Transient self-templating assembly of M13 bacteriophage for enhanced biopiezoelectric devices

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Nano Energy
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Transient self-templating assembly of M13 bacteriophage for enhanced
biopiezoelectric devices
Kwang Heoa,b,c,1, Hyo-Eon Jina,b,c,2, Han Kima,b,c, Ju Hun Leea,b,c, Eddie Wanga,b,c,
Seung-Wuk Leea,b,c,⁎
a Department of Bioengineering, University of California, Berkeley, Tsinghua Berkeley Shenzhen Institute, Berkeley, USA
b Berkeley Nanoscience and Nanoengineering Institute, Berkeley, Applied Science & Technology, College of Engineering, University of California, Berkeley, USA
c Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, CA 94720 USA
A R T I C L E I N F O
Keywords:
Biopiezoelectricity
Phage engineering
Self-assembly
Energy harvesting
Self-templating assembly
Biosensor
A B S T R A C T
Many biogenic materials are hierarchically structured across several length scales. Attaining similar control over
bottom-up assembly would allow for efficient synthesis of materials for enhanced electrical or energy generating
devices. Here we developed a transient self-templating assembly process inspired by nature for the synthesis of
enhanced biopiezoelectric devices. We used M13 bacteriophage (phage) as a piezoelectric nanofiber building
block and created hierarchically organized nano- and micro-structures for enhanced piezoelectric devices. We
controlled the self-assembly of M13 phage by exploiting transiently forming, periodic menisci. Each micron-
sized meniscus directed the local formation of hexagonally packed nanostructures that further assembled into
macroscopic, hierarchical structures. By tuning deposition parameters during the transient self-templating as-
sembly process, we created diverse array of phage nanostructures. The resulting hierarchically organized phage
nanostructure exhibited enhanced piezoelectric energy generation performance. Our transient self-templating
assembly process can assemble many other energy generating colloidal nanoparticles into ordered nanos-
tructures for the development of optical, mechanical, and electrical materials in the future in an energy efficient
manner.
1. Introduction
Many natural biomaterials have exquisite physical, optical, and
mechanical properties that scientists strive to replicate. These features
are often attributable to hierarchical ordering: organized structure at
the nano-, micro-, and macro- length scales. Some examples include
butterfly wings, beetle exocuticles, diatom skeletons, and mantis
shrimp hammers [1–4]. Achieving similar complexity by bottom-up
self-assembly in the laboratory is highly desirable because it would
enable to synthesize advanced materials and devices at large scales in
an energy efficient manner. Recent advances in material engineering
involving self-assembled small molecules, polymers, and particles have
led to the creation of functional optical and electrical structures and
devices [5–18]. In addition, techniques that exploit some of nature's
strategies such as inducing chiral liquid crystalline organization and
self-assembly have achieved a degree of hierarchical ordering with
desirable properties [19,20]. However, limited control over the tem-
poral and spatial resolution during self-assembly has limited the
structural complexity and material properties to fabricate functional
nanodevices. Here we developed a transient self-templating assembly
process that is inspired by butterfly wing morphogenesis process for the
synthesis of enhanced biopiezoelectric devices. We used M13 bacter-
iophage (phage) as a biopiezoelectric nanofiber model system and
fabricated hierarchically organized phage structures for the develop-
ment of enhanced piezoelectric devices. We exploited transiently
forming, periodic menisci to control the self-assembly of M13 phage.
Each micron-sized meniscus directed the local formation of self-as-
sembled nanostructures that further assembled into macroscopic,
hierarchical structures. By tuning deposition parameters during the self-
assembly process, we created a diverse array of nanostructures in large
scale. The resulting hierarchically organized phage nanostructure ex-
hibited enhanced piezoelectric performance. Our newly developed
https://doi.org/10.1016/j.nanoen.2018.11.084
Received 4 October 2018; Received in revised form 11 November 2018; Accepted 27 November 2018
⁎ Corresponding author at: Department of Bioengineering, University of California, Berkeley, Tsinghua Berkeley Shenzhen Institute, Berkeley, USA.
E-mail address: leesw@berkeley.edu (S.-W. Lee).
1 Present Addresses Department of Nanotechnology and Advanced Materials Engineering, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 05006, Republic
of Korea.
2 College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea.
Nano Energy 56 (2019) 716–723
Available online 04 December 2018
2211-2855/ Published by Elsevier Ltd.
T
transient self-templating assembly process can potentially be expanded
to self-assemble many other energy generating colloidal nanoparticles
into ordered structures for the development of optical, mechanical, and
electrical materials in an energy efficient manner.
2. Experimental section
2.1. Construction of engineered phage
M13 phage was genetically engineered using recombinant DNA
engineering methods as previously described [19]. In short, two, three
and four glutamate (2E, 3E and 4E peptides) were expressed at the N-
terminus of the M13 major coat protein pVIII, replacing residues 2–4
(Ala-Glu-Gly-Asp-Asp with Ala-(Insert)-Asp). To facilitate ligation, a
PstI restriction site (CTGCAG) was modified by mutating the nucleic
acid at position 1372 of the M13KE vector (NEBiolabs, Ipswich, MA)
from T to A by mutagenesis (Quick Change® Kit, Stratagene, La Jolla,
CA). To create pVIII insertions, insertion primer was designed to insert
their respective peptide, 5′-ATATATCTGCAGGAAGAAGAGGAGCCCGC
AAAAGCGGCCTTTAACTCCC-3′ for 4E-phage (insert GAA or GAG
codon for glutamate, insert underlined, EEEE shown in italics). 5′-GCT
GTCTTTCGCTGCAGAGGGTG-3′ was used as the linearization primer
(IDT, Coralville, IA). We incorporated the desired gene sequences by
polymerase chain reaction (PCR) using Phusion™ High-Fidelity DNA
polymerase (NEBiolabs, Ipswich, MA), two primers (insertion and lin-
earization), and the M13KE vector with the engineered PstI site as the
template. Purified PCR product was digested with PstI (NEBiolabs,
Ipswich, MA) and ligated at 16 °C with T4 DNA ligase (NEBiolabs,
Ipswich, MA) and transformed into XL1-Blue electrocompetent cells
(Stratagene, La Jolla, CA). The sequences of the products were con-
firmed by DNA sequencing at the University of California, Berkeley
DNA sequencing facility (Berkeley, CA).
2.2. Phage mass amplification
A single plaque of the desired phage was selected from the phage
plate, and cultured at 37 °C for 5 h in 1mL LB media with 10 μL of
overnight cultured E. coli (XL1-Blue). 0.5 mL of the amplified phage was
transferred to 50mL LB media with 500 μL of overnight cultured E. coli
and the mixture was incubated at 37 °C for approximately 8 h. The
supernatant was then added to larger volumes of LB and incubated for
14 h to further amplify the phage. Afterwards, the bacteria were sepa-
rated by centrifugation. The remaining phages in the supernatant were
precipitated by adding 20% v/v of 20% w/v poly(ethylene glycol)
(PEG) 8000 Da in 2.5M NaCl solution. The phage could then be isolated
by centrifugation and resuspended in the buffer of choice at ~30% of
the original volume. PEG precipitation was then repeated twice to
further purify the phage. Finally, the amplified phage suspensions were
filtered through 0.45 µm pore size membranes to remove remaining
bacterial cells. The concentration of phage was calculated by the for-
mula below as previously reported (3): mg of phages/mL =(A269 –
A320)/3.84 (A269: UV absorbance at 269 nm, A320: UV absorbance at
320 nm).
2.3. Transient self-templating assembly of phage
A computer controlled home-built transient self-templating as-
sembly apparatus was constructed by modifying a syringe pump. A
gold-coated SiO2 wafer was vertically dipped into a phage suspension
and pulled out at specific speeds using a syringe pump controlled by a
computer-programmed system (C++ or Labview).
2.4. Optical microscopy imaging
Two different methods were used for real-time imaging of the self-
assembly process at the meniscus. To visualize the meniscus on the
substrates as they were pulled from solution, a Sony a57 DSLR camera
equipped with a macro lens (Tamron SP G005 Macro Lens, 60mm, F/
2.0), extension tubes (Kenko automatic extension tube set DG for 3
ring), and camera flash (METZ 15 MS-1 Digital mecablitz Wireless
Macro Camera Flash) was used to take a photograph every 30 s. The
camera was controlled via iPhone App (DSLR.Bot, DSLR camera control
by IR). Images were analyzed using Adobe Photoshop (Adobe Inc, San
Jose, CA). For visualizing the assembly of phage microstructures, an
optical microscope (AxioPlan 2 Microscope System, Zeiss) was laid
horizontally on an optical table and positioned to image the substrate in
real time as it was pulled from phage suspension. A CCD camera
(QImaging Retiga 2000R Scientific CCD Camera, Qimaging, Canada)
mounted on the microscope was used to record a series of snapshots
(time frame: 1 s - 1 min) for the duration of the pulling process.
2.5. Fluorescence microscopy
In order to investigate the phage self-assembly process, we used
fluorescence microscopy (Olympus® IX71 inverted microscope) to vi-
sualize phage aggregation at the meniscus after staining with fluor-
escein isothiocyanate (FITC). FITC powder was obtained from
Calbiochem® and used without further modification. 10 μL of FITC in
DMSO was added to a 4E-phage suspension (6mg/mL in 0.1M sodium
carbonate buffer, pH 9). The solution was gently stirred and then stored
at 2 - 6 °C overnight to allow FITC conjugation to free carboxyl groups
on the phage coat. After the reaction, four cycles of PEG/NaCl pre-
cipitation and resuspension were performed to remove excess FITC.
Phage self-assembly on the meniscus was recorded as series of snap-
shots (1 - 30 per second) using a QImaging® Retiga 2000R CCD camera
mounted on an Olympus® IX71 inverted microscope.
2.6. Atomic force microscopy
Atomic force microscopy (AFM) images were collected using an
MFP3D AFM (Asylum Research, Santa Barbara, CA) and analyzed using
Igor Pro 6.0 (Wave Metrics, Inc. Lake Oswego, OR) and the Asylum
software package (Asylum Research, Santa Barbara, CA).
2.7. Scanning electron microscopy
Scanning electron microscopy (SEM) images were collected using a
scanning electron microscope (ESEM, Hitachi-ESEM, Lawrence
Berkeley National Laboratory, Berkeley CA).
2.8. Grazing incidence small angle X-ray scattering
To characterize the structure of the phage films, we performed
grazing incidence small-angle X-ray scattering (GISAXS) experiments.
The GISAXS data were collected on beamline 7.3.3 in the Advanced
Light Source (ALS) at Lawrence Berkeley National Laboratory. X-rays
with a wavelength of 1.240 Å (10 keV) were used, and the scattering
spectra were collected on an ADSC Quantum 4 u CCD detector with an
active area of 188mm×188mm (2304× 2304 pixels). The scattering
profiles were obtained after a 20 s collection time by integrating the 2D
scattering pattern. The sample to detector distance was 1.5m, and the
incidence angle was 0.16 degrees. Line-averaged intensities were re-
ported as I versus q, where q = (4π/λ) × sin(θ/2), λ is the wavelength
of incident X-rays, and θ is the scattering angle.
2.9. Laser diffraction
Laser diffraction was performed with a He-Ne laser (632.8 nm;
Melles Griot, Albuquerque, NM), and the diffraction patterns were
analyzed after collection with a Sony digital camera (Sony Inc, Japan).
K. Heo et al. Nano Energy 56 (2019) 716–723
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2.10. Phage-based piezoelectric generator fabrication
Phage micro-patterns were fabricated on gold and chromium-coated
SiO2 wafers through the transient self-templating assembly process.
After drying, another gold-coated SiO2 wafer was overlaid on the phage
micro-pattern. The effective device area, where the phage films and
electrodes overlap, was ~1 cm2. The device was then embedded be-
tween two ~2.5mm thick PDMS films for structural stability. Lastly,
signal wires were soldered onto the gold electrodes.
2.11. Phage-based piezoelectric generator characterization
Phage-based piezoelectric generators were mounted onto a dynamic
mechanical test system (Electroforce 3200, Bose, MN) and a predefined
displacement was applied while monitoring the total force on the de-
vices with a load cell. The actual displacement was monitored by a
linear variable differential transformer (LVDT) installed in the
Electroforce 3200. The current/voltage signals and charge produced by
the device were then measured in response to the applied mechanical
load using a semiconductor parameter analyzer (4200-SCS, Keithley,
OH, USA). To measure the open circuit voltage of the phage-based
generator, the semiconductor parameter analyzer was used in zero-
current (I=0) mode. In this mode, the observed current level was less
than ~1.5 pA during the measurement. To measure the short circuit
current of the phage-based generator, the semiconductor parameter
analyzer was used in zero-voltage (V=0) mode. In this mode, the ob-
served voltage level was less than ~1.0 μV during the measurement.
2.12. Powering a liquid crystal display
Phage-based piezoelectric generators were used to power a liquid
crystal display (LCD). The LCD was taken from a laboratory timer
(Traceable® 4- channel timer, Fisher Scientific, Waltham, MA), and
several pins were selected so that the word “VIRUS” or “LEE LAB”
would be displayed on the LCD. Two thin copper wires were attached to
the pins using soldering and silver paint (SPI Supplies, West Chester,
PA) and then connected to the device wires using alligator clips. When
we applied a mechanical stimulus using the Electroforce 3200 or a
finger, we were able to display the desired word “VIRUS” or “LEE LAB”
on the LCD panels.
3. Result and discussion
We fabricated the hierarchically organized phage nanostructures
inspired by the butterfly wings biosynthesis process. Butterfly wings are
covered with numerous arrays of scales [1]. The scales themselves are
made mostly of chitin and have an intricate three-dimensional
architecture of parallel and regularly spaced ridges (Fig. 1A–C). The
butterfly wings are also known to exhibit piezoelectric properties [33].
These hierarchical ridge nanostructures (Fig. 1C) are organized with the
help of transient structures composed of F-actin [21]. Initially, actin
filaments form scaffold-like structures within the scale forming cells.
During the metamorphosis process, the interspacing between the actin
scaffolds act as sites for chitin self-assembly. After the synthesis of the
chitin structures, the actin scaffolds are broken down, leaving organized
chitin nano- and microstructures behind. Thus, the transient scaffolds
are essential for creating the intricate hierarchically ordered chitinous
structures of butterfly wing scales. To mimic the transient scaffold-
based self-assembly process used during butterfly wing metamorphosis,
we developed a novel self-assembly process termed ‘transient self-
templating assembly process’ to exploit M13 bacteriophage (phage) as
model colloidal nanoparticle and transiently formed meniscus as a
scaffold (Fig. 2A). Phage are bacterial viruses that can easily produce
identical copies of themselves in large quantities [22]. They have a
long, rod-shape (880 nm in length and 6.6 nm in diameter) covered by
2700 copies of pVIII major coat proteins (Fig.2B). Through genetic
engineering, phage have been engineered to produce materials for op-
tical, electrical, and biomedical applications [23–26]. Furthermore,
phage go through chiral liquid crystalline self-assembly processes si-
milar to natural building blocks, such as chitin, collagen and cellulose
[19,27,28]. Previously, we developed self-templating assembly process
to organize the M13 phage into hierarchically organized nano- and
micro-structure to synthesize various phage nanostructures [19]. We
also applied this process to fabricate phage-based colorimetric bio-
sensor matrix [29], peptide amphiphile based tissue engineering ma-
trices [30], and FF-peptide based piezoelectric thin film fabrications
[31]. In this manuscript, we greatly expanded the ability of self-tem-
plating assembly using the transiently forming meniscus scaffold with
thermodynamic and kinetic parameter controls.
The transient self-templating assembly process relies on the ma-
nipulation of micrometer-sized meniscus features using a computer-
controlled dynamic pulling process (Fig. 2A). As substrates are pulled
from phage suspensions at programmed speeds and durations, sponta-
neous evaporation of solvents at the air–liquid–solid interface contact
line results in the local deposition of phage on the substrate at the
meniscus. During the pulling process, the phage deposition along the
dynamically changing menisci can be controlled by altering various
thermodynamic and kinetic parameters (i.e., concentration of the
phage, ionic concentration, phage surface charge, and pulling speed).
More importantly, under certain conditions, the meniscus can sponta-
neously divide into tunable, wavy patterns due to fingering instabilities
[32], and each meniscus can serve as a transient scaffold to guide self-
assembly in a spatially and temporally controlled manner (Fig. 2).
We visualized the formation of the transient scaffold and local
Fig. 1. Hierarchical chitin structure of butterfly wing. (A) Photograph ofMorpho butterfly. (B) Optical microscopy image of a portion of a butterfly wing showing the
rows of colored scales. (C) Scanning electron microscope image of a single wing scale to show hierarchically organized chitin-based nanostructures.
K. Heo et al. Nano Energy 56 (2019) 716–723
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phage accumulation using fluorescence microscopy after labeling the
phage with fluorescein isothiocyanate (FITC). Phage assembled uni-
formly along the meniscus with little characteristic micro-pattern at
high ionic strength (> 100mM). When the ionic strength of the sus-
pension was decreased to 10mM, periodic wave-shaped micro-
structures began to appear along the meniscus (Fig. 3A). Further de-
creasing the ionic concentration increased the wavelength of the
periodic menisci. Therefore, ionic strength controlled the phage de-
position periodicity along the meniscus line. At the same time as peri-
odic meniscus division, phage deposition was extended vertically along
the pulling direction at controlled speeds and durations to create ad-
ditional levels of hierarchy (Fig. 3B and C). Real-time observation of the
meniscus showed that, during slow pulling, phage were pinned and
deposited at the crests of the periodic menisci (Supplementary Movie 1
and red arrow in Fig. 3C), while the trough regions (blue arrows in
Fig. 3C) continually slipped downwards. At this point, switching to
faster pulling speeds induced necking in the meniscus scaffold due to
the surface tension of water. This cohesive force pinched off the crest
regions as the scaffold amplitude returned to its initial state. Increasing
the speed of the fast pulling step increased the severity of the necking.
At lower speeds, the degree of necking was minimal: crest regions
where phage accumulate in the scaffold were continuous without se-
paration during the pulling cycles, and the resulting structures were
elongated along the vertical direction (blue arrow in Fig. 3B). In-
creasing to intermediate speeds induced partial necking (red arrows in
Fig. 3B); finally, the highest speeds resulted in complete separation of
the scaffold crest from the rest of the meniscus (yellow arrows in
Fig. 3B), resulting in structures periodically deposited along the pulling
direction. Thus, controlling the speed of the pulling and its duration
time provides a way to modulate the interspacing along the pulling
direction by determining when and to what degree scaffold necking
occurs. Finally, the concentration of the phage suspension determines
the liquid crystal formation and the amount of phage deposited to the
substrate, with higher concentrations producing thicker films. The si-
multaneous control of these thermodynamic and kinetic parameters
occurring at the menisci can kinetically trap phages in far-from-equi-
librium nano- and microstructures that would be challenging to achieve
through either microfabrication or directed self-assembly processes.
Supplementary material related to this article can be found online at
doi:10.1016/j.nanoen.2018.11.084.
A wide range of transient self-templating assembly structures were
formed by tuning of multiple variables. A structural transition diagram
(Fig. 4A) shows how various hierarchical micro-patterns arise as a
function of ionic strength of suspension, phage concentration, and
pulling speeds. Continuous, uniform films are formed by pulling sub-
strates from a phage suspension at a constant speed (black box in
Fig. 4A and Supplementary Fig. S1). Introducing kinetic control by al-
ternating fast and slow pulling steps produced horizontal line patterns
(blue box in Fig. 4A) at high ionic concentrations (> 10mM). At ionic
concentrations ranging from 1 μM to 10mM, 2D-dot patterned struc-
tures (red box in Fig. 4A) ranging from 0 to 90 µm in spacing formed
(Fig. 4B and Supplementary Fig. S2). At ionic strengths greater than
10mM, meniscus instability was not observed, so phage deposition
occurred in a continuous horizontal line (blue rectangular box in
Fig. 4A). We also tuned the interspacing of phage micropatterns by
controlling phage-phage repulsion using genetically engineered phage
with different amounts of negative charges on their major coat protein
surfaces (two, three, or four glutamates; 2E-, 3E- and 4E-phage, re-
spectively) (Supplementary Fig. S3). When we increased surface charge
of the phage from the 2E- to 4E-phage, the interspacing of the hor-
izontal patterns increased. Therefore, the charge-charge interactions
based on ionic-strength or phage sequence play a critical role in the
microstructure formation. We also tuned vertical interspacing between
100 and 300 µm by altering pulling speed and duration (Fig. 4C).
Pulling speeds exceeding 5mm/s failed to produce periodic structures,
presumably because it provided insufficient time to allow phage coa-
lescence and deposition. Extended duration of fast pulling times in-
creased the vertical spacing (Supplementary Fig. S4). Similarly, in-
creasing the slow pulling speeds and pulling time extended the vertical
interspacing of the patterns (green box in Fig. 4A and Supplementary
Fig. S5). Varying the phage concentration from 0.3 to 1mg/mL con-
trolled the pattern thickness between 30 and 50 nm (Fig. 4D). Phage 2D
micro-patterning was rarely observed for concentrations less than
0.3 mg/mL, due to the reduced liquid crystalline behavior at these
concentrations. In addition, concentrations greater than 1.2mg/mL
produced poorly defined micro-patterns as a result of heavy adsorption
Fig. 2. Transient self-templating assembly process. (A) Schematic illustration of the transient self-templating assembly process. As a substrate is pulled vertically from
a phage suspension at precisely controlled speeds and duration times, evaporation proceeds fastest near the air–liquid–solid contact line. This results in the local
accumulation and deposition of phage nanofilaments on the substrate at the meniscus. The morphologyof the meniscus can be controlled by altering thermodynamic
and kinetic parameters such as the ionic strength of the phage suspension, phage concentration, pulling speed, and pulling time; in this way, the meniscus acts as a
tunable transient scaffold. (B) Schematic structure of the M13 phage (880 nm x 6.6 nm). We can control the phage surface charge by genetic engineering of the major
coat proteins. In this work, we used phage with two, three, and four glutamates (2E, 3E and 4E).
K. Heo et al. Nano Energy 56 (2019) 716–723
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of phage to the substrate (Supplementary Fig. S6).
The transient self-templating assembly process-induced phage films
possess multiple levels of hierarchies from nanoscopic to macroscopic
dimensions. For example, 2D-dot patterned phage films were composed
of smectic ordered phage nanofiber bundles at the molecular level
(Inset of Fig. 4A). Tiled AFM images show that the smectic nanofila-
ments formed periodic arch structures with a pitch (P) guided by the
transiently formed periodic menisci (Inset Figure (i), (ii), and (iii) of
Fig. 4). GISAXS analysis showed that the smectic ordered phage films
are pseudo-hexagonally packed crystalline structures exhibiting (100),
(110) and (200) inter-viral spacing peaks (Inset Figure (iv) of Fig. 4).
The horizontally ordered phage nanofilaments possessed much higher
crystallinity than those of the vertically organized phage structures
(Supplementary Fig. S7) mainly because they have more time to enter
the smectic phase during slow pulling speeds. The macroscopic peri-
odicity of the film was confirmed by laser diffraction analysis, which
showed two-dimensional periodicity (Supplementary Fig. S8). Thus, the
transient self-templating assembly process can create controlled hier-
archical structures from nanoscopic to macroscopic levels.
In nature, the chitin-based hierarchical structures in butterfly wings
and crab shells exhibits piezoelectric properties [33,34]. Inspired by
that fact, we fabricated hierarchically organized phage based
piezoelectric devices using the transient self-templating assembly pro-
cess. Previously, we reported that M13 phage exhibited piezoelectric
properties [35]. The liquid crystalline thin film of M13 phage exhibited
6 nA and 400mV of piezoelectric performance. Using hierarchically
organized phage micropatterns, we fabricated piezoelectric devices and
characterized their structure-dependent piezoelectric properties
(Fig. 5). We used 2D-dot, line and continuous phage film patterns to
compare the structure dependent piezoelectric device performance. We
fabricated the ~ 1-cm2-sized phage thin film with micro-patterned
structures on an Au-coated SiO2 substrate (Au/Cr/SiO2/Si = 30 nm/
10 nm/500 nm/400 µm) (Fig. 5A and B). The SiO2 layer was used for
the deposition of additional metal electrodes (Au and Cr) on top of the
PDMS substrates. The phage micro-patterns were then sandwiched
between two gold electrodes and encapsulated in PDMS to prevent the
destruction of the micro-pattern from applied mechanical force. Peri-
odic mechanical loads were applied to the phage-based piezoelectric
generators using a dynamic mechanical test system, and the resulting
electrical output signal was characterized by measuring short-circuit
current and open-circuit voltage. When we applied a rectangular
compressive load at 10 s intervals on the devices, the signal showed two
peaks of reverse polarity, which corresponds to the pressing and re-
leasing motion of the mechanical test system. A connection polarity
Fig. 3. Transient self-templating assembly process and effects of deposition parameters. (A) Images of FITC-labeled phage at the air-solid-liquid interface shows
alterations in the morphology of the transient meniscus scaffold at different ionic strengths ranging from 0.01 to 100mM. (B) Images of the transient scaffold at
different pulling speeds ranging from 0.1 to 4mm/min. (C) Time-lapse images of the transient scaffold during the transient self-templating assembly process. By
combining the kinetic and thermodynamic parameters controlling transient scaffold modulation, we were able to fabricate hierarchically structured phage films. At
low ionic strength, phage nanofilaments tended to coalesce locally at the crest of the meniscus as a result of its undulating morphology. As the substrate was pulled
out at a slow speed, the amplitude of the undulating meniscus gradually increased. The crests of the meniscus (red arrow) remained pinned at the same position on
the substrate due to the local accumulation of phage, while the troughs (blue arrow) continuously slipped downwards. Once the pulling speed was switched to the
fast speed, scaffold necking separated the crest regions from the rest of the scaffold and the meniscus returned to its initial shape. Under optimized conditions,
repeated fast/slow pulling cycles induced diverse hierarchical microstructures. Red star indicates the same position on the substrate during the pulling process.
K. Heo et al. Nano Energy 56 (2019) 716–723
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reversal test showed that the current and voltage signals were reversed
when the device polarity was switched, indicating that the electrical
signals originate from the phage micro-pattern (Supplementary Fig.
S9). The 2D-dot patterns showed the highest piezoelectric performance
exhibiting peak values of 94 nA for the short-circuit current and 0.95 V
for the open circuit voltage (Fig. 5C). Control piezoelectric devices with
the line- and continuous film patterns outputted 56 and 6.3 nA, and
0.75 and 0.36 V, respectively (Supplementary Fig. S10 and S11). The
electric power generated by 2D-dot pattern phage piezoelectric struc-
tures is ~40 times higher than that of the continuous phage film and
two times higher than the line patterned phage piezoelectric generators
[35] (Fig. 5D). Many organic and inorganic piezoelectric materials
exhibit enhanced piezoelectric performance when designed with better
crystallinity [36–39]. Therefore, we believe that these enhanced pie-
zoelectric properties of phage generators from the 2D-dot patterns are
mainly due to the enhanced crystallinity of the phage nanofilaments
that are periodically organized in an active array than those of line
pattern and continuous films (Fig. 3). GISAXS characterization showed
that phage-based 2D-dot micro-patterned phage films exhibit enhanced
hexagonal closed-packed structures with single-domain and directional
alignment compared with that of line pattern and continuous film
(Supplementary Fig. S7). We tested and confirmed the stability and
durability of the devices by continuously applying 5000 cycles of me-
chanical force and monitoring their electrical performance
(Supplementary Fig. S12). As a demonstration, we generated enough
power from these enhanced piezoelectric devices to display the words
"VIRUS" and “LEE LAB” on a liquid crystal display by simply pushing
the device using a finger-tip (Fig. 5E and Supplementary Movie 2). We
also used the resulting piezoelectric generators for detecting body
movements such as bending arm motions (Supplementary Fig. S13).
Supplementary material related to this article can be found online at
doi:10.1016/j.nanoen.2018.11.084.
4. Conclusion
In summary, we have developed a novel, biomimetic process termed
‘transient self-templating assembly process’ to create hierarchically
organized nanostructures using M13 phage as building blocks. We
exploited the piezoelectric property of M13 phage to fabricate piezo-
electric devices with enhanced energy conversion efficiencies. By con-
trolling interparticle interactions at transiently forming menisci, and
controlling the kinetics of meniscus movement, we were able to self-
assemble M13 phage into diverse, two- and three-dimensionally or-
dered functional nanostructures. The resulting 2D-dot micro-patterns
exhibited ~40 times higher piezoelectric power performance compared
with continuous phage thin film devices[35]. Using the resulting de-
vices, we powered multiple LCD display panel devices upon the appli-
cation of mechanical stimulus. We also sensed the bending motion of a
Fig. 4. Structural variety of transient self-templating assembly produced films depending on thermodynamic and kinetic parameters. (A) Structural transition
diagram of phage films depending on concentration, ionic concentration and pulling speed. Inset shows the hierarchical structures of the phage-based films from
nanoscale to microscale. Tiled AFM image (i) shows the arch-shaped phage microstructures with a pitch (P) composed of smectic nanofilments (ii) and (iii). GISAXS
analysis (iv) shows that phage are pseudo-hexagonally packed in the phage nanofilaments to exhibit (100), (110) and (200) peaks (white, yellow and red arrows,
respectively). (B) Inter-spacing control through ionic strength of phage suspension. (C) Inter-spacing control by controlling pulling speed and time. (D) Thickness
control through phage concentration.
K. Heo et al. Nano Energy 56 (2019) 716–723
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human arm by attaching a device on the elbow. Our newly developed
transient self-templating assembly process can potentially be expanded
to self-assemble many other energy generating colloidal particles into
ordered structures for the development of optical, mechanical, elec-
trical and biomedical materials in the future.
Acknowledgements
This work was supported by the U.S. Army Engineering Research
Development Center (W912HZ-14-2-0027). This work is also supported
in part by the Samsung Advanced Institute of Technology (SAIT)’s
Global Research Outreach (GRO) Program and Tsinghua Berkeley
Shenzhen Institute. For GiSAXS measurement, beamline 7.3.3 of the
Advanced Light Source is supported by the Director of the Office of
Science, Office of Basic Energy Sciences, of the U.S. Department of
Energy under Contract No. DE-AC02–05CH11231.
Conflict of interest
The authors declare no competing financial interests.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.nanoen.2018.11.084.
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Dr. Kwang Heo is currently a professor at Department of
Nanotechnology and Advanced Materials Engineering,
Sejong University. Dr. Heo received his B.S and Ph.D. from
Seoul National University. Then, he worked as a postdoc in
Department of Bioengineering, University of California,
Berkeley from 2012 to 2015. His research interests include
the formation of hierarchical structures based on nano-bio
hybrid materials and the development of their functional
devices, such as chemical/biosensors, energy harvesting
devices, biocompatible/biodegradable electronic devices
and tissue engineering applications.
Dr. Hyo-Eon Jin is a professor at College of Pharmacy,
Ajou University, Korea. Dr. Jin carried out her under-
graduate studies in Pharmacy at Seoul National University
(SNU), Korea. She received her M.S. and Ph.D. in the
Department of Pharmaceutics from SNU. Dr. Jin was a
postdoctoral fellow at UC Berkeley and a scientist at
Lawrence Berkeley National Laboratory. Her research in-
terests include the design and development of functional
biomaterials for biopharmaceuticals, tissue engineering and
biosensor to treat various diseases.
Han Kim is pursuing Ph.D. degree under the supervision of
Prof. Seung-Wuk Lee at the Department of Applied Scienceand Technology, University of California, Berkeley. His re-
search interests include the surface science of virus en-
gineering system in charge analysis.
Dr Ju Hun Lee is a postdoctoral fellow at University of
California (UC), Berkeley. He earned his B.S. and M.S. de-
grees in Materials and Science Engineering (MSE) from
Korea University and his Ph.D. degree in MSE from UC, San
Diego. His current research interests are the design of bio-
mimetic self-assembled nanostructures and their applica-
tions
Dr. Eddie Wang is currently an engineer at Emerald Cloud
Lab. He earned his B.S. and Ph.D. in department of
Bioengineering, University of California. Berkeley. His
major research focus is to develop precisely defined na-
nostructured material using biological material design for
tissue engineering and soft robots.
Dr. Seung-Wuk Lee is a professor at Department of
Bioengineering, University of California, Berkeley and a
faculty scientist at Lawrence Berkeley National Laboratory.
Dr. Lee received his B.S and M.S. from Korea University
(Seoul) and his Ph.D. from the University of Texas at Austin
(2003). Dr. Lee's major research interest is to create pre-
cisely defined nanomaterials, to investigate complex phe-
nomena at their interfaces, and to develop novel, biomi-
metic, functional materials for bioenergy, biosensor and
biomedicine. He is also an adjunct professor at Tsinghua
Berkeley Shenzhen Institute, Tsinghua University.
K. Heo et al. Nano Energy 56 (2019) 716–723
723
	Transient self-templating assembly of M13 bacteriophage for enhanced biopiezoelectric devices
	Introduction
	Experimental section
	Construction of engineered phage
	Phage mass amplification
	Transient self-templating assembly of phage
	Optical microscopy imaging
	Fluorescence microscopy
	Atomic force microscopy
	Scanning electron microscopy
	Grazing incidence small angle X-ray scattering
	Laser diffraction
	Phage-based piezoelectric generator fabrication
	Phage-based piezoelectric generator characterization
	Powering a liquid crystal display
	Result and discussion
	Conclusion
	Acknowledgements
	Conflict of interest
	Supporting information
	References