt% H2O2, the synthesized tubules
are expected to use H2O2 as a propulsion fuel in the hollow
cylinder and also as an oxidizing agent on the outside enzyme
surface.
The PB solution of HRP was added to wrap the exterior
surface of Cat MTs. After incubation for 30 min, the MTs werecollected using a Nd-magnet. The supernatant was removed,
yielding HRP/PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs(HRP/Cat MTs) (Fig. 1). The TEM image of the air-dried sample
exhibited 281 nm wall thickness (Fig. 5A), which is similar tovalues found for ConA/Cat MT and aGD/Cat MT in a swelledstate. To ascertain the surface covering rate with HRP, we
exploited Cy5.5-labeled HRP (CyHRP). From the mixture of
Cat MTs and CyHRP, the CyHRP/Cat MTs were excluded usinga Nd-magnet. Then the fluorescence of the supernatant wasmeasured. The fluorescence intensity declined to 34% of thatfor the identically treated CyHRP solution without the tubes.
The number of adhered CyHRP molecules per tube wasascertained as approximately 4.8  106. The coverage rate of
the MT surface with CyHRP was 59%. The zeta potentials of theAvi MT and Cat MT were observed respectively at 48.0 mV and
52.0 mV. These results manifested that the exterior surfaces of
the cylinders indeed consist of positively charged PLA layer.
In marked contrast, the zeta potential of HRP/Cat MT wasfound to be 10.0 mV, implying that the wrapping by HRP
inverted the surface charge of the tube to negative. Although
HRP used for this study was a mixture of isoforms with wide
range of pI, 3.0–9.0, the acidic components are likely to bindthe tube by electrostatic interaction. The obtained HRP/Cat
MTs swam smoothly in 2 wt% aqueous H2O2 medium with aturning motion (Fig. 5B and Video S3, ESI†). The swimming
speed was 66  13 mm s1 (2.8 body-lengths per s). The
difference in exterior surface (ConA, aGD, or HRP) did notaffect the catalytic performance of the inside wall of the
MT motor.
Numerous substrates for HRP have been developed andcommercialized. As the first chromogenic substrate, we chooseo-phenylenediamine (OPD, Fig. 5C), which is used extensively
in HRP-mediated ELISA.50 The soluble product is an orangebrown 2,3-diaminophenazine (DAP) with strong absorption at
417 nm. Upon addition of OPD to the swimming HRP/Cat MTs
dispersion (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100), thecolor changed immediately to yellow. The H2O2 in the medium
was consumed as both fuel for autonomous propulsion andoxidizing agent. After 20 min, the tubules were collected using a
Nd-magnet. The visible absorption spectrum of the supernatant
was measured. The absorption intensity at 417 nm suggested
that the DAP concentration reached 38  2 mM, which is 62% of
the value observed for the free HRP solution in an identicalsetup (Fig. 5D). This restriction percentage is exactly the same© 2021 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2021, 2, 6428–6438 | 6433

Fig. 5 (A) TEM image of HRP/Cat MT. (B) Snapshot taken from Video S3(ESI†) of microscopic observations of self-propelled HRP/Cat MT by jetting
O2 bubbles in PB solution (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100) at
25 1C under bright field mode. (C) Oxidation reactions of chromogenic
substrates, OPD and OD. (D) Product DAP concentrations in the PB
solution (pH 7.0, 2 wt% H2O2, 0.1 wt% Triton X-100) of OPD withswimming HRP/Cat MTs and non-swimming HRP/HSA MTs at 25 1C.
(E) Product BAB concentrations in the PB solution (pH 7.0, 2 wt% H2O2,
0.1 wt% Triton X-100) of OD with swimming HRP/Cat MTs and nonswimming HRP/HSA MTs at 25 1C.

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Paper

Not only lectin, enzymes, but also metal nanoparticles are able tofunctionalize the Cat MT motors. AuNPs possess photothermal
heating ability. For example, they are developed as suitableagents for thermal destruction of cancer cells.52 The photothermal property of AuNP rely on its ability to convert theabsorbed light into heat. The red color of AuNPs is attributed
to a localized surface plasmon resonance (LSPR) in which theconduction electrons on the surface of the NP oscillate inresonance with incident light. He et al. reported theultrasound-driven AuNP functionalized NTs for photochemical
perforation of a cancer cell.31 The Cat demonstrates maximum
activity at 37 1C, which is the optimal temperature of the
enzyme.53 We previously reported that the velocity of Cat MT
motors was regulated by varying the enzyme activity with pH andtemperature of bulk aqueous solution.26 If one can enwrap
the exterior surface of Cat MTs with AuNPs, light irradiationengenders heat generation to increase the enzyme activity of Cat,
which might then accelerate the swimming tubes.

The citrate-reduced AuNPs with 40  3 nm diameter weresynthesized according to our earlier reported procedure.54 The
aqueous AuNP solution showed a characteristic SPR band at
530 nm (Fig. S3, ESI†), which is well consistent with datareported elsewhere.55 The zeta potential of the AuNP was34.0 mV, implying the surface charge is suﬃciently negative.
The (PLA/HSA)7PLA/PLG/Avi/bCat MTs (Cat2 MTs) without
MNP layer were prepared from (PLA/HSA)7PLA/PLG/Avi MTs(Fig. S4, ESI†) and used as parent tubes. The MNP layer was
excluded to prevent unfavorable adsorption of the generated
heat. The Cat2 MTs were suspended in aqueous AuNP solution
to wrap the outside surface of the tube. After incubation for
30 min, the unbound AuNP was removed by centrifugation,yielding AuNP(PLA/HSA)7PLA/PLG/Avi/bCat MTs (AuNP/Cat
MTs) (Fig. 1). From the absorption spectrum of the supernatant
including unbound free AuNP (Fig. S3, ESI†) the average
numbers of the AuNPs immobilized onto the one tube were
estimated as 2.0  104. The coverage rate of the MT surface
with AuNPs was calculated as 30%. The TEM image of the
AuNP/Cat MT showed homogeneous distribution of AuNPs on
the cylindrical wall (Fig. 6A). Careful inspection of the TEM
picture revealed that the wrapping rate of the tube surface withthe particles was 33% (Fig. 6B), which is almost equivalent tothe value from the UV-vis absorption measurement. The densityof the AuNP distribution in Fig. 6B appears to be higher than
33%, probably because the picture shows all particles absorbed
on both-side of the flattened tube on the TEM grid. The AuNP/Cat MTs were self-propelled in PB solution with H2O2 (pH 7.0,
2 wt% H2O2, 0.1 wt% Triton X-100) by jetting O2 microbubbles(Video S4A, ESI†). The average velocity of 53  12 mm s1(2.2 body-lengths per s) was markedly less than that of parent
Cat2 MTs (66  11 mm s1, 2.8 body-lengths per s). Wrapping of
the surface with heavy AuNPs increased the tube weight andslowed it down.
Movement of the AuNP/Cat MT was observed under brightfield mode with light irradiation using a Hg lamp through afluorescence mirror unit (Ex, 530–550 nm/Em, 575 nm) at room
temperature. The light intensity was modulated using ND
filters (0–193 mW cm2). As expected, the AuNP/Cat MTs swim
much faster under light irradiation: 92  11 mm s1 (3.8 bodylengths per s) at 139 mW cm2 (Fig. 6C, D and Video S4B, ESI†).
Photothermal property of the AuNPs on the exterior surface ofthe MTs induced heat generation and raised the hierarchicalwall temperature. Thereby, the increased enzyme activity
enhanced the swimming speed of the tube. Indeed, the mobilityof the AuNP/Cat MTs was changed in response to the opticalintensity. A maximum peak appeared at 139 mW cm2, where thewall temperature might reach the optimum temperature of theCat (Fig. 6C and D).53 Further strong irradiation at 193 mW cm2
reduced the velocity, implying that the wall became warmer thanthe optimum temperature. In striking contrast, the swimming
speed of the Cat2 MT motor without AuNP was unaffected by
optical intensity. We confirmed that the bulk aqueous medium
temperature was unaltered under light irradiation; it remained
constant at 26.4–27.6 1C (line chart in Fig. 6D). These accelerationand deceleration by visible light were fully reversible. The Cat

6434 | Mater. Adv., 2021, 2, 6428–6438© 2021 The Author(s). Published by the Royal Society of Chemistry

as that of the carbohydrase reaction with aGD/Cat MT motors
described above. Furthermore, we conducted similar experiments
using non-swimming HRP-covered HRP/PLA/HSA/MNP(PLA/
HSA)5PLA/PLG/Avi/bHSA MTs (HRP/HSA MTs) with and without
stirring using a magnetic stirrer bar (300 rpm). The concentrationof product DAP after 20 min in the non-stirring condition was
20  3 mM (Fig. 5D). This somewhat higher than expected value
might have arisen from (i) high catalytic activity of HRP and (ii)the technical limits to prevent the unavoidable partial mixing of
the heterogeneous solution occurred by adding the substratesolution and by collecting the supernatant. Again, it is remarkablethat the stirring of the mixture using a magnetic stirrer did not
facilitate the reaction. Similar DAP concentration was observed tothat of non-stirring condition (Fig. 5D).
Second, o-dianisidine (OD, Fig. 5C), which is also typicalsubstrate for HRP, was examined.51 The stable product is abrown bisazobiphenyl (BAB), which is regarded as produced by
self-coupling of two quinonediimines at neutral pH. The BAB
shows strong absorption at 455 nm. The product concentrationin the HRP/Cat MT motor dispersion was 49% of that for thefree HRP solution (Fig. 5E). In the case of non-swimming HRP/
HSA MTs, the forced stirring did not improve the reaction
compared to that of the swimming MTs. The trend closely
resembled the results observed for OPD. All data of HRP/Cat
MT motors supported strongly our mechanism proposed in theswimming aGD/Cat MTs. Overall, these findings manifested
that the enzyme/Cat MT motors act as ultrasmall biocatalyst
with self-stirring motion to promote the enzyme reaction.
In this study, self-stirring motion can be defined as the MT’sunique swimming movement, which causes suﬃcientcollisions between surface enzymes and substrates without
denaturing the enzymes. The motors are expected to be usefulin a very narrow channel in a microfluidic device and a verysmall aqueous droplet, where a magnetic stirrer cannot
be used.
AuNP-covered Cat MTs (AuNP/Cat MTs) and velocity control
with light irradiation

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sonication. The MNPs and AuNPs show no cytotoxicity to living
cells.56,57 We inferred that the four Cat MTs having diﬀerent
exterior surfaces possess suﬃcient biodegradability.

Conclusions
Protein MT motors with Cat engine layer on the interior wall anddiﬀerent functional material layers on the exterior wall weresynthesized using a combination of LbL build-up assemblies in
PC membrane and aqueous bulk solution. The MTs are selfpropelled catalytically in aqueous H2O2 medium by ejection of
O2 bubbles from the open-end terminus. The swimming tubes
demonstrated interesting biofunctionalities with self-propulsive
motion. The E. coli was captured specifically on the ConA outersurface of the tubules, providing green-emitting MTs. The lectin–
LPS bonding is sufficiently strong to hold the bacteria on theswimming tube. Moreover, the aGD-wrapped MTs act as a selfstirring biocatalyst that promotes the enzyme reaction bycontinuous movement. This enhancement was observed similarly on HRP/Cat MT motors. Phototermal heating of AuNP
outside surface of the capillary enables control of the tube’s
velocity by visible light irradiation. All MTs were decomposed byprotease, signifying biodegradability. It is apparently possible toimmobilize other proteins or antibodies on the exterior surface
and to create diverse MTs that can capture and transport
targeted drugs, viruses, and pathogenic bacteria. Although therelatively high H2O2 concentration limits the scope of theexperimentation and implementation design,5,58–60 the tailormade protein MT motors with biofriendly nature are anticipatedas promising biotools used in various biological and biomedical
applications.

Materials and methods
Fig. 6 (A and B) TEM images of AuNP/Cat MT. (C) Schematic illustration oftemperature increase in self-propelled AuNP/Cat MT by light irradiation.
(D) Influence of optical intensity on the average velocity of self-propelled
AuNP/Cat MTs and the parent Cat2 MTs (pH 7.0, 2 wt% H2O2, 0.1 wt%
Triton X-100) and on the temperature of the bulk aqueous solution.

Materials and apparatus

Biodegradability is the most important feature for biologicaland biomedical applications. The cylindrical shapes of ConA/Cat MTs, aGD/Cat MTs, HRP/Cat MTs, and AuNP/Cat MTs
became short and slender in the aqueous solution of Pronase,a cocktail of several proteases, within 2 h at 37 1C (Fig. S5, ESI†).
It can be inferred that proteases digested the protein andpolypeptide layers in the tubular walls. The remaining
aggregates of MNPs or AuNPs were dispersed immediately byPoly(L-arginine) hydrochloride (PLA; Mw, 470 000); poly(Lglutamic acid) sodium salt (PLG; Mw, 50 000–100 000), horseradish
peroxidase (HRP), 4-methylumbelliferyl-a-D-glucopyranoside(MUGlc), o-phenylenediamine (OPD), and o-dianisidine dihydrochloride (OD) were purchased from Sigma-Aldrich Co. LLC.
Catalase from bovine liver (Cat), a-glucosidase (aGD), andpolyoxyethylene(10) octylphenyl ether (Triton X-100) were purchased from Fujifilm Wako Pure Chemical Corp. Human serum
albumin (HSA) was purchased from the Japan Blood Products
Organization. Avidin (Avi) was purchased from Calzyme Laboratories, Inc. Concanavalin A (ConA) from Canavalia ensiformis(Jack-bean) was purchased from J-Oil Mills, Inc. The magnetite(Fe3O4) nanoparticle (MNP) (EMG607, ca. 10 nm diameter covered
with cationic surfactant) was purchased from Ferrotec Corp.
Biotinylated catalase (bCat) and biotinylated HSA (bHSA) wereprepared according to a procedure reported earlier.26 Fluoresceinlabeled Cat (FCat) and aGD (FaGD) were prepared by generalprocedure using fluorescein isothiocyanate (FITC; Sigma-Aldrich
Co. LLC). Cyanine dye-labeled ConA (CyConA) and HRP (CyHRP)
were prepared using a general procedure with cyanine5.5 NHS© 2021 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2021, 2, 6428–6438 | 6435

engine layer and AuNP photothermal layer are robustly
immobilized respectively on the interior and exterior walls. We
inferred from these findings that the AuNP/Cat MT motor’s
velocity can be tuned by the regulation of enzyme activity withphotothermal heat generation of the outside AuNPs mantle.
Biodegradability

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Materials Advancesester (Cy5.5; Lumiprobe Corp.). Citrated Au nanoparticles (AuNP,
ca. 40 nm diameter) were synthesized as described in an earlier
report.54 Deionized water (18.2 MQ cm) was prepared using
a water purification system (Elix UV and Milli Q Reference;
Millipore Corp.). UV-vis absorption spectra were measured using
a UV/Vis spectrophotometer (8453; Agilent Technologies Inc. or
V-650; Jasco Corp.). Fluorescence emission spectra were recorded
using a spectrofluorometer (FP-8300; Jasco Corp.).
Template synthesis of precursor PLA/HSA/MNP(PLA/HSA)5PLA/
PLG/Avi microtubes (Avi MTs)
The precursor Avi MTs were synthesized according to ourearlier reported procedure using a track-etched polycarbonate(PC) membrane (isopore membrane, 25 mm diameter, 1.2 mm
pore-diameter, 4.94  107 pores per piece; Millipore Corp.)26,37
with some modifications. The PC membrane was set in astainless steel syringe holder (25 mm diameter; Advantec Toyo
Kaisha, Ltd). First, the sodium phosphate buﬀer (PB) solution(pH 7.2, 10 mM, +0.1 M NaCl, 10 mL) of PLA (1 mg mL1) was
filtered through the membrane (15 mL h1) using a syringe
pump (PHD-2000; Harvard Apparatus). The excessively
absorbed PLA was rinsed by water filtration (12 mL, 60 mL h1).
The membrane was dried under vacuum for 10 min. Second, thePB solution (pH 7.2, 10 mM, 10 mL) of HSA (2 mg mL1) was
passed through (30 mL h1). After washing by water filtration(12 mL, 60 mL h1), the membrane was dried in vacuo for 10 min.
Third, a lid of the syringe holder was removed and the aqueous
solution (10 mL) of MNP (1 mg mL1) was dropped evenly onto
the PC membrane with suction from the opposite side. After
closing the lid and rinsing by water filtration (12 mL, 60 mL h1),
the pale-brown membrane was dried under vacuum for 10 min.
Subsequently, the pressure infiltrations of PLA and HSA describedabove were repeated in turn for 5.5 cycles to deposit the fourth tofourteenth layers. Then the PLG layer was made onto the pore wallusing PB solution (pH 7.2, 10 mM, 10 mL) of PLG (1 mg mL1)(30 mL h1). Finally, the PB solution (pH 7.2, 10 mM, 10 mL) of
Avi (2 mg mL1) was passed through (15 mL h1) two times tocreate the Avi innermost layer. The PC membrane was washed bywater dropping (10 mL) onto the membrane with suction from the
opposite side and drying in vacuo for 30 min.
The surface of the PC membrane including multilayered
tubules was wiped using a cotton swab with deionized water
and was immersed into a DMF solution using tweezers todissolve the PC flame. The precipitated MTs were washed twicewith DMF and 1,4-dioxane, and were freeze-dried in vacuo,
yielding the lyophilized PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi
MTs (Avi MTs) as pale-yellow powder.

Paper
solution (pH 7.0, 10 mM, 1.3 mL) of bCat (26.2 mM) was injected
into the dispersion. The resultant mixture (ca. 2.06 
106 tubes per mL, [bCat] = 17 nM, [bCat]/[Avi] = 1.8 (mol/mol),
PBS +150 mM NaCl, 2 mL) was incubated for 3 h at 25 1C under
darkness. After centrifugation (500  g, 3 min), the supernatant,including unbound bCat, was discarded. The precipitatedMTs, PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs (Cat MTs),
were resuspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES) buﬀer solution (pH 6.8, 10 mM, +1 mM CaCl2, +1 mM
MnCl2, 2 mL) and were centrifuged again (500  g, 3 min). Afterremoving the supernatant, HEPES solution was added to adjust thevolume of 0.35 mL. Subsequently, the HEPES solution of ConA
(1 mg mL1, 0.15 mL) was added. The resultant mixture wasincubated for 30 min at 25 1C. Then, after the dispersion wascentrifuged (500  g, 3 min), the supernatant was discarded,followed by addition of HEPES solution (2 mL). By repeating thiswashing twice, we obtained the HEPES solution (2 mL) of ConA/
PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs (ConA/Cat MTs).
Using the same procedure, ConA/PLA/HSA/MNP(PLA/HSA)5PLA/
PLG MTs (ConA/PLG MTs) and HSA/PLA/HSA/MNP(PLA/HSA)5
PLA/PLG/Avi/bCat MTs (HSA/Cat MTs) were also prepared.
Coverage rate estimation with ConA
The coverage rate of the outer surface of Cat MTs with ConA
was estimated using a fluorescent CyConA. The Cat MTs wereprepared in the manner described above (ca. 2.06  106 tubes
per mL, [bCat] = 17 nM, [bCat]/[Avi] = 1.8 (mol/mol), PBS+150 mM NaCl, 2 mL). The dispersion was centrifuged(500  g, 3 min) to remove the supernatant including freebCat. The precipitated Cat MTs were suspended in PB solution(pH 7.0, 10 mM, 2 mL) and were centrifuged again (500  g,
3 min). After removing the supernatant, PB solution was added
to adjust the volume of 0.9 mL. Subsequently, the PB solutionof CyConA (0.4 mM, 0.1 mL) was added and the mixture wasincubated for 30 min at 25 1C. The dispersion of the CyConA/Cat MTs was centrifuged (4000  g, 10 min). Then the fluorescence spectrum of the supernatant (0.8 mL) was measured (lex,684 nm/lem, 710 nm) to ascertain the concentration of theunbound free CyConA. An identically treated control CyConA
sample without MT was prepared; its fluorescence intensity was
regarded as a 100% CyConA concentration. CyConA/FCat MTs
were also prepared using the same synthetic procedure for
CLSM observations.
E. coli capture by ConA/Cat MTs

The Avi MTs (ca. 8.23  106 tubes, one-sixth of the powder
prepared using one PC membrane) were dispersed in deionizedwater (3.41 mL) using an ultrasonic cleaner for 1 min. Thisaqueous dispersion was divided into two. After the 20
phosphate-buﬀered saline (PBS) solution (pH 7.4, 0.1 mL) and
9% NaCl solution (195 mL) were added to each dispersion, themixture was incubated for 30 min at 25 1C. Then, the PB

The green fluorescent protein (GFP) secreted E. coli (F-E. coli)
was prepared using an HB101 (K-12) competent cell and pGLOt
Bacterial Transformation Kit (Bio-Rad Laboratories Inc.). The
HEPES dispersion (pH 6.8, 10 mM, +1 mM CaCl2, +1 mM MnCl2,
2 mL) of ConA/Cat MTs (ca. 2.06  106 tubes per mL), prepared
as described above, was divided by four into 2 mL-volume plastic
tubes. Using a neodymium (Nd)-magnet (468 mT, 30 mm
diameter  15 mm), the MTs were collected at the bottom andthe supernatant was removed. The obtained ConA/Cat MTs wereredispersed into HEPES solution (0.8 mL) containing H2O2
(2.5 wt%) and Triton X-100 (0.125 wt%) to promote self-propulsion

6436 | Mater. Adv., 2021, 2, 6428–6438© 2021 The Author(s). Published by the Royal Society of Chemistry

Synthesis of ConA-covered Cat MTs (ConA/Cat MTs)

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Paperof the tubes. Thereafter, the HEPES suspension (0.2 mL) of F-E. coli
(OD600 = 0.05, 2.5  107 cells per mL) was added slowly. The
resultant mixture (ca. 1.03  106 tubes per mL, [F-E. coli] = 5.0 
106 cells per mL, [H2O2] = 2 wt%, [Triton X-100] = 0.1 wt%, 1 mL) wasleft for 15 min without vibration at 25 1C. By exposure to a magnetic
field using a Nd-magnet, the F-E. coli bound ConA/Cat MTs werecollected at the bottom. The supernatant was transferred to otherplastic tubes. This supernatant was centrifuged (4000  g, 10 min).
The precipitated F-E. coli was redispersed in HEPES solution (1 mL).
Fluorescence measurement of this dispersion (lex, 395 nm/lem,
508 nm) was used to estimate the concentration of the unboundfree F-E. coli. An identically treated control sample without MT was
prepared. Its fluorescence intensity was regarded as a 100% F-E. coli
concentration. The same experiments were conducted using the
ConA/PLG MTs and HSA/Cat MTs.
Enzyme reaction measurements of aGD/Cat MTs
Synthesis of aGD/Cat MTs was described in ESI.† The PB
dispersion (pH 7.0, 10 mM, 0.5 mL) of aGD/Cat MTs (ca.
1.65  107 tubes per mL) was diluted to 1.37 mL and put it in
a 10-mm path length optical quartz cuvette. After the PB
solution (pH 7.0, 10 mM) of Triton X-100 (5 wt%, 30 mL) was
added, the mixture was incubated for 10 min at 25 1C. ThenNMP solution of MUGlc (5 mM, 15 mL) was injected into thedispersion, followed by addition of H2O2 solution (35.6 wt%,
85 mL) immediately to the cuvette (ca. 5.5  106 tubes per mL,[H2O2] = 2 wt%, [Triton X-100] = 0.1 wt%, [MUGlc] = 50 mM).
An increase in fluorescence of the product 4-methylumbelliferone(MU) (lex, 370 nm/lem, 447 nm) was monitored at 25 1C for
50 min. The MU concentration was ascertained from the fluorescence intensity. The appearance of emission was observed visually
under irradiation of 365 nm UV light. Identical experiments wereconducted with non-swimming aGD/PLG MTs with stirring(300 rpm) and without stirring.
Enzyme reaction measurements of HRP/Cat MTs
Synthesis of HRP/Cat MTs was described in ESI.† The PB
dispersion (pH 7.0, 10 mM, 0.5 mL) of HRP/Cat MTs (ca. 1.65 
107 tubes per mL) was diluted to 1.0 mL and put it in a 5 mLvolume glass bottle. Freshly prepared PB solution (pH 7.0, 10 mM,
0.5 mL) including H2O2 (3 wt%), Triton X-100 (0.15 wt%),
and substrate (o-phenylenediamine (OPD) or o-dianisidine
dihydrochloride (OD), 225 mM) was added to the solution. Thenthe mixture was left for 20 min at 25 1C (5.5  106 tubes per mL,[H2O2] = 2 wt%, [Triton X-100] = 0.1 wt%, [substrate] = 150 mM). By
exposure of a Nd-magnet, the HRP/Cat MTs were collected. The
supernatant was transferred to a 10 mm path length opticalquartz cuvette to measure the UV-vis absorption spectrum of theoxidized product [2,3-diaminophenazine (DAP; lmax, 417 nm) or
bisazobiphenyl derivative (BAB; lmax, 455 nm)]. Identicalexperiments were also conducted using non-swimming HRP/
HSA MTs with stirring (300 rpm) and without stirring.

Materials Advances
or PB dispersion (pH 7.0, 10 mM, ca. 1.06  106–4.12  106
tubes per mL, [H2O2] = 2 wt%, [Triton X-100] = 0.1 wt%) ofswimming MTs were prepared in a 24-well glass bottom microplate (Iwaki EZVIEW Culture Plate LB; AGC Techno Glass., Co.,
Ltd). The moving behavior was observed using a research inverted
microscope (IX73; Olympus Corp.) under bright field mode
equipped with a digital high-speed camera (HAS-220; Detect
Corp.) at 200 frames per s. Editing of the movies and determination of the tube velocity were performed using an image processing program (Image J; NIH).
Optical microscopy observations of swimming AuNP/Cat MTs
under light irradiation
Synthesis of AuNP/Cat MTs was described in ESI.† Movementsof the AuNP/Cat MT were observed under bright field modewith light irradiation using a Hg lamp through a fluorescencemirror unit (U-FGW, Ex, 530–550 nm/Em, 575 nm) at room
temperature. The irradiation light intensity was regulated using
ND filters (0–193 mW cm2). The solution temperature wasmeasured using a digital thermometer (TF-2100; As One Corp.).

Author contributions
T. K. designed and initiated this study. All the authors
conducted experiments and analysed the data. M. U., N. S.,
K. M., and T. K. drafted the manuscript.

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research (B) (No. 18H01833 and No. 21H01767) from JSPS.

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Typically, the HEPES dispersion (pH 6.8, 10 mM, ca. 1.06  106–4.12  106 tubes per mL, [H2O2] = 2 wt%, [Triton X-100] = 0.1 wt%)

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