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Cite this: Mater. Adv., 2022,
3, 6988

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Swimming protein microtube motors capture
virus-shaped fluorescent nanoparticles†
Yushi Akashi, Natsuho Sugai, Ryo Kato and Teruyuki Komatsu*

Received 20th May 2022,
Accepted 9th August 2022
DOI: 10.1039/d2ma00566b
rsc.li/materials-advances

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† Electronic supplementary information (ESI) available: Detailed experimental
procedures; the fluorescence spectra of HA-FNP solution after treatment with selfpropelled Fet/Cat MT motors (Fig. S1); the TEM image of HA-FNP bound Fet/Cat
MT (Fig. S2) (all PDF); self-propelled motion of a Fet/Cat MT motor (Video S1)(AVI); self-propelled motion of a FIgG-HA-Fet/Cat MT motor (Video S2) (AVI); selfpropelled motion of a HA-FNP bound Fet/Cat MT motor (Video S3) (AVI). See DOI:
https://doi.org/10.1039/d2ma00566b

micrometer size with autonomous propulsion have attracted considerable attention because of their versatility, with diverse
potential applications5–24 that include as drug carriers,12,14,17,18,20
environmental remediators,13,21,24 and analytical sensors.22,23 One
beneficial procedure to synthesize these hollow swimmers is
template synthesis using layer-by-layer (LbL) assembly in a tracketched polycarbonate (PC) membrane.12,14,16,17,20,24 Such fabricated unique microtube (MT) motors able to capture influenza
viruses would strongly benefit not only nanomaterials science, butalso diagnostic applications. Fetuin from fetal calf serum (Fet, Mw:
48.4 kDa, pI: 3.3), a glycosylated protein with tribranched oligosaccharides containing Neu5Ac residues,25 interacts with influenza
viruses.26,27 This paper is the first to describe the synthesis of
catalase (Cat)-driven protein MT motors with a fetuin layer as anexternal wall and to explain their efficient capture of molecular
HAs and spherical HA-FNPs.
The stratiform Fet/PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/
bCat MTs (Fet/Cat MTs) were prepared using our earlier
reported procedures of LbL assembly using a PC membrane
template [PLA, poly(L-arginine); HSA, human serum albumin;
MNP, magnetite nanoparticle; PLG, poly(L-glutamic acid); Avi,avidin; bCat, biotinylated catalase] (Fig. 1).24 Phosphate buffered (PB) solution of Fet was added to decorate the outersurface of the precursor PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/
bCat MTs (Cat MTs), yielding the designed Fet/Cat MTs. The
transmission electron microscopy (TEM) images of the airdried sample revealed that the MTs swelled considerably inwater. They had 1.15 mm outer diameter and 24 mm tube length.
The wall thickness was 281 nm (Fig. 2A). The Fet coverage rateof the tube surface was ascertained using fluorescent Cy5.5labeled Fet (CyFet). After centrifugation of the mixture of Cat
MTs and CyFet, the fluorescence intensity of the supernatant
was 33% of that observed for the identically treated CyFet
solution without the tubes. This result signifies that the number of adhered CyFet molecules onto one tube was approximately 3.9  106 per tube. Thereby, the coverage rate of the MT
surface with CyFet was estimated as 58%. Similar tubules havebeen produced using fluorescein-labeled Cat (FCat) as an

6988 | Mater. Adv., 2022, 3, 6988–6992© 2022 The Author(s). Published by the Royal Society of Chemistry

Hemagglutinins (HAs), spike proteins of influenza virus, were fixedon luminous polystyrene beads to make HA-immobilized fluorescent nanoparticles (HA-FNPs). The protein MTs with an exterior
surface of fetuin and an interior surface of catalase (Fet/Cat MTs)
were self-propelled in aqueous H2O2 solution by spouting O2
bubbles. They captured molecular HAs and virus-shaped HA-FNPs
eﬃciently.

Influenza viruses are annoying pathogens for human beings.
Thucydides reported an infectious disease that struck Athens in
430 B.C. Presumably, that is the earliest recorded outbreak ofwhat we know as influenza.1 Among the three influenza virus
types of A, B and C, type A tends to engender severe diseases.2 It
belongs to the Orthomyxoviridae family, with a negative-sense
single-stranded RNA genome. In 2009, swine-origin A (H1N1)
virus caused a human pandemic.3 The initial infection of
influenza A virus starts from the specific binding of spike
protein hemagglutinin (HA) to a cellular surface receptor containing sialyl oligosaccharide with N-acetyl neuraminic acid (Neu5Ac)
terminuses.4 Currently, great interest has arisen in the study of
infectious viral particles. It is nonetheless diﬃcult to handle thesepathogens in a common experimental laboratory. Moreover, analyses of viruses must be sensitive. To develop virus researchextensively, we created a non-infectious and highly detectable fakevirus, an HA-immobilized fluorescent nanoparticle (HA-FNP), on
which the HAs are fixed by Ni-histidine (His)-tag complexation.
The functionality of HA coordinated on the particle has beenchanged as little as possible. Swimming cylindrical motors ofView Article Online

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Materials Advancesinternal wall. The CLSM images of the CyFet/FCat MTs showedboth fluorescence colors (Fig. 2B), implying that the FCat
molecules are adhered on the interior wall and that the CyFet
molecules are bound onto the exterior surface of the hollow
cylinder. The Fet/Cat MTs were self-propelled in aqueous H2O2
solution by ejecting O2 bubbles from the open end terminus
with an average velocity of 64  5 mm s 1 (2.7 body-lengths
per s) (pH 7.0, 2 wt% H2O2, and 0.1 wt% Triton X-100) (Fig. 2C
and D and Video S1, ESI†). The swimming behavior of Fet/Cat
MT was similar to that of the parent Cat MT.16
After HA solution was added to the PB dispersion of selfpropelled Fet/Cat MTs (pH 7.0, 2 wt% H2O2, and 0.1 wt% Triton
X-100), the mixture was left for 15 min without vibration in thedark at 25 1C. By bringing a Nd-magnet closer to the cuvette, the

HA bound Fet/Cat MTs (HA-Fet/Cat MTs) were attracted to thebottom. The supernatant was analyzed using direct enzymelinked immunosorbent assay (ELISA) to ascertain the concentration of the unbound free HA. The HA concentration was only
8% of that observed for the identically treated HA without the
Fet/Cat MTs (Fig. 3A). This result indicated that 92% of HA wasremoved from the sample solution by a single treatment withthe swimming MTs. This finding enables us to calculate theaverage number of HA molecules absorbed onto one tubule as
1.2  106 per tube.
To clarify that the fetuin surface of the tube indeed captures
the HA, identical experiments were conducted using similar
MTs: HSA/PLA/HSA/MNP(PLA/HSA)5PLA/PLG/Avi/bCat MTs(HSA/Cat MTs). Incubation with the HSA/Cat MTs, in which
HSA was used instead of Fet, showed only 5% reduction of the
HA amount (Fig. 3A). Furthermore, incubation with the nonswimming Fet/PLA/HSA/MNP(PLA/HSA)5PLA/PLG MTs (Fet/
PLG MTs) decreased 3% relative to the control value. Our
results proved that self-propelled Fet/Cat MTs capture HAeﬃciently and that their motion plays an important role inaccelerating the contact frequency to catch the HA.
To ensure HA binding onto the Fet surface, we used fluorescent anti-hemagglutinin IgG antibody (FIgG). Upon additionof FIgG to the PB solution of the swimming HA-Fet/Cat MTs(pH 7.0, 2 wt% H2O2, and 0.1 wt% Triton X-100), the antibody
adhered immediately on the tubes’ exterior surface. Optical
microscopy observation under fluorescence mode revealed thatthe swimming FIgG-bound HA-Fet/Cat MTs (FIgG-HA-Fet/Cat
MTs) emitted green fluorescence (Fig. 3B–D and Video S2,
ESI†). The average velocity was 71  9 mm s 1 (3.0 bodylengths per s). In light of these findings, we inferred that (i)specific interaction between HA and the sialyl oligosaccharide
chain of the Fet wall is responsible for HA capture and that (ii)
propulsion is crucially important to promote HA capture.
Next, we investigated the capture of virus-shaped fluorescentNPs with the swimming Fet/Cat MT motors. Moulès et al.
reported the morphology of the influenza A virus [Puerto Rico/
8/34 (PR8, H1N1)] virion using cryogenic TEM (cryoTEM).28 The
PR8 viruses comprised a mixture of spherical particles (94 
15 nm diameter) and elongated particles. We chose carboxylated
fluorescent polystyrene NPs (FNPs, 100 nm diameter) as aluminous core. First, Ni2+-nitrilotriacetic acid (Ni-NTA) groups
were introduced onto the FNP surface (Scheme 1). Subsequently, the HA having a His-tag was complexed with the NiNTA groups on the bead, thereby yielding HA-FNPs. After
dialysis of the mixture, the HA concentration in the external
dialysate was negligible, as revealed by ELISA; the amount was
below the detection limit. We inferred that all HA molecules
bound to the particle as a spike protein in our experimental
protocol. The number of HAs on one NP was ascertained as
approximately 400, which is equivalent to the value of thenatural influenza A virus.28 TEM observation clearly revealed amonomolecular HA layer with 10 nm thickness on the particles(Fig. 4A). The positive contrast of HA can be attributed to uranyl
acetate (UO2+) staining. An earlier report described that thisnegative contrasting agent works as a positive staining material© 2022 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2022, 3, 6988–6992 | 6989

Fig. 1 Preparation and stratiform structure of the protein MT motor with a
Cat engine layer as an internal wall and Fet exterior surface: Fet/PLA/HSA/
MNP(PLA/HSA)5PLA/PLG/Avi/bCat MT (Fet/Cat MT).

Fig. 2 (A) The TEM image of Fet/Cat MT. (B) The CLSM images of CyFet/
FCat MT (left – lex, 633 nm for CyFet; right – lex, 488 nm for FCat). (C andD) Snapshots taken from Video S1 (ESI†) of microscopic observations ofself-propelled Fet/Cat MT by ejecting O2 bubbles in PB solution (pH 7.0,
2 wt% H2O2, and 0.1 wt% Triton X-100) at 25 1C under bright field mode.

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Fig. 3 (A) Residual rate of HA after incubation with self-propelled Fet/Cat MTs. (B and C) Snapshots taken from Video S2 (ESI†) of microscopic
observations of self-propelled FIgG bound HA-Fet/Cat MT by spouting O2 bubbles in PB solution (pH 7.0, 2 wt% H2O2, and 0.1 wt% Triton X-100) at 25 1C
under fluorescence mode. (D) Schematic illustration of HA capture and subsequent FIgG binding on the exterior surface of a bubble-propelled Fet/Cat
MT.

Scheme 1

Synthetic route of HA-FNPs.

for certain proteins.29 This reverse eﬀect caused the dark imageof the HAs. Several diﬀerent methods are currently available tocharacterize the nanostructure of protein-immobilized
particles.30 However, the detailed physicochemical features ofthe HA-FNP surface are not very important at this stage. This
will be revealed in the near future. The luminous HA-FNPs are a
non-infectious fake influenza A virus, which can be detectedwith high sensitivity using fluorescence spectroscopy.
Later, the HA-FNPs were injected into the PB dispersion ofself-propelled Fet/Cat MTs. The mixture (pH 7.0, 2 wt% H2O2,
and 0.1 wt% Triton X-100) was left for 2 h without vibration in
the dark at 25 1C. Using a Nd-magnet, the HA-FNP-bound Fet/
Cat MTs were removed carefully. As expected, the fluorescenceintensity of the supernatant was only 54% of that of the control
solution without the tubes (Fig. S1A, ESI†). It became apparent
that ca. 2.0  103 particles bound to one tube (Fig. 4B). The
swimming Fet/Cat MTs with bound HA-FNP fluoresced
strongly; their velocity was 72  8 mm s 1 (3.0 body-lengths
per s) (Fig. 4C and Video S3, ESI†). It is noteworthy that the HAFNPs were absorbed tightly onto the Fet exterior wall. The HAbinding to the Fet layer is suﬃciently strong to retain the NPs,
which were never released. Subsequent TEM observations
indicated definitively that HA-FNPs were immobilized onto
the cylindrical surface (Fig. S2, ESI†). The image shows theparticles absorbed on both sides of the flattened tube as 2.0 
103 pieces (85 pieces per mm-tube-length), which is exactly

6990 | Mater. Adv., 2022, 3, 6988–6992© 2022 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Conflicts of interest
Open Access Article. Published on 09 August 2022. Downloaded on 9/24/2024 4:06:56 AM.
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The authors declare no competing financial interest.

References

T. K. designed and initiated this study. All the authors conducted experiments and analysed the data. Y. A., N. S. and T. K.
drafted the manuscript.

1 A. D. Langmuir, T. D. Worthen, J. Solomon, C. G. Ray and
E. Peterson, New Engl. J. Med., 1985, 313, 1027–1030.
2 R. A. Medina and A. Garcia-Sastre, Nat. Rev. Microbiol., 2011,
9, 590–603.
3 H. V. Fineberg, New Engl. J. Med., 2014, 370, 1335–1342.
4 W. Weis, J. H. Brown, S. Cusack, J. C. Paulson, J. J. Skeheland D. C. Wiley, Nature, 1988, 333, 426–431.
5 Y. Mei, G. Huang, A. A. Solovev, E. B. Ureña, I. Mönch,
F. Ding, T. Reindl, R. K. Y. Fu, P. K. Chu and O. G. Schmidt,
Adv. Mater., 2008, 20, 4085–4090.
6 W. Gao, S. Sattayasamitsathit, J. Orozco and J. Wang, J. Am.
Chem. Soc., 2011, 133, 11862–11864.
7 Y. Mei, A. A. Solovev, S. Sanchez and O. G. Schmidt, Chem.
Soc. Rev., 2011, 40, 2109–2119.
8 H. Wang and M. Pumera, Chem. Rev., 2015, 115, 8704–8735.
9 Z. Wu, X. Lin, T. Si and Q. He, Small, 2016, 12, 3080–3093.
10 B. Xu, B. Zhang, L. Wang, G. Huang and Y. Mei, Adv. Funct.
Mater., 2018, 28, 1705872.
11 M. Safdar, S. U. Khan and J. Jänis, Adv. Mater., 2018, 30, 1703660.
12 T. Komatsu, Chem. Lett., 2020, 49, 1245–1255.
13 L. Soler, V. Magdanz, V. M. Fomin, S. Sanchez andO. G. Schmidt, ACS Nano, 2013, 11, 9611–9620.
14 Z. Wu, W. Lin, X. Zou, J. Sun and Q. He, ACS Appl. Mater.
Interfaces, 2015, 7, 250–255.
15 Y. Yoshizumi and H. Suzuki, ACS Appl. Mater. Interfaces,
2017, 9, 21355–21361.
16 N. Sugai, Y. Nakai, Y. Morita and T. Komatsu, ACS Appl.
Nano Mater., 2018, 2, 3080–3085.
17 Y. Nakai, N. Sugai, H. Kusano, Y. Morita and T. Komatsu,
ACS Appl. Nano Mater., 2019, 2, 4891–4899.
18 B. Esteban-Fernández de Ávila, P. Angsantikul, X. Li, W. Gao,
L. Zhang and J. Wang, Adv. Funct. Mater., 2018, 28, 1705640.
19 C. Liang, Z. Zhan, F. Zeng, D. Xu, Y. Wang, W. Zhao,
J. Zhang, J. Guo, H. Feng and X. Ma, ACS Appl. Mater.
Interfaces, 2018, 10, 35099–35107.
20 W. Wang, Z. Wu, X. Lin, T. Si and Q. He, J. Am. Chem. Soc.,
2019, 141, 6601–6608.
21 X. Bing, X. Zhang, J. Li, D. H. L. Ng, W. Yang and J. Yang,
J. Mater. Chem. A, 2020, 8, 2809–2819.
22 C. C. Mayorga-Martinez and M. Pumera, Adv. Funct. Mater.,
2020, 30, 1906449.
23 Á. Molinero-Fernández, M. Á. López and A. Escarpa, Anal.
Chem., 2020, 92, 5048–5054.
24 M. Umebara, N. Sugai, K. Murayama, T. Sugawara, Y. Akashi,
Y. Morita, R. Kato and T. Komatsu, Mater. Adv., 2021, 2,
6428–6438.
25 W. M. Brown, N. R. Saunders, K. Mollgard and K. M.
Dziegielewska, BioEssays, 1992, 14, 749–755.
26 D. K. Takemoto, J. J. Skehel and D. C. Wiley, Virology, 1996,
217, 452–458.

© 2022 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2022, 3, 6988–6992 | 6991

Fig. 4 (A) TEM images of (top) unmodified FNPs and (bottom) HA-FNPs(bar: 50 nm). (B) Schematic illustration of HA-FNPs capturing a bubblepropelled Fet/Cat MT. (C) Snapshots taken from Video S3 (ESI†) of microscopic observations of self-propelled HA-FNP bound Fet/Cat MT byejecting O2 bubbles in PB solution (pH 7.0, 2 wt% H2O2, and 0.1 wt%
Triton X-100) at 25 1C under (top) bright field mode and (bottom)
fluorescence mode.

equal to the value obtained from the fluorescence measurements. By contrast, when adding Ni-NTA-FNPs instead of HAFNPs, the fluorescence change was negligibly small (Fig. S1B,
ESI†). This finding implies that Fet/Cat MT motors recognize
the HA surface of the particle.
In conclusion, Cat-driven MT motors composed of blood
serum proteins (HSA and fetuin) captured influenza virusshaped FNPs, which can be used easily in a normal laboratory
without restriction. This system involves no antigen–antibody
reaction. Therefore, virus-shaped FNPs of various kinds can becreated using His-tagged spike proteins of desired viruses anddifferent size fluorescent NPs. These particles are expected tobe effective for virus research in common laboratory environments. The swimming Fet/Cat MTs might be applicable asdetectors for several viruses. They are expected to be of diagnostic importance for use in advanced biotechnology.
This work was supported by Grants-in-Aid for Scientific
Research (B) (No. 18H01833 and No. 21H01767) from JSPS.

Author contributions

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27 B. Meng, A. C. Marriott and N. J. Dimmock, Influenza Other
Respir. Viruses, 2010, 4, 147–153.
28 V. Moulès, O. Terrier, M. Yver, B. Riteau, C. Moriscot,
O. Ferraris, T. Julien, E. Giudice, J.-P. Rolland, A. Erny,
M. Bouscambert-Duchamp, E. Frobert, M. Rosa-Calatrava,

Y. P. Lin, A. Hay, D. Thomas, G. Schoehn and B. Lina,
Virology, 2011, 414, 51–62.
29 X. Qu and T. Komatsu, ACS Nano, 2010, 4, 563–573.
30 P. M. Kelly, C. Åberg, E. Polo, A. O’Connell, J. Cookman, J. Fallon,
Ž. Krpetić and K. A. Dawson, Nat. Nanotechnol., 2015, 10, 472–479.

6992 | Mater. Adv., 2022, 3, 6988–6992© 2022 The Author(s). Published by the Royal Society of Chemistry