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Title

High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based
Assemblies with a Smectic Liquid Crystalline Orientation

Authors

Toshiki Sawada, Yuta Murata, Hironori Marubayashi, Shuichi Nojima,Junko Morikawa, Takeshi Serizawa

Citation

Viruses, Vol. 10, Issue 11

Pub. date

2018, 11Creative Commons

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viruses
ArticleHigh Thermal Diffusivity in Thermally TreatedFilamentous Virus-Based Assemblies with a Smectic
Liquid Crystalline Orientation
Toshiki Sawada 1,2, * , Yuta Murata 1 , Hironori Marubayashi 1 , Shuichi Nojima 1 ,Junko Morikawa 3 and Takeshi Serizawa 1, *
1

2
3

*

Department of Chemical Science and Engineering, School of Materials and Chemical Technology,
Tokyo Institute of Technology, Tokyo 152-8550, Japan; ymurata@polymer.titech.ac.jp (Y.M.);
marubayashi@polymer.titech.ac.jp (H.M.); snojima@polymer.titech.ac.jp (S.N.)
Precursory Research for Embryonic Science and Technology, Japan Science and Technology,
Saitama 332-0012, Japan
Department of Materials Science and Engineering, School of Materials and Chemical Technology,
Tokyo Institute of Technology, Tokyo 152-8550, Japan; morikawa.j.aa@m.titech.ac.jp
Correspondence: tsawada@polymer.titech.ac.jp (To.S.); serizawa@polymer.titech.ac.jp (Ta.S.);
Tel.: +81-3-5734-3655 (To.S. & Ta.S.)

Received: 6 August 2018; Accepted: 31 October 2018; Published: 2 November 2018


Abstract: Polymers are generally considered thermal insulators because the amorphous arrangementof the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies
reveal that individual chains of polymers with oriented structures could have high thermal
conductivity, because such stretched polymeric chains effectively conduct phonons through polymericcovalent bonds. Previously, we have found that the liquid crystalline assembly composed of oneof the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity
even though the assembly is based on non-covalent bonds. Despite such potential applicabilityof biopolymeric assemblies as thermal conductive materials, stability against heating has rarely
been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic
liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 ◦ C) treatment.
The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability
against heating processes. Our results provide insight into the future use of biomolecular assemblies
for reliable thermal conductive materials.
Keywords: filamentous virus; self-assembly; liquid crystal; thermophysical property; structural stability

1. Introduction
The increasing interest in flexible electronics has facilitated the development of new organic
polymers that provide seamless integrated structure-property relationships required for a variety ofefficient configurations in electronic devices, such as organic solar cells and organic light emitting
diodes. Generally, the thermal conductivity of bulk organic polymers is very low due to strong phonon
scattering caused by various defects and interfaces [1]. Typical methods for improving polymer
thermal conductivity have often focused on the construction of composite materials, which containadditives (so-called fillers) such as metals or ceramics [2]. However, large amounts of fillers are
required to sufficiently increase the thermal conductivity, which can significantly increase the materials
cost and may change the original characteristics of the polymer, such as electrical, optical, andphysical properties [3,4]. In contrast, the alignment of the polymer chains can greatly enhance their
thermal conductivities along the axis of the polymer chains without the use of other additives [5,6].
Viruses 2018, 10, 608; doi:10.3390/v10110608

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Not only synthetic polymers [4,6,7] but also biomacromolecular proteins [8] can have high thermal
conductivities, due to efficient phonon propagation along the oriented polymeric covalent bonds.
Nevertheless, the practical applications of thermal conductive polymeric materials are still limited dueto the inherent difficulties in processing the materials [9].
M13 bacteriophage (M13 phage), a filamentous virus, has a regularly assembled structurecomposed of multiple proteins and genomic DNA. In the past two decades, M13 phage has beenused as various material components such as sensors, electronics, and devices [10–18]. M13 phage
is an attractive biomaterial because of the ease by which its surface can be functionalized via genetic
engineering [19] and/or chemical methods [20]. Furthermore, due to their uniform size, uniformity ofdimensions, high aspect ratio (4.5 nm width and 900 nm length), dipole properties, and charge densities,
M13 phage showed liquid crystalline properties with various phases in concentrated solutions [21–24].
Such liquid crystalline properties have been utilized for the construction of M13 phage-based materials
with ordered structures [11,14,25–33]. Recently, we developed M13 phage-based assemblies withordered structures in various scale (so called hierarchical assemblies) with high thermal diffusivity,
even though the assemblies were based on non-covalent bonds, and found that macroscopic phageorientation in the assemblies is essential for high thermal diffusivity [34]. Despite the promisingpotential of biomacromolecular assemblies as thermal conductive materials, the stability against
heating of the assemblies, which are generally regarded as low heat resistance objects, have rarely
been investigated.
Biomacromolecular proteins are well known to denature upon heating, and typical denaturing
temperature in an aqueous phase is approximately 60 ◦ C, except for hyperthermophile proteins,
viral assembled capsids, and similar systems [35,36]. To utilize M13 phage-based assemblies forreliable thermal conductive materials, the characterization of their thermal stabilities is an important
requirement. Here we demonstrate the high structural stability against heating of M13 phage-based
assemblies with high thermal diffusivities. Microscopic observations, infrared (IR) spectroscopic
measurements, and small angle X-ray scattering (SAXS) measurements revealed that the molecular
packing, secondary structures, molecular assembled structures, and macroscopic orientation of theassemblies had a smectic liquid crystalline orientation with little change upon thermal treatment at
150 ◦ C. Importantly, the high thermal diffusivity of the assemblies was maintained after the thermal
treatment. The thermal diffusivities of assemblies with nematic liquid crystalline orientation or with
non-orientation are significantly decreased by thermal treatment; therefore, smectic liquid crystalline
orientation plays an essential role in providing stability against heating. These results provide insight
into the future use of biomolecular assemblies for reliable thermal conductive polymeric materials.
2. Materials and Methods
2.1. Preparation of M13 Phage-Based Films
The M13 phages were expressed using the Ph.D. Peptide Display Cloning System (New England
Biolabs, Inc., Ipswich, MA, USA). Phagemid DNA was heat-shocked into competent Escherichia coli
ER2738 cells. The M13 phages expressed were amplified using the Escherichia coli and purified by
precipitation and re-dispersion procedures using a mixed solution composed of 5 w/v% poly(ethylene
glycol) (average molecular weight of 7000–10,000) and 2.5 M NaCl. M13 Phage solutions (15 mg/mL,
500 µL) were mounted on glass plates circularly patterned with a highly water-shedding coatingbased on fluororesin (glass diameter: 15 mm, Matsunami Glass Ind., Ltd., Osaka, Japan) followed byincubation for 24 h at 25 ◦ C in a dry atmosphere [34].
2.2. Polorized Optical Microscopy (POM) Observation
The phage films were set onto the stage of a polarized optical microscope (Eclipse LV100ND,
Nikon). Then, the samples were observed at ambient temperature. Images with a sample rotation of45◦ were recorded to identify the liquid crystalline-oriented structures.

Viruses 2018, 10, 608

3 of 102.3. Atomic Force Microscopy (AFM) Observation
The phage films were set onto the stage of an AFM. Then, the samples were observed. The AFM
images were obtained using an SPM-9600 (Shimadzu, Kyoto, Japan) in tapping mode with standard
silicon cantilevers. All images were scanned at a scan rate of at least 1 Hz with a maximum number ofpixels (512 × 512).
2.4. Thermal Diffusivity Measurements
Thermal diffusivity (regarded as the ratio of the thermal conductivity of the material to thespecific heat capacity) of the films in the thickness direction was measured using an ai-Phase mobile
1u (ai-Phase Co. Ltd., Tokyo, Japan) based on the temperature wave analysis method [37]. Thermal√
diffusivity (α) was calculated from the relationship of the square root of the angular frequency ( ω)and the phase delay (∆θ) of the temperature wave as shown in the following Equation (1):∆θ = −

r

ω
π
d−
2α
4

(1)

where d is the thickness of the film.
2.5. SAXS Measurements
SAXS measurements of the phage films were performed at BL-10C (λ = 0.1488 nm) and 6A(λ = 0.1500 nm) of Photon Factory in KEK (Japan). The two-dimensional SAXS intensity was detectedusing PILATUS3 2M and 1M. A series of X-ray structure analyses were performed using homemade
GUI software [38]. Full width at half maximum (FWHM) was determined by fitting with thepseudo-Voigt function to calculate the degree of orientation, which indicates the orientation states ofthe phage molecule in entire film, as in the following equation:
Degree of orientation =180 − FWHM
180

(2)

3. Results and Discussion
M13 phage-based films of highly oriented liquid crystalline assembled structures with highthermal diffusivity were prepared according to a previously reported flow-induced method on a
glass plate that had been circularly patterned with a hydrophobic coating based on fluororesin [34](Figure 1b). We have previously found that the film thickness at the outside edge of the circularpattern (outside) is thicker than at the center (center) or part-way between the center and the outside(midpoint). This observation can be explained due to solute condensation that occurred by capillary
flow induced by the differential evaporation rates across the drop at the outside of the pattern (so-called
coffee-ring effect), resulting in different assembled structures [39]. It is noted that near the center,the film breaks when it is peeled off the glass substrate in order to measure the thermal diffusivity.
In a control experiment, a non-oriented film composed of M13 phages was also prepared by a simple
solution casting method. In military electronics, the desired high temperature target is 125 ◦ C, whereas
in automotive applications, it is 140 ◦ C [40]. Therefore, the films were thermally treated at 150 ◦ C for
30 min to evaluate their thermal stability. Rare differences were visually observed before and afterthermal treatment (Figure 1c).
To characterize the effects of the thermal treatment on the thermal conductive properties of the M13
phage-based assemblies, thermal diffusivity values of the phage films at the three positions (outside,
midpoint, and center) were measured by temperature wave analysis (Figure 2a). It was previously
found that the thermal diffusivity value on the outside of the as-prepared film was approximately 10
times greater than that of non-oriented films due to the formation of extremely oriented structures in theassemblies, which might lead to a decrease in phonon scattering at structural defects [34]. The thermal

Viruses 2018, 10, 608

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diffusivity value at the outside of the thermally treated films was the same within experimental error
as the as prepared films (that is, untreated films). On the other hand, the thermal treatment caused
Viruses
2018, 10, x FOR
PEER REVIEW
4 of 10 wellthermaldiffusivity
valuesto slightly decrease at the midpoint and center of the oriented films
Viruses
2018, 10, x FOR
PEER REVIEW
4 of as10
as across the non-oriented film (Figure 2a, inset). Importantly, thermal diffusivity values showed noand center of the oriented films as well as across the non-oriented film (Figure 2a, inset). Importantly,
andchanges
center of when
the oriented
as wellas across thenon-oriented(Figure 2a,demonstrating
inset). Importantly,
further
cyclicfilms
testingof thermal
treatments
wasfilm
performed,
thermalthermal diffusivity values showed no further changes when cyclic testing of thermal treatments wasthermaldiffusivity
valuesshowednofurtherchangeswhencyclictestingofthermaltreatments
wasstabilityof the films(Figure 2b).
Therefore,
wasthat
the assembled
structures at theperformed,
demonstrating
thermalstabilityitof
the demonstrated
films (Figure 2b).
Therefore,
it was demonstrated
performed, demonstrating thermal stability of the films (Figure 2b). Therefore, it was demonstrated
outside
of
the
film
are
essential
tomaintainthethermaldiffusivity
after
the
thermaltreatment(details
that the assembled structures at the outside of the film are essential to maintain the thermal diffusivity
that the assembled structures at the outside of the film are essential to maintain the thermal diffusivity
are discussed
later). treatment (details are discussed later).
after the thermal
after the thermal treatment (details are discussed later).

Figure1. Schematic
illustrationof (a)
bacteriophage(M13
phage)
and(b)(b)
preparation
Figure1. Schematic
illustrationof M13(a) M13
bacteriophage(M13
phage)
andpreparationofofthe
thephage
Figure 1. Schematic illustration of (a) M13 bacteriophage (M13 phage) and (b) preparation of thefilm. phage(c) Opticalphotographsof the as prepared(left) and(left)
the andthermallytreatedtreated(right)(right)
films composed
film. (c)Optical photographsof the as prepared
the thermallyfilms
phage film. (c) Optical photographs of the as prepared (left) and the thermally treated (right) filmsof liquid
crystalline
oriented
M13oriented
phages.M13 phages.
composedof liquid
crystalline
composed of liquid crystalline oriented M13 phages.

Figure(a) Comparison
thermaldiffusivity
diffusivity values
values ofandandthermallytreatedM13 M13
Figure2. (a)2.Comparison
of ofthermalofthe
theasaspreparedpreparedthermallytreatedFigure 2. (a) Comparison of thermal diffusivity values of the as prepared and thermally treated M13
phagefilms.
Thermaldiffusivity
valuesof theaspreparedfilmswere
weretaken
takenfromfromref.
ref. 34.
34. Pink
Pink andphagephagefilms.films.
Thermaldiffusivity
valuesoftheaspreparedfilms
and blue
Thermal diffusivity values of the as prepared films were taken from ref. 34. Pink andblue bars represent
thermaldiffusivity
valuesofthe thermallytreatedandtheas-preparedM13
M13phage
phage films.
bars represent
thermaldiffusivity
valuesofthethermallytreatedandtheas-preparedblue bars represent thermal diffusivity values of the thermally treated and the as-prepared M13 phagefilms.
Inset represents
expandedview for the midpoint,
center,and non-oriented
M13 phage
films.
Inset films.
represents
expandedexpanded
view for thecenter, andnon-oriented
M13 phage
films.films.
(b) Cyclic
Inset represents
viewmidpoint,
for the midpoint,
center,and non-oriented
M13 phage(b) Cyclic testing of thermal diffusivity of the films against thermal treatment. Thermal treatments
testing
thermaldiffusivityof diffusivity
the films against
thermaltreatment.
Thermal treatments
and thermal(b)ofCyclictestingof thermalof the films
againstthermal treatment.
Thermal treatments
and thermal diffusivity measurements were repeated for 5 times.
and thermal
diffusivity were
measurements
diffusivity
measurements
repeatedwerefor 5repeatedtimes. for 5 times.

POM was performed to evaluate the ordered structures of the M13 phages in the films before

performed
to evaluate
orderedstructuresofofthe
theM13M13phages
phages inin thethe films
POMPOMwas wasperformed
to evaluate
thetheorderedstructures
films before
before andand after thermal treatment. In the POM images before thermal treatment, it was observed that theafter thermal
treatment.
In
the POM
imagesbeforethermaltreatment,
it wasobserved
observedthat
that thetheafter andthermaltreatment.
In
the
POM
imagesbeforethermaltreatment,itwas
M13 phage films possessed differently oriented structures at the different positions on the film, as M13
M13
phage
films
possessed
differently
oriented
structuresatthedifferent
positions
on
the
film,
as
phagepreviously
films possessedoriented
structures
the phages
differenton theshowed
film, ashighly
previously
reporteddifferently[34] (Figure
3a–c, inset).
In brief,atM13
onpositions
the film outside
previously reported [34] (Figure 3a–c, inset). In brief, M13 phages on the film outside showed highly
reported[34]smectic(Figureliquid
3a–c,crystal
inset).structures
In brief, throughM13 phageson layered
the filmbirefringenceoutside showed
highlyorientedorienteda clearly(Figure3a, inset).
oriented smectic liquid crystal structures through a clearly layered birefringence (Figure 3a, inset).
On liquid
the otherhand,oriented throughstructuresa consistingof nematic
liquid crystals
weretheothersmectic
crystalstructures
clearly layeredbirefringence(Figure3a, observed
inset). Onatthe
On the other hand, oriented structures consisting of nematic liquid crystals were observed at the
midpoint and center positions of the film due to the absence of layered structures (Figure 3b,c, inset).
midpoint and center positions of the film due to the absence of layered structures (Figure 3b,c, inset).

Viruses 2018, 10, 608

5 of 10

hand, oriented structures consisting of nematic liquid crystals were observed at the midpoint andcenter positions of the film due to the absence of layered structures (Figure 3b,c, inset). The orientation
regularity was higher at the outside of the film. Importantly, the POM images after thermal treatment
Viruses 2018, 10, x FOR PEER REVIEW
5 of 10
exhibited the same oriented states at all three positions (Figure 3). Therefore, thermal treatment under◦ C for 30 min) did not affect the liquid crystalline oriented states of
the conditions
appliedhere, (150
The orientation
regularity
was higher
at the outside of the film. Importantly, the POM images afterM13 phages
the exhibitedwhole of thethe same
film atthe POM
scale.
thermal acrosstreatmentorientedstatesat all three positions (Figure 3). Therefore,The
assembled
structures
of
the
M13
phagesinthecharacterizedbytheatomicthermal treatment under the conditions applied here, (150films°C forwere
30 min)
did not affectliquidforce
crystalline
orientedstatesthermalof M13 phages
across layered
the wholedomainsof the filmwith
at thea POM
scale.
microscopy(AFM).
Beforetreatment,
width
of approximately 1
Thecorresponds
assembled structuresof theof
M13
in the films
characterized
by atomicofforceµm, whichto the length
thephages
M13 phage,
werewereobserved
at the outside
the film,
microscopy(AFM).
Beforethermaltreatment, on
layereddomains withscale,a widthapproximately
1 µm, [34]supporting
smectic
liquidcrystalorientation
the micrometer
asofpreviously
reported
which
to the length
the M13 phage,
were observedat thewith
outsideof the(Figure3d). corresponds
Similarly, nematic
crystaloforientationof the assembled
domainsa widthof film,
0.5–1 µm
supporting smectic liquid crystal orientation on the micrometer scale, as previously reported [34]and a length of approximately 1 µm was observed at the midpoint and center positions (Figure 3e,f).
(Figure 3d). Similarly, nematic crystal orientation of the assembled domains with a width of 0.5–1 µm
Therefore, the M13 phages in the film were oriented in a plane direction and the orientation regularity
and a length of approximately 1 µm was observed at the midpoint and center positions (Figure 3e,f).
was higher
at thetheM13outsideofintheas observed
the POM
studies.
thermaltreatment,
Therefore,
phagesthefilm,
film wereoriented inina planedirectionand theAfterorientation
regularity
the layered
domainsattheoutsidewere
maintainedeventhoughthebendingofthewas higher at the outside of the film, as observed in the POM studies. After thermal treatment,domains
thedisappeared(Figure 3g),
suggestingthat maintained
the M13 phages
still formed
smectic
crystalline
layered domains
at theoutside wereeven though
the bendingof liquid
the domains
disappeared(Figure 3g),that the M13 domains
phages still
smectic andliquidcrystalline
orientation.
In contrast,
thesuggestingnematically-oriented
at formed
the midpoint
centerpositions
In contrast,
the nematically-oriented
at themidpoint
center positions
were ofwere orientation.
more packed
or aggregatedafter the thermaldomains
treatment(Figure3h,i),andindicatingthat relaxation
more packed or aggregatedafter the thermal
treatment (Figure3h,i),
indicating that
relaxationof thethe smectic-oriented
domain structures
was suppressed.
These
observations
suggestedthatsmectic
smectic-oriented
domainstructureswassuppressed.
These
observations
suggestedthatsmectic
liquid crystalline oriented states of the assembled M13 phages on the film outside appears to affect theliquid crystalline oriented states of the assembled M13 phages on the film outside appears to affectthermal diffusivity values.
the thermal diffusivity values.

Figure 3. Microscopic observations of the M13 phage films prepared by flow-induced orientation
Figure3. Microscopic observations of the M13 phage films prepared by flow-induced orientation
methods. (a–c) Polarized optical microscopy (POM) images of the thermally treated M13 phage films.
methods. (a–c) Polarized optical microscopy (POM) images of the thermally treated M13 phage films.
(a) Outside, (b) midpoint, and (c) center positions of the film were observed. Insets represent the POM(a) Outside, (b) midpoint, and (c) center positions of the film were observed. Insets represent the POM
images of the as prepared M13 phage films at the same positions. (d–i) Atomic force microscopy
imagesof theas prepared
phage
films
the sametreatedpositions.
(d–i)phage
Atomicforce
microscopy
(AFM)
(AFM)
imagesof the asM13
prepared(d–f)
andatthermally(g–i) M13
films.
Images(d,g) wereimagesoftheasprepared(d–f)
andthermallytreated
(g–i)
M13
phage
films.
Images(d,g)
were
taken from the outside, (e,h) the midpoint, and (f,i) the center of the film. Some of the observedtakenfrom domains
the outside,
the midpoint,
and oblongs(f,i) theincenterof(d–f).
the film.
Somethe observed
domainshave (e,h)
been highlighted
by white
imagesScale bars
areofshownin the images.
have been highlighted by white oblongs in images (d–f). Scale bars are shown in the images.
The secondary structures of the M13 phages in the film before and after the thermal treatment
were characterized by attenuated total refraction (ATR)/IR spectroscopy measurements (Figure 4a–c). The IR spectra revealed two sharp peaks at approximately 1535 and 1630 cm−1 in all cases, which

Viruses 2018, 10, 608

6 of 10The secondary structures of the M13 phages in the film before and after the thermal treatment
were characterized by attenuated total refraction (ATR)/IR spectroscopy measurements (Figure 4a–c).
The IR spectra revealed two sharp peaks at approximately 1535 and 1630 cm−1 in all cases, which wereprimarily assigned to the α-helix structures of N-H bending (amide II) and C=O stretching (amide
Viruses 2018, 10, x FOR PEER REVIEW
6 of 10I) [41], respectively. After thermal treatment, peaks assignable to other secondary structures, such as
the β-sheet
conformation
ortorandom
coil structures
structures,
not observed.
the positions
were primarily
assigned
the α-helix
of wereN-H bending(amide Importantly,II) and C=O stretching(amide I) [41],respectively.
After thermal
treatment, peaks
assignable
other secondary
structures,and half-band
widthsof the peaks
were unchangedby thethermaltotreatment,suggestingthat the
such
as
the
β-sheetconformationor
randomcoil
structures,were
notobserved.
Importantly,
theoriginal α-helical conformation was maintained. These results indicated that the slight structural
positionsandhalf-band
widths
of
the
peaks
were
unchangedbythethermaltreatment,suggestingchanges, observed on the micrometer scale after thermal treatment, were not caused by changes inthat the original α-helical conformation was maintained. These results indicated that thesecondarystructures. Such structural stability in proteins against high temperatures (150 ◦ C)slight
generally
structural changes, observed on the micrometer scale after thermal treatment, were not caused bycannot be explained by the structural and/or conformational stability of proteins. On the other hand,changes in secondary structures. Such structural stability in proteins against high temperatures (150
the thermal
stability of the secondary structures of M13 phage molecules in solution was characterized°C) generally cannot be explained by the structural and/or conformational stability of proteins. On
by circulardichroism
spectroscopy4d,e). The
CD spectra
negativeinCotton
effects
the other hand, the(CD)thermalstability of(Figure
the secondary
structuresof M13revealed
phage molecules
solutionat 208wasandcharacterized
222 nm (Figure4d), whichwere (CD)assigned
to α-helicalstructures
corresponding
to the major
by circulardichroism
spectroscopy(Figure4d,e). The
CD spectra revealed
coat proteinsof M13effects
phages.
Cotton4d),
effects
decreased
withtoincreasing
temperature,negative Cotton
at 208The
andnegative
222 nm (Figure
whichwere assigned
α-helical structures
corresponding
to the major
M13 phages.ItThe
negative
Cottondecreasedwithdemonstrating
deformation
ofcoatthe proteins
α-helicalofstructures.
is well
known
thateffects
the CDsignal at
222 nmincreasing
temperature,demonstratingof astheaα-helical
It is well known
that
correlates
to α-helicalformation;
thus, the deformation
signal values,
functionstructures.
of the temperature,
were obtained
the 4e).
CD signal
at 222 nm incorrelates
to α-helicalthus,signal values, asof the(FigureThe decrease
the Cotton
effect formation;is saturated
atthe
approximately
80a◦function
C, demonstrating
temperature, were obtained (Figure 4e). The decrease in the Cotton effect is saturated atthat deformation of the M13 phage structures (i.e., denaturing) in a solution progressed sufficiently
approximately 80 °C, demonstrating that deformation of the M13 phage structures (i.e., denaturing)by heating at 80 ◦ C. Therefore, the formation of liquid crystalline ordered structures in a solid state
in a solution progressed sufficiently by heating at 80 °C. Therefore, the formation of liquid crystalline
yieldedstructural stability against heating and resulted in the maintenance of high thermal diffusivity
ordered structures in a solid state yielded structural stability against heating and resulted in theafter maintenancethermal treatment.
of high thermal diffusivity after thermal treatment.

Figure4. Secondary
structural
analysesofofM13
M13 phages.
phages. (a–c)
totalreflection/infrared
Figure4. Secondary
structural
analyses(a–c)AttenuatedAttenuated
total
reflection/infrared(ATR/IR)
spectraof theoutside,(b)(b)midpoint,
midpoint, andfilms.
Solid
andanddottedlines lines(ATR/IR)
spectraof the(a)(a)
outside,and(c)(c)center
centerofofthe
thefilms.
Solid
dottedrepresent
thethermallytreatedandaspreparedfilms,
respectively.
(d,e)
Circulardichroism(CD)represent the thermally treated and as prepared films, respectively. (d,e) Circular dichroism (CD)
spectra of M13 phages in an aqueous solution. (d) CD spectra of M13 phages at different temperature
spectra of M13 phages in an aqueous solution. (d) CD spectra of M13 phages at different temperature(5–95 °C, every 5 °C). (e) CD intensity at 222 nm as a function of temperature. Phage concentration is(5–95 ◦ C, every 5 ◦ C). (e) CD intensity at 222 nm as a function of temperature. Phage concentration is
5 nM.
5 nM.

SAXS experiments were performed to examine the stabilities based on a detailed structural

SAXS experiments were performed to examine the stabilities based on a detailed structuralcharacterization of the films (Figure 5a–c). In the resulting scattering profiles of the outside of thecharacterization of the films (Figure 5a–c). In the resulting scattering profiles of the outside of thefilm, intense peaks at 8.21, 4.94, and 4.18 nm were observed with a reciprocal d-spacing ratio of 1:√3:2 √film, (Figure
intense5d),
peaks
at 8.21,
4.94,hexagonallyand 4.18 nm
werestructures
observedofwith
a reciprocal
d-spacingratio
of 1: 3:2
which
indicates
packedthe M13
phages. The
scatteringprofiles(Figureindicates
hexagonallystructuresof the ofM13
phages. The
scatteringprofilesfor5d),
the whichmidpoint
and centerof the filmpacked
were alsocharacteristic
hexagonally
packedstructures(Figure 5e,f). This packing structure of M13 phages at all positions on the thermally treated films wasViruses 2018, 10, 608

7 of 10for the midpoint and center of the film were also characteristic of hexagonally packed structures(Figure 5e,f). This packing structure of M13 phages at all positions on the thermally treated films was
the samethat
thePEERas preparedfilms, demonstrating that the packing of the M13 phages
Virusesto
2018,
10, ofx FOR
REVIEW
7 of 10 on amolecular level was essentially the same during the thermal treatment.
the sameto ofthatof the as preparedfilms, demonstratingthat theorientation
packing of the
M13wasphageson a toThe
degreeorientation,
which represents
the macroscopic
states,calculated
molecular level was essentially the same during the thermal treatment.
quantitatively investigate the macroscopic orientation of the structures by full width at half maximum
The degree of orientation, which represents the macroscopic orientation states, was calculatedof the azimuth scan of the primary peaks at approximately 8 nm (Figure 5g–i), fitting with theto quantitatively investigate the macroscopic orientation of the structures by full width at half
pseudo-Voigt
function
accordingprevious[34].
The degree8ofnm
orientationof theM13
phagesmaximumof the azimuth
scantoofatheprimaryreportpeaks atapproximately(Figure 5g–i),
fittingwithin thetheentirefilm
ranged
from
0
to
1
(a
highervalue
representsahigherorientation
state).
The
resultantpseudo-Voigt function according to a previous report [34]. The degree of orientation of the M13
degreeof orientation
atfilmthe ranged
outsidefromof theafter thermal
treatment(0.76)orientation
was comparable
to thatphagesin the entire
0 tofilm
1 (a higher
value represents
a higherstate). The
orientation
the outsidefilmstructural
after thermal
treatment (0.76) wasof theresultant
outsidedegreeof theofas
preparedatfilms(0.78).of theThesecharacterizationsof comparable
the assemblies
to that of thatthe outsideof theliquid
as preparedfilmsoriented(0.78). These
structuralcharacterizationsof outsidethedemonstrated
the smectic
crystalline
assembliesof M13
phages at thesmectictreatment,
liquid crystalline
orientedassembliesof M13 phages
at the theof theassemblies
film weredemonstrated
not affectedthat
bythe
thermalindicating
structural
stability.
In contrast,
outsideof the film values
were notataffectedby thermal
treatment,
indicating
structural
stability.treatment
In contrast,weredegreeof orientation
the midpoint(0.18)and center(0.06)
after thermal
the degree of orientation values at the midpoint (0.18) and center (0.06) after thermal treatment werecomparable or lower than those before treatment (0.19 and 0.01), respectively. The observed structuralcomparable or lower than those before treatment (0.19 and 0.01), respectively. The observed
properties in the nematically oriented assemblies led to the scattering of phonons, possibly due tostructural properties in the nematically oriented assemblies led to the scattering of phonons, possibly
the presenceof structural
Therefore,
the hierarchicalassemblyassemblyof smectic
liquid crystalline
due to thepresence ofdefects.
structuraldefects. Therefore,
the hierarchicalof smectic
liquidoriented
M13
phagesplays
animportantrole
inprovidingstability
againstheatinginorder
to achieve
crystalline oriented M13 phages plays an important role in providing stability against
heatinginreliablethermally
conductive
soft materialsof biomolecularassemblies.
order
to achieve
reliable thermallyconductive
soft materials of
biomolecular assemblies.

Figure5. 5.
Smallscattering
analysesof thermallythe thermally
M13
phage
FigureSmallangle
angle X-ray
X-ray scattering
analysesof thetreated treated
M13 phage
films.
Two-films.
Two-dimensional(2D)
patterns
for
the
(a)
outside,(b)
midpoint,and(c)center
of
the
film.
The
scattering
dimensional (2D) patterns for the (a) outside, (b) midpoint, and (c) center of the film. The scattering
curvesfor the(e) (e)midpoint,
thefilm.
film.Azimuth
Azimuthscanof theprimary
peaks
curvesfor(d)
theoutside,(d) outside,
midpoint,andand(f)(f)center
center of thescanof theprimary
peaks
for thefor(g)(h) midpoint,and(i)(i)center
Azimuthisiszero
zero
upper
direction
theoutside,(g) outside,(h) midpoint,andcenterof
ofthe
the film.
film. Azimuth
in in
thetheupper
direction
fromfrom
the centerand increases
a clockwise
directionin
inthe
the 2D
scattering(SAXS)
images.
the centerand increases
in ainclockwise
direction
2D smallsmallangle
angleX-ray
X-ray
scattering(SAXS)
images.

4. Conclusions
4. Conclusions
We investigated
the thermal
diffusivityof thermallytreated
M13M13
phage-based
assemblies(films).
We investigated
the thermal
diffusivityof thermallytreatedphage-based
assemblies(films).
Thermal measurements
diffusivity measurements
demonstrated
thethermalhigh thermal
diffusivityof smectic
Thermaldiffusivity
demonstrated
that thethat
highdiffusivityof smectic
liquidliquid oriented
crystallineassemblies
oriented assembliesof M13 (edgephagesof(edgeof thecouldfilm) could
be preparedthroughcrystallineof M13 phages
the film)
be preparedthroughsuitablesuitableassembly,
convectiveremained
assembly, remained
unchanged
by thermal
treatment°C formin)
andwere◦ C forconvective
unchanged
by thermal
treatment(150 (150
30 30
min)
and
were approximately 10 times greater than those of nematic liquid crystalline oriented assemblies
approximately 10 times greater than those of nematic liquid crystalline oriented assemblies (midpoint(midpoint and center of the film) and a non-oriented cast film. Thermal diffusivity values at theand center of the film) and a non-oriented cast film. Thermal diffusivity values at the midpoint andmidpoint and center positions, as well as for the non-oriented film, slightly decreased upon thermal
centertreatment.
positions,Structural
as well as
for the non-oriented
film, slightly
decreased
upon thermal
characterization
by microscopic
observation,
IR spectroscopy,
and treatment.
SAXS

Viruses 2018, 10, 608

8 of 10Structural characterization by microscopic observation, IR spectroscopy, and SAXS indicated that thestructures of the smectic liquid crystalline assemblies with high thermal diffusivity were unaffected by
the thermal treatment, which was in contrast to the nematic liquid crystalline oriented assemblies (withlower thermal diffusivity), in which the packing or aggregated states of the assembled domains
changed over the micrometer scale. Even though the M13 phage is composed of biomolecular
proteins, the smectic liquid crystalline orientation caused remarkable thermal stability. Additionally,
our results indicating that ordered biomolecular assemblies were related to their thermal stability
might help explain the evolution of life in high temperature environments, such as thermal vents.
This biomolecule-based structurally regular assembly will open up attractive opportunities for the
next generation of reliable thermal conductive materials composed of biomacromolecular assemblies.
Author Contributions: T.S. (Toshiki Sawada) and T.S. (Takeshi Serizawa) directed and designed the project. T.S.
(Toshiki Sawada) conceived and designed the experiments. T.S. (Toshiki Sawada) and Y.M. analyzed the data.
Y.M. performed the experiments. J.M. directed the thermal diffusivity measurements. H.M. and S.N. directed the
SAXS measurements. T.S. (Toshiki Sawada) and T.S. (Takeshi Serizawa) co-wrote the paper.
Funding: This research was funded by the Japan Science and Technology Agency (JST) through the Precursory
Research for Embryonic Science and Technology (PRESTO) Grant Number JPMJPR17I4 to Toshiki Sawada.
Acknowledgments: Toshiki Sawada is grateful to the Challenging Research Award from Tokyo Insitute ofTechnology. The SAXS measurements were performed under the approval of Photon Factory Advisory Committee(Nos. 2014G011 and 2015G076).
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.
4.

5.
6.
7.

8.
9.
10.
11.
12.

13.

Chae, H.G.; Kumar, S. Materials science. Making strong fibers. Science 2008, 319, 908–909. [CrossRef][PubMed]Winey, K.I.; Kashiwagi, T.; Mu, M.F. Improving electrical conductivity and thermal properties of polymers
by the addition of carbon nanotubes as fillers. MRS Bull. 2007, 32, 348–353. [CrossRef]
Wang, X.; Ho, V.; Segalman, R.A.; Cahill, D.G. Thermal conductivity of high-modulus polymer fibers.
Macromolecules 2013, 46, 4937–4943. [CrossRef]
Singh, V.; Bougher, T.L.; Weathers, A.; Cai, Y.; Bi, K.; Pettes, M.T.; McMenamin, S.A.; Lv, W.; Resler, D.P.;
Gattuso, T.R.; et al. High thermal conductivity of chain-oriented amorphous polythiophene. Nat. Nanotechnol.
2014, 9, 384–390. [CrossRef] [PubMed]
Lim, C.T.; Tan, E.P.S.; Ng, S.Y. Effects of crystalline morphology on the tensile properties of electrospun
polymer nanofibers. Appl. Phys. Lett. 2008, 92, 141908. [CrossRef]
Shen, S.; Henry, A.; Tong, J.; Zheng, R.; Chen, G. Polyethylene nanofibres with very high thermal
conductivities. Nat. Nanotechnol. 2010, 5, 251–255. [CrossRef] [PubMed]
Kim, G.-H.; Lee, D.; Shanker, A.; Shao, L.; Kwon, M.; Gidley, D.; Kim, J.; Pipe, K.P. High thermal conductivity
in amorphous polymer blends by engineered interchain interactions. Nat. Mater. 2015, 14, 295–300.
[CrossRef] [PubMed]
Huang, X.; Liu, G.; Wang, X. New secrets of spider silk: Exceptionally high thermal conductivity and its
abnormal change under stretching. Adv. Mater. 2012, 24, 1482–1486. [CrossRef] [PubMed]
Henry, A. Thermal transport in polymers. Annu. Rev. Heat Transf. 2014, 17, 485–520. [CrossRef]
Wu, L.; Lee, A.L.; Niu, Z.; Ghoshroy, S.; Wang, Q. Visualizing cell extracellular matrix (ECM) deposited bycells cultured on aligned bacteriophage M13 thin films. Langmuir 2011, 27, 9490–9496. [CrossRef] [PubMed]
Yang, S.; Chung, W.-J.; McFarland, S.; Lee, S.-W. Assembly of bacteriophage into functional materials.
Chem. Rec. 2013, 13, 43–59. [CrossRef] [PubMed]
Courchesne, N.-M.M.; Klug, M.T.; Chen, P.-Y.Y.; Kooi, S.E.; Yun, D.S.; Hong, N.; Fang, N.X.; Belcher, A.M.;
Hammond, P.T. Assembly of a bacteriophage-based template for the organization of materials into
nanoporous networks. Adv. Mater. 2014, 26, 3398–3404. [CrossRef] [PubMed]
Bardhan, N.M.; Ghosh, D.; Belcher, A.M. Carbon nanotubes as in vivo bacterial probes. Nat. Commun. 2014,
5, 4918. [CrossRef] [PubMed]

Viruses 2018, 10, 608

14.

15.
16.
17.
18.
19.
20.
21.
22.
23.
24.

25.
26.
27.
28.
29.

30.

31.

32.
33.
34.
35.

36.

9 of 10

Moghimian, P.; Srot, V.; Rothenstein, D.; Facey, S.J.; Harnau, L.; Hauer, B.; Bill, J.; Aken, P.A.V. Adsorption
and self-assembly of M13 phage into directionally organized structures on C and SiO2 films. Langmuir 2014,
30, 11428–11432. [CrossRef] [PubMed]
Lee, J.; Jin, H.-E.; Desai, M.S.; Ren, S.; Kim, S.; Lee, S.-W. Biomimetic sensor design. Nanoscale 2015, 7,
18379–18391. [CrossRef] [PubMed]
Sawada, T.; Asada, M.; Serizawa, T. Selective rare earth recovery employing filamentous viruses withchemically conjugated peptides. ChemistrySelect 2016, 1, 2712–2716. [CrossRef]
Moghimian, P.; Harnau, L.; Srot, V.; de la Peña, F.; Bafi, N.; Facey, S.J.; van Aken, P.A. Controlled self-assemblyof biomolecular rods on structured substrates. Soft Matter 2016, 12, 3177–3183. [CrossRef] [PubMed]
Devaraj, V.; Han, J.; Kim, C.; Kang, Y.-C.C.; Oh, J.-W.W. Self-assembled nanoporous biofilms fromfunctionalized nanofibrous M13 bacteriophage. Viruses 2018, 10, 322. [CrossRef] [PubMed]
Smith, G.P.; Petrenko, V.A. Phage display. Chem. Rev. 1997, 97, 391–410. [CrossRef] [PubMed]
Mohan, K.; Weiss, G.A. Chemically modifying viruses for diverse applications. ACS Chem. Biol. 2016, 11,1167–1179. [CrossRef] [PubMed]
Dogic, Z.; Fraden, S. Ordered phases of filamentous viruses. Curr. Opin. Colloid Interface Sci. 2006, 11, 47–55.
[CrossRef]
Dogic, Z.; Sharma, P.; Zakhary, M.J. Hypercomplex liquid crystals. Annu. Rev. Condens. Matter Phys. 2014, 5,
137–157. [CrossRef]
Zhang, Z.; Grelet, E. Tuning chirality in the self-assembly of rod-like viruses by chemical surface
modifications. Soft Matter 2012, 9, 1015–1024. [CrossRef]
Zan, T.; Wu, F.; Pei, X.; Jia, S.; Zhang, R.; Wu, S.; Niu, Z.; Zhang, Z. Into the polymer brush regime throughthe “grafting-to” method: Densely polymer-grafted rodlike viruses with an unusual nematic liquid crystal
behavior. Soft Matter 2015, 12, 798–805. [CrossRef] [PubMed]
Chung, W.-J.; Oh, J.-W.; Kwak, K.; Lee, B.; Meyer, J.; Wang, E.; Hexemer, A.; Lee, S.-W. Biomimetic
self-templating supramolecular structures. Nature 2011, 478, 364–368. [CrossRef] [PubMed]
Lee, B.; Zhang, J.; Zueger, C.; Chung, W.-J.; Yoo, S.; Wang, E.; Meyer, J.; Ramesh, R.; Lee, S.-W. Virus-based
piezoelectric energy generation. Nat. Nanotechnol. 2012, 7, 351–356. [CrossRef] [PubMed]
Sawada, T.; Serizawa, T. Immobilization of highly-oriented filamentous viruses onto polymer substrates.
J. Mater. Chem. B 2013, 1, 149–152. [CrossRef]
Sawada, T.; Kang, S.; Watanabe, J.; Mihara, H.; Serizawa, T. Hybrid hydrogels composed of regularly
assembled filamentous viruses and gold nanoparticles. ACS Macro Lett. 2014, 3, 341–345. [CrossRef]
Sawada, T.; Chen, H.; Shirakawa, N.; Kang, S.; Watanabe, J.; Serizawa, T. Regular assembly of filamentous
viruses and gold nanoparticles by specific interactions and subsequent chemical crosslinking. Polym. J. 2014,
46, 511–515. [CrossRef]
Sawada, T.; Otsuka, H.; Yui, H.; Serizawa, T. Preparation and characterization of hybrid hydrogels composedof physically cross-linked gelatin and liquid-crystalline filamentous viruses. Polym. Bull. 2015, 72, 1487–1496.
[CrossRef]
Sawada, T.; Yanagimachi, M.; Serizawa, T. Controlled release of antibody proteins from liquid crystallinehydrogels composed of genetically engineered filamentous viruses. Mater. Chem. Front. 2017, 1, 146–151.
[CrossRef]
Sawada, T. Filamentous virus-based soft materials based on controlled assembly through liquid crystalline
formation. Polym. J. 2017, 49, 639–647. [CrossRef]
Sawada, T.; Serizawa, T. Filamentous viruses as building blocks for hierarchical self-assembly towardfunctional soft materials. Bull. Chem. Soc. Jpn. 2018, 91, 455–466. [CrossRef]
Sawada, T.; Murata, Y.; Marubayashi, H.; Nojima, S.; Morikawa, J.; Serizawa, T. Filamentous virus-based
assembly: Their oriented structures and thermal diffusivity. Sci. Rep. 2018, 8, 5412. [CrossRef] [PubMed]
Poklar, N.; Lah, J.; Salobir, M.; Maček, P.; Vesnaver, G. pH and temperature-induced molten globule-like
denatured states of equinatoxin II: A study by UV-melting, DSC, far- and near-UV CD spectroscopy, and
ANS fluorescence. Biochemistry 1997, 36, 14345–14352. [CrossRef] [PubMed]
Borzova, V.A.; Markossian, K.A.; Chebotareva, N.A.; Kleymenov, S.; Poliansky, N.B.; Muranov, K.O.;
Stein-Margolina, V.A.; Shubin, V.V.; Markov, D.I.; Kurganov, B.I. Kinetics of thermal denaturation andaggregation of bovine serum albumin. PLoS ONE 2016, 11, e0153495. [CrossRef] [PubMed]

Viruses 2018, 10, 608

37.
38.
39.
40.
41.

10 of 10Morikawa, J.; Hashimoto, T. Thermal diffusivity of aromatic polyimide thin films by temperature wave
analysis. J. Appl. Phys. 2009, 105, 113506. [CrossRef]
Marubayashi, H.; Asai, S.; Sumita, M. Complex crystal formation of poly(L-lactide) with solvent molecules.
Macromolecules 2012, 45, 1384–1397. [CrossRef]
Deegan, R.D.; Bakajin, O.; Dupont, T.F.; Huber, G.; Nagel, S.R.; Witten, T.A. Capillary flow as the cause ofring stains from dried liquid drops. Nature 1997, 389, 827–829. [CrossRef]
Lu, G.; Hong, W.; Tong, L.; Bai, H.; Wei, Y.; Shi, G. Drying enhanced adhesion of polythiophene nanotubule
arrays on smooth surfaces. ACS Nano 2008, 2, 2342–2348. [CrossRef] [PubMed]
Haris, P.I.; Chapman, D. The conformational analysis of peptides using Fourier transform IR spectroscopy.
Biopolymers 1995, 37, 251–263. [CrossRef] [PubMed]© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).