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applied
sciences
Article
Mechanical and Electrical Properties of DNA
Hydrogel-Based Composites Containing Self-Assembled
Three-Dimensional Nanocircuits
Ming Gao 1, Abhichart Krissanaprasit 1, Austin Miles 2, Lilian C. Hsiao 3and Thomas H. LaBean 1, *
Citation: Gao, M.; Krissanaprasit, A.;
Miles, A.; Hsiao, L.C.; LaBean, T.H.
Mechanical and Electrical Properties
of DNA Hydrogel-Based Composites
Containing Self-Assembled
Three-Dimensional Nanocircuits.
Appl. Sci. 2021,11, 2245. https://
doi.org/10.3390/app11052245
Academic Editor: Alexander Marras
Received: 5 February 2021
Accepted: 24 February 2021
Published: 3 March 2021
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1Department of Materials Science and Engineering, College of Engineering, North Carolina State University,
Raleigh, NC 27695, USA; mgao4@ncsu.edu (M.G.); akrissa@ncsu.edu (A.K.)
2North Carolina School of Science and Mathematics, Durham, NC 27705, USA; austinmiles57@gmail.com
3Department of Chemical and Biomolecular Engineering, College of Engineering,
North Carolina State University, Raleigh, NC 27695, USA; lilian_hsiao@ncsu.edu
*Correspondence: thlabean@ncsu.edu; Tel.: +1-919-515-2204
Abstract:
Molecular self-assembly of DNA has been developed as an effective construction strategy
for building complex materials. Among them, DNA hydrogels are known for their simple fabrication
process and their tunable properties. In this study, we have engineered, built, and characterized a
variety of pure DNA hydrogels using DNA tile-based crosslinkers and different sizes of linear DNA
spacers, as well as DNA hydrogel/nanomaterial composites using DNA/nanomaterial conjugates
with carbon nanotubes and gold nanoparticles as crosslinkers. We demonstrate the ability of this
system to self-assemble into three-dimensional percolating networks when carbon nanotubes and
gold nanoparticles are incorporated into the DNA hydrogel. These hydrogel composites showed
interesting non-linear electrical properties. We also demonstrate the tuning of rheological properties
of hydrogel-based composites using different types of crosslinkers and spacers. The viscoelasticity
of DNA hydrogels is shown to dramatically increase by the use of a combination of interlocking
DNA tiles and DNA/carbon nanotube crosslinkers. Finally, we present measurements and discuss
electrically conductive nanomaterials for applications in nanoelectronics.
Keywords:
DNA hydrogel; DNA hydrogel-based composites; self-assembly; biomaterials; DNA
nanotechnology; carbon nanotubes; gold nanoparticles
1. Introduction
Self-assembly by molecular recognition is a fundamental property of soft matter
that can be utilized as a building tool to construct nanoscale to macroscale materials via
bottom-up approaches. Using programmed assembly of nucleic acid molecules, structural
DNA nanotechnology has rapidly expanded to construct sophisticated biomaterials [
1
–
5
].
Beyond self-assembly, DNA is also biocompatible and can be readily conjugated with other
bio-/nanomaterials including proteins and conductive polymers [
6
–
9
]. Leveraging these
capabilities, DNA-based hydrogels have drawn a lot of attention starting with basic research
and moving to applications such as biomedicine, biosensing, and drug delivery [10–15].
The most common strategies to form DNA-based hydrogels are through complemen-
tary strand hybridization, enzyme-catalyzed assembly, and molecular entanglement [
16
].
Studies based extensively upon hybridization focused mainly on pure DNA hydrogels with
three-dimensional (3D) hydrophilic networks crosslinked via complementary basepairing.
These hydrogels typically employed multistrand DNA tiles to construct the multivalent,
crosslinking structural members (crosslinkers) as well as the spacer units (spacers) de-
signed to assemble and control spacing between adhesive arms of the crosslinkers [
17
,
18
].
DNA hydrogels contain available free volume between their polymeric chains in which
other nanomaterials can be trapped, thus providing them the capacity to non-specifically
incorporate functional components. Recently, studies have described strategies for coating
Appl. Sci. 2021,11, 2245. https://doi.org/10.3390/app11052245 https://www.mdpi.com/journal/applsci
Appl. Sci. 2021,11, 2245 2 of 15
and crosslinking nanomaterials such as quantum dots, nanoparticles, and nanotubes with
DNA to create a variety of water soluble heterostructured conjugates [
19
–
21
]. Using these
DNA/nanomaterial conjugates as integral building blocks of crosslinked molecular net-
works has led to interesting DNA hydrogel composites assembled with oligonucleotides
and other nanomaterials.
Integration of nanomaterial conjugates makes it possible to modify the hydrogel
properties to engineer mechanically and electrically adjustable materials. The mechanical
properties of hydrogel-based composites can be fine-tuned by adjusting the concentrations
and branch architectures of initial DNA tiles and embedded nanomaterials. However, the
electrical properties of hydrogel composites bearing embedded conductive nanomaterials
have rarely been studied, and there is a need for a deeper understanding of DNA as a build-
ing material to assemble 3D nanocircuits with conductive nanomaterials. One such material
is carbon nanotubes (CNTs). CNTs are known for their utility in reinforcing nanofiber
networks due to their excellent mechanical strength and stiffness [
22
]. They are chemically
stable and have high aspect ratios that contribute to electrical percolation in nanocom-
posites, which makes CNTs a promising material in nanoelectronics applications [
23
].
However, bare CNTs naturally have low solubility in aqueous solutions in the absence
of surfactants or sidewall functionalizations [
24
]. One method to effectively solubilize
CNTs is via biomolecular dispersion, where single-stranded DNA (ssDNA) wraps around
individual nanotubes via the strong non-covalent hydrophobic interactions between CNT
walls and DNA nucleobases to form water-soluble supramolecular complexes [
25
–
28
].
Besides improving solubility and manageability, the DNA–CNT hybrids also combine the
advantageous electrical and mechanical properties of CNTs and the molecular recognition
capabilities of DNA. Moreover, CNTs have been a promising material for the development
of non-volatile memory with short switching times [
29
]. Some CNT-based memory has
been shown to operate using electromechanical interactions of nanotubes with each other
under the influence of an applied voltage [
30
]. For this reason, researchers have chosen
CNTs to build memristive structures; these are structures that display variable electrical
resistance based upon their ability to remember recent current/voltage activity. Another
classic example of integrating biomolecules with nanomaterials is DNA-functionalized
gold nanoparticles (AuNPs). These hybrids have been shown to be useful for many appli-
cations from biosensing to use as building blocks with which to fabricate more complicated
nanostructures [
31
,
32
]. The original approach to make DNA–AuNP conjugates is to bind
thiolated DNA to AuNPs with the formation of gold–thiolate bonds [
33
,
34
]. More recently,
another strategy has been reported to adsorb non-thiolated DNA strands onto AuNPs via
polyadenine bases that interact strongly with the gold surface [
35
]. Since this method covers
AuNP surfaces quickly and effectively with unmodified ssDNA and provides an acceptable
loading capacity, it was adapted in our study for synthesis of DNA-functionalized AuNPs.
Here, we present the construction and analysis of DNA hydrogels, some with em-
bedded percolating networks using DNA-wrapped CNTs and DNA-attached AuNPs as
crosslinkers. We started at the molecular level by designing DNA sequences and moved
up to macroscale hydrogels realized by bottom-up fabrication. We applied the crosslinker
plus spacer design, where oligonucleotides were designed to form molecular networks
by sequence-directed hybridization with sticky ends on crosslinker units (i.e., DNA tiles
or DNA/nanomaterial conjugates) and coupling components (i.e., spacers) to construct
3D networks (Figure 1). We were able to improve the mechanical strength of the formed
hydrogels using a variety of strategies including adjusting the spacer length and mixing
different types of crosslinkers. We also showed that the 3D structures of nanomaterials
can be programmed efficiently via nucleic acid sequence, and that it is possible to direct
the formation of percolating networks with DNA self-assembly. In addition, using inspi-
ration from biological neural networks that display extraordinary signal dynamics and
processing abilities, we aimed to mimic some aspects of the morphology of natural neural
networks using DNA self-assembly to fabricate nanoelectronic devices with measurable
function. Non-linear electrical properties of nanocomposites that integrate DNA-modified
Appl. Sci. 2021,11, 2245 3 of 15
CNTs are reported. Our eventual goal is to harness molecular recognition to precisely
control the configuration and connection of nanomaterials to self-assemble into controllable
nanostructures and, thus, to engineer, fabricate, and characterize DNA-based hydrogels for
desired applications. Future DNA hydrogel composites may find impactful application
as building blocks in artificial computer hardware, with architectures inspired by natural
neural systems for memory and information-processing applications.
Appl. Sci. 2021, 11, 2245 3 of 15
from biological neural networks that display extraordinary signal dynamics and pro-
cessing abilities, we aimed to mimic some aspects of the morphology of natural neural
networks using DNA self-assembly to fabricate nanoelectronic devices with measurable
function. Non-linear electrical properties of nanocomposites that integrate DNA-modified
CNTs are reported. Our eventual goal is to harness molecular recognition to precisely
control the configuration and connection of nanomaterials to self-assemble into controlla-
ble nanostructures and, thus, to engineer, fabricate, and characterize DNA-based hydro-
gels for desired applications. Future DNA hydrogel composites may find impactful appli-
cation as building blocks in artificial computer hardware, with architectures inspired by
natural neural systems for memory and information-processing applications.
Figure 1. Schematic illustrations of DNA-based hydrogel formation. (a–d) Various types of cross-
linkers: (a) Y-shaped DNA tile, (b) X-shaped DNA tile, (c) DNA–carbon nanotube (CNT) conju-
gate, and (d) DNA–gold nanoparticle (AuNP) conjugate. (e) Spacers of different lengths: Ss, Sm,
and Sl (short (33 nt), medium (44 nt), and long (55 nt), respectively). (f) Pure DNA hydrogel con-
structed by combining Y-shaped or X-shaped DNA tiles with spacers. (g–h) DNA hydrogel com-
posites: (g) DNA–CNT hydrogel and (h) DNA–AuNP hydrogel.
2. Materials and Methods
Materials. Oligonucleotides were purchased from Integrated DNA Technologies
(IDT, Coralville, IA, USA). Oligonucleotides were ordered with standard desalting, and
no further purification was performed prior to use. Nucleotide sequences are listed in
Supplementary Table S1. Single-walled carbon nanotubes (SWNTs; 0.78 nm average di-
ameter; 1 μm median length) were purchased from Sigma-Aldrich (773735, St. Louis, MO,
USA), and multi-walled carbon nanotubes (MWNTs) were purchased from Sigma-Al-
drich (698849). Milli-Q deionized (DI) water (>18 MΩ.cm resistivity) was used for all ex-
periments. Nitric acid (ACS reagent, Sigma-Aldrich) and hydrochloric acid (ACS reagent,
Sigma-Aldrich) were used to make aqua regia. Hydrogen tetrachloroaurate (III) trihydrate
(99.9%, Sigma-Aldrich) and sodium citrate tribasic dihydrate (ACS reagent, >99.0%,
Sigma-Aldrich) were purchased for the synthesis of gold nanoparticles. In addition, Tris
base (tris(hydroxymethyl)aminomethane, Fisher Scientific), sodium chloride (NaCl,
>99.5%, Sigma-Aldrich, St. Louis, MO, USA), HEPES (4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid) (>99.5%, Sigma-Aldrich, St. Louis, MO, USA), and sodium hy-
droxide (NaOH, >98%, Sigma-Aldrich, St. Louis, MO, USA) were used to make buffer so-
lutions.
Synthesis of gold nanoparticles (AuNPs). AuNPs were synthesized based on a
method adapted from the standard citrate reduction procedures [36]. First, all glassware
was cleaned with aqua regia and then rinsed with DI water. After the glassware was dried
+
Complementary
basepairing
Spacers
Crosslinkers Composites
Pure DNA
hydrogel
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(d)
Figure 1.
Schematic illustrations of DNA-based hydrogel formation. (
a
–
d
) Various types of crosslink-
ers: (
a
) Y-shaped DNA tile, (
b
) X-shaped DNA tile, (
c
) DNA–carbon nanotube (CNT) conjugate,
and (
d
) DNA–gold nanoparticle (AuNP) conjugate. (
e
) Spacers of different lengths: Ss, Sm, and Sl
(short (33 nt), medium (44 nt), and long (55 nt), respectively). (
f
) Pure DNA hydrogel constructed
by combining Y-shaped or X-shaped DNA tiles with spacers. (
g
,
h
) DNA hydrogel composites:
(g) DNA–CNT hydrogel and (h) DNA–AuNP hydrogel.
2. Materials and Methods
Materials.
Oligonucleotides were purchased from Integrated DNA Technologies
(IDT, Coralville, IA, USA). Oligonucleotides were ordered with standard desalting, and
no further purification was performed prior to use. Nucleotide sequences are listed in
Supplementary Table S1. Single-walled carbon nanotubes (SWNTs; 0.78 nm average
diameter; 1
µ
m median length) were purchased from Sigma-Aldrich (773735, St. Louis,
MO, USA), and multi-walled carbon nanotubes (MWNTs) were purchased from Sigma-
Aldrich (698849). Milli-Q deionized (DI) water (>18 M
Ω·
cm resistivity) was used for all
experiments. Nitric acid (ACS reagent, Sigma-Aldrich) and hydrochloric acid (ACS reagent,
Sigma-Aldrich) were used to make aqua regia. Hydrogen tetrachloroaurate (III) trihydrate
(99.9%, Sigma-Aldrich) and sodium citrate tribasic dihydrate (ACS reagent, >99.0%, Sigma-
Aldrich) were purchased for the synthesis of gold nanoparticles. In addition, Tris base
(tris(hydroxymethyl)aminomethane, Fisher Scientific), sodium chloride (NaCl, >99.5%,
Sigma-Aldrich), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (>99.5%,
Sigma-Aldrich), and sodium hydroxide (NaOH, >98%, Sigma-Aldrich) were used to make
buffer solutions.
Synthesis of gold nanoparticles (AuNPs).
AuNPs were synthesized based on a
method adapted from the standard citrate reduction procedures [
36
]. First, all glassware
was cleaned with aqua regia and then rinsed with DI water. After the glassware was
dried completely, 500 mL of 1 mM hydrogen tetrachloroaurate (III) trihydrate in DI water
was prepared in a round-bottom flask and heated to a vigorous boil with stirring. Then,
50 mL of 38.8 mM sodium citrate tribasic dihydrate in DI water was added to the gold
solution flask and the reaction was allowed to proceed for 15 min. The solution turned
from yellow to clear, to black, to purple, and finally to deep red. Lastly, the solution was
cooled down to room temperature. Synthesized AuNPs were characterized by transmission
Appl. Sci. 2021,11, 2245 4 of 15
electron microscopy (TEM). The concentration of AuNPs was estimated with a UV–Vis
spectrophotometer and calculated using the Beer–Lambert equation, A=εbC [37].
Construction of DNA tiles and spacers.
To construct Y-shaped DNA tiles, 10
µ
L of
10 mM Y
1
, Y
2
, and Y
3
precursor strands for the building blocks was added to a folding
buffer solution (20 mM Tris-HCl (pH 7.5) and 100 mM NaCl) to obtain a final concentration
of 1 mM for each strand. Then, the mixture went through a heat-annealing process where it
was heated to 95
°C
for 5 min and then cooled to room temperature over 30 min. Similarly,
the X-shaped DNA tiles were assembled by mixing 8
µ
L of 10 mM precursor strands X
1
, X
2
,
X
3
, and X
4
in the folding buffer solution to obtain a final concentration of 1 mM for each
strand. The mixture went through the same heat-annealing process as described above. To
construct spacers, 15
µ
L of the two 10 mM precursor strands for the spacers was mixed in
the folding buffer (to obtain a final concentration of 1.5 mM for each strand). The mixture
then went through the same heat-annealing process described above for DNA tiles. Spacers
were also made to different concentrations to pair with different types of crosslinkers.
All pH values of the buffers were measured with a standard pH meter (Mettler Toledo
SevenEasyTM, Columbus, USA).
DNA-assisted solubilization of CNTs and DNA–CNT conjugate formation.
We
constructed DNA–CNT conjugates by wrapping CNTs with DNA based on a previously
reported method [
38
]. Briefly, 120
µ
L HEPES (final concentration 50 mM, pH 7.6), 1.2 mg
CNTs, and 15
µ
L of 10 mM DNA strand C
1
were mixed together. The mixture was sonicated
in an ice-water bath for 30 min using a 100-W bath sonicator. Then, 15 µL of 10 mM DNA
strand C
2
was added. The solution was incubated at room temperature overnight and then
stored at 4 °C.
DNA–AuNP conjugates.
DNA-decorated AuNPs were synthesized with a previously
reported method [
39
]. First, 16
µ
L of DNA stock solution of sequence G (100
µ
M in 5 mM
HEPES buffer, pH 7.4) was added to 1.6 mL AuNP solution (10 nM). The solution was
mixed by brief vortexing. Then, 32
µ
L of 500 mM citrate
·
HCl buffer, pH 3 (final 10 mM),
was added to the AuNP solution (1
µ
L of buffer per 50
µ
L of AuNP solution). The solution
once again went through vortex mixing and was incubated at room temperature for 3 min.
Then, the pH of the AuNP solution was adjusted back to neutral by adding 96
µ
L of
500 mM HEPES buffer (pH 7.6, 3
µ
L of buffer per 50
µ
L of AuNP solution). The solution
was then incubated for 5 to 10 min at room temperature. The DNA–AuNP mixture was
centrifuged at 13,300 rpm for 6 min, and the supernatant was removed and discarded. The
pellet was washed four times with 5 mM HEPES buffer (pH 7.6) and centrifuged to remove
any unbound DNA strands. The final DNA–AuNP conjugate was redispersed in 100
µ
L of
5 mM HEPES buffer (pH 7.6) for further use.
Construction of pure and DNA hydrogel composites.
To make hydrogels using
the self-assembled DNA tiles or DNA/nanomaterials conjugates, and spacers, desired
volumes of crosslinker and spacer stocks were combined on a piece of parafilm using
the concentrations and ratios listed in Table S2. For example, 10
µ
L of Y-shaped DNA
tiles stock and 10
µ
L of spacer stock were added on a piece of parafilm and immediately
mixed. DNA hydrogels were formed within one minute, and the DNA gel samples were
immediately tested.
TEM and analysis.
The dimensions and morphology of AuNPs, DNA–AuNP conju-
gates, DNA–CNT conjugates, and dehydrated DNA hydrogel composites were imaged
using an FEI Talos F200X scanning/transmission electron microscope (Hillsboro, OR, USA)
at an accelerating voltage of 200 kV. CNT samples were drop cast and dried onto 300-mesh
copper grids with lacey formvar support film reinforced by a heavy coating of carbon (Ted
Pella, 01883, Redding, USA). AuNP samples were prepared on 200-mesh copper grids with
a formvar film covered with a light layer of carbon (Ted Pella, 01800-F). Dimensions of the
imaged samples were measured with ImageJ software (Bethesda, MD, USA).
Measurement of rheological properties of hydrogels.
A TA Instruments DHR-2
stress-controlled rheometer (New Castle, DE, USA) was used to perform small-amplitude
oscillation frequency sweeps at room temperature. An aluminum plate of 20-mm diameter
Appl. Sci. 2021,11, 2245 5 of 15
was used as the top plate. We prepared DNA hydrogel samples by pipetting the compo-
nents onto parafilm; following gel formation, the samples were transferred to the rheometer
plate without pipetting, to avoid shearing the gels. For a ~30-
µ
L hydrogel sample, the gap
distance was set as 35
µ
m. The applied strain was set to 1%, while the angular frequency
was decreased from 100 to 0.1 rad/s. Five points were collected per decade.
Electrodes and electrical measurement setup.
Gold electrodes of parallel lines with
defined widths and gap distances were fabricated via thin film vapor deposition. To
perform current–voltage (IV) curve measurement, the sample was connected to a socket
board in a Faraday cage (Hewlett Packard Test Fixture Analyzer 16058A, Palo Alto, CA,
USA) connected to a 2-channel (medium power) source/monitor unit module (Agilent
Technologies E5272A, Santa Clara, CA, USA).
3. Results
3.1. Sequence Design of Crosslinker and Spacer Strands for DNA Gel Formations
We constructed DNA hydrogels by mixing crosslinker and spacer modules that asso-
ciate based on DNA–DNA hybridization, as shown in Figure 1. Four types of crosslinkers
(Y-shaped DNA tiles, X-shaped DNA tiles, DNA–CNT conjugates, and DNA–AuNP conju-
gates) and three different lengths of spacers (Ss, Sm, and Sl; short (33 nt), medium (44 nt),
and long (55 nt), respectively) were tested. With these building blocks, we produced
two major types of hydrogels: pure DNA hydrogels and DNA/nanomaterial hydrogel
composites. For DNA crosslinkers, we used two types of branching crosslinkers called
Y-shaped and X-shaped DNA tiles that were assembled from three and four ssDNA strands,
respectively. Each arm in the DNA tile carried a sticky end complementary to sticky ends
on the spacers. We adopted sequences of strands D
1
, D
2
, and D
3
from Xing et al. to
construct Y-shaped DNA tiles [
40
]. Similarly, we modified the sequences of strands X
01
,
X
02
, X
03
, and X
04
reported by Um et al. [
41
] by swapping in our sticky end to construct
X-shaped DNA tiles.
To construct DNA–CNT conjugates, we applied a DNA sequence containing multiple
repeated GT units that wrap around CNTs. Specifically, we adopted Sequence C as reported
by Cheng et al. [
38
] and modified their Sequence D with our sticky end to make it compati-
ble with our spacers. The repeated GT units have been proven to efficiently wrap around
CNTs well due to strong
π
–
π
interactions between the CNT sidewall and the nucleobase
aromatic rings [
38
,
42
]. Zheng et al. also demonstrated a systematic study showing that
among all DNA sequences that wrap around CNTs, (GT)
n
gives the highest dispersion
efficiency and only requires 30 min sonication to obtain well-dispersed DNA-wrapped
individual nanotubes [
26
]. A longer sonication treatment breaks CNTs and decreases their
high aspect ratio [
43
], which is a property essential to our objective of building percolating
networks. Therefore, we chose to make DNA–CNT conjugates with (GT)
20
repeat units
to allow a shorter sonication time. Strands of this length tightly wrap around nanotubes,
while a longer DNA strand wraps around nanotubes more loosely and may also entangle
multiple nanotubes [
44
]. We examined both single-walled CNTs (SWNTs) and multi-walled
CNTs (MWNTs) in this study. According to the vendor’s specifications, the SWNTs have
an average diameter of 0.78 nm with a median length of 1
µ
m. We chose these dimensions
because previously reported atomic force microscopy (AFM) studies observed that (GT)
20
can form multiple wraps around SWNTs with an average diameter around 1 nm [
44
]. We
purchased MWNTs of a much larger size with an average outer diameter of 8.7–10 nm
and an average length of 10
µ
m. The same study showed that DNA wrapping loosens
as the nanotubes become larger and mostly only wrap with one turn around MWNTs of
greater diameters.
Lastly, we utilized DNA–AuNP conjugates as the fourth type of crosslinker. We
synthesized AuNPs with an average diameter of 13 nm using a method adapted from the
standard citrate reduction procedure [
36
]. As-synthesized AuNPs were characterized by
TEM, as shown in Figure 2a. DNA–AuNP conjugates were created by attaching ssDNA
to the surfaces of AuNPs using the method reported by Zhang et al. [
39
]. Briefly, 13-nm
Appl. Sci. 2021,11, 2245 6 of 15
AuNPs at neutral pH adsorb DNA strands with polyadenine (A
n
) as the anchoring block
due to the strong interaction between adenine and gold. We designed a DNA sequence
to contain 13-mer of polyadenine (A
13
) that is connected with a 12-nt sticky end. The
polyadenine sequence strongly adsorbed onto AuNPs with high loading capacity. This
method produced DNA–AuNP conjugates with much higher concentrations of DNA
and AuNPs compared to another method prepared by polymerase chain reaction (PCR)
elongation [45].
Appl. Sci. 2021, 11, 2245 6 of 15
diameter of 8.7–10 nm and an average length of 10 μm. The same study showed that DNA
wrapping loosens as the nanotubes become larger and mostly only wrap with one turn
around MWNTs of greater diameters.
Lastly, we utilized DNA–AuNP conjugates as the fourth type of crosslinker. We syn-
thesized AuNPs with an average diameter of 13 nm using a method adapted from the
standard citrate reduction procedure [36]. As-synthesized AuNPs were characterized by
TEM, as shown in Figure 2a. DNA–AuNP conjugates were created by attaching ssDNA
to the surfaces of AuNPs using the method reported by Zhang et al. [39]. Briefly, 13-nm
AuNPs at neutral pH adsorb DNA strands with polyadenine (A
n
) as the anchoring block
due to the strong interaction between adenine and gold. We designed a DNA sequence to
contain 13-mer of polyadenine (A
13
) that is connected with a 12-nt sticky end. The polyad-
enine sequence strongly adsorbed onto AuNPs with high loading capacity. This method
produced DNA–AuNP conjugates with much higher concentrations of DNA and AuNPs
compared to another method prepared by polymerase chain reaction (PCR) elongation
[45].
Figure 2. TEM images of (a) as-synthesized AuNPs, (b) DNA–AuNP conjugates, (c) DNA–AuNP
hydrogel; (d) DNA–single-walled CNT (SWNT) conjugates, (e) DNA–SWNT hydrogel; (f) DNA–
multi-walled CNT (MWNT) conjugates on lacey film, (g) DNA–MWNT hydrogel on lacey film,
and (h) a zoomed-in image of DNA–MWNT hydrogel, showing areas of the CNT wall wrapped
by DNA and the bare wall without DNA wrapping. DNA binding between CNT junctions is also
shown.
Spacers are linear duplexes formed by two ssDNAs that each contains a sticky end
that is complementary to the sticky ends of crosslinkers. We used three different lengths
of spacers 33 nt (Ss), 44 nt (Sm), and 55 nt (Sl). The spacer sequences were inspired by Xing
et al. [40] and adapted to the other components of our system. All the spacer strands have
Figure 2.
TEM images of (
a
) as-synthesized AuNPs, (
b
) DNA–AuNP conjugates, (
c
) DNA–AuNP
hydrogel; (
d
) DNA–single-walled CNT (SWNT) conjugates, (
e
) DNA–SWNT hydrogel; (
f
) DNA–
multi-walled CNT (MWNT) conjugates on lacey film, (
g
) DNA–MWNT hydrogel on lacey film, and
(
h
) a zoomed-in image of DNA–MWNT hydrogel, showing areas of the CNT wall wrapped by DNA
and the bare wall without DNA wrapping. DNA binding between CNT junctions is also shown.
Spacers are linear duplexes formed by two ssDNAs that each contains a sticky end
that is complementary to the sticky ends of crosslinkers. We used three different lengths
of spacers 33 nt (Ss), 44 nt (Sm), and 55 nt (Sl). The spacer sequences were inspired by
Xing et al. [
40
] and adapted to the other components of our system. All the spacer strands
have the same sticky ends that are complementary to the sticky ends on crosslinkers. All
the DNA sequences were examined using NUPACK online software (Pasadena, CA, USA)
to predict their most stable folded structures to avoid unwanted secondary structures. The
final DNA sequences offer a minimum free energy of secondary structure.
3.2. Characterization of Conjugates and Hydrogels
To understand and compare the morphologies of conjugates and hydrogels, we used
TEM imaging techniques to visualize AuNP, SWNT, and MWNT conjugates with and
Appl. Sci. 2021,11, 2245 7 of 15
without DNA spacers to show how the crosslinked DNA networks connect and arrange the
structures of these nanomaterials. We first imaged the original AuNPs right after synthesis,
then the DNA–AuNP conjugates and DNA–AuNP hydrogel constructed by combining
DNA–AuNP conjugates with Sl. With the same concept, we also imaged DNA–SWNT
conjugates and DNA–MWNT conjugates and then took a look at hydrogels made of these
conjugates with the long spacers (Sl). The TEM images of the DNA–CNT hydrogels showed
that amorphous materials with hierarchical structures were formed. We can clearly see the
larger-scale CNT networks as well as DNA binders on the surfaces of nanotubes, especially
around the junctions of CNTs (Figure 2g,h).
3.3. Rheological Properties of DNA Hydrogels
Polymeric hydrogels generally demonstrate robust mechanical strength because of
their dense, entangled, and crosslinked networks with small mesh sizes. Unlike these
conventional hydrogels, pure DNA hydrogels are more thixotropic and can display poor
mechanical strength [
46
]. Although the mechanical properties of DNA hydrogels can
be fine-tuned by adjusting the type and concentration of initial DNA tiles with different
numbers of branches, even the toughest DNA hydrogel only exhibits a storage modulus
of a few thousand Pa [
46
]. Because of this property, the applications of DNA hydrogels
are limited to only certain fields. To explore further enhancement of DNA hydrogels’
mechanical strength, we implemented two strategies: modifying the DNA building blocks
and fortifying the structure with novel nanomaterials that confer mechanical rigidity.
We performed small-amplitude oscillatory rheology to understand the gelation prop-
erties of pure and DNA hydrogel composites. The storage modulus (G’) and loss modulus
(G”) represent the elastic and viscous contributions to the total stress. Viscoelastic materi-
als with solid-like properties are formed due to internal crosslinks within the materials;
crosslinking can come from chemical bonds or physical–chemical interactions between
individual molecules [47,48].
Using the testing conditions described in the methods session, we performed oscil-
lation measurements on hydrogels formed by Y-shaped DNA tiles, X-shaped DNA tiles,
DNA–SWNT conjugates, DNA–MWNT conjugates, and, finally, DNA–AuNP conjugates
with spacers of three different lengths. The goals of this group of tests were to study the
influence of length of spacers on different types of crosslinkers, as well as to compare the
influence of different crosslinkers. For all tested samples, we saw a higher storage modulus
(G’) than loss modulus (G”) across the tested angular frequencies, as shown in Figure 3,
demonstrating solid-like behavior, which is typically observed for hydrogels constructed
with DNA [49,50].
As Figure 3a shows, when constructing pure DNA hydrogels with the same crosslink-
ers (X or Y), using shorter spacers gives more solid-like hydrogels as indicated by the
higher storage modulus. The X-shaped DNA tiles also construct more solid-like hydrogels
than the Y-shaped DNA tiles with all types of spacers. However, when using conjugates as
crosslinkers (see below), longer spacers construct more solid-like hydrogels, opposite to the
behavior observed from pure DNA hydrogels. The mechanical strengths of pure hydrogels
are also improved by integrating nanomaterials. With the same spacers (Sl), DNA–SWNT
conjugates also construct more solid-like hydrogels than DNA–MWNT conjugates, and
both DNA–CNT conjugates make more solid-like hydrogels than DNA–AuNP conjugates
(Figure 4).
Appl. Sci. 2021,11, 2245 8 of 15
Appl. Sci. 2021, 11, 2245 8 of 15
Figure 3. Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheo-
logical properties of (a) pure DNA hydrogels, (b) DNA–SWNT and DNA–MWNT hydrogels, and
(c) DNA–AuNP hydrogels constructed with spacers of different lengths.
Figure 4. Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheological properties of (a)
hydrogel composites constructed with DNA–SWNT conjugates, X- (or Y-) shaped DNA tiles, and long spacer (Sl); and (b)
hydrogel composites constructed with DNA–MWNT conjugates, X- (or Y-) shaped DNA tiles, and Sl.
Figure 3.
Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheological
properties of (
a
) pure DNA hydrogels, (
b
) DNA–SWNT and DNA–MWNT hydrogels, and (
c
) DNA–
AuNP hydrogels constructed with spacers of different lengths.
Appl. Sci. 2021, 11, 2245 8 of 15
Figure 3. Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheo-
logical properties of (a) pure DNA hydrogels, (b) DNA–SWNT and DNA–MWNT hydrogels, and
(c) DNA–AuNP hydrogels constructed with spacers of different lengths.
Figure 4. Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheological properties of (a)
hydrogel composites constructed with DNA–SWNT conjugates, X- (or Y-) shaped DNA tiles, and long spacer (Sl); and (b)
hydrogel composites constructed with DNA–MWNT conjugates, X- (or Y-) shaped DNA tiles, and Sl.
Figure 4.
Storage modulus and loss modulus (G’, G”) vs. angular frequencies showing the rheological properties of
(
a
) hydrogel composites constructed with DNA–SWNT conjugates, X- (or Y-) shaped DNA tiles, and long spacer (Sl); and
(b) hydrogel composites constructed with DNA–MWNT conjugates, X- (or Y-) shaped DNA tiles, and Sl.
Appl. Sci. 2021,11, 2245 9 of 15
Our next set of tests was to use mixed crosslinkers, combining DNA–CNT conju-
gates and DNA tiles. The objective of these tests was to show the influence of different
crosslinker compositions and to see how mixed crosslinkers of different length scales
change the mechanical properties of the final hydrogels. Specifically, we substituted 25%,
50%, and 75% of the DNA–CNT conjugates from the previous test with X- or Y-shaped
DNA tiles while keeping the total concentration of sticky ends from all crosslinkers the
same. Only long spacer (Sl) was used for these mixed crosslinker tests. The oscillatory
measurements showed that hydrogels constructed using Sl and containing a crosslinker
mixture of 75% DNA–CNT conjugates and 25% DNA tiles exhibited the highest values of
G’. This composition formed hydrogels with G’ above 50 kPa, over 100-fold higher than the
G’ of pure DNA hydrogels. It is followed by using Sl with 100% DNA–CNT conjugates as
crosslinkers. Then, the mechanical strength dropped even further when using Sl with 50%
DNA–CNT conjugates and 50% DNA tiles crosslinkers, and hydrogels constructed by Sl
with 25% DNA–CNT conjugates and 75% DNA tiles had the lowest storage modulus. All
DNA hydrogel composites still had higher mechanical strengths than pure DNA hydrogels.
Moreover, using DNA–SWNT conjugates always gave more solid-like hydrogels than
using DNA–MWNT conjugates in the above compositions, and using X-shaped DNA tiles
resulted in more solid-like hydrogels than using Y-shaped DNA tiles in these compositions.
3.4. Electrical Characterization
In order to minimize undesired complications due to ionic conduction associated
with performing electrical measurements on nanocircuits embedded within hydrogels, we
performed two-dimensional measurements of dehydrated hydrogels instead. We used a
two-terminal current–voltage (IV) characterization setup with parallel line-shaped gold
electrodes. As shown in Figure S5, the gold microelectrodes were fabricated with 200
µ
m
spacing and were wire-bonded to a commercial ball grid array (BGA) board connected to
the setup. The hydrogel was placed across the gap between microelectrodes and then dried
completely before IV curves were recorded. IV characterization allows the measurement
of small conductivity as a response to an applied voltage. During the test, the current
was measured during 10 consecutive pulses of 10 V. The goal of IV characterization is to
investigate whether or not DNA crosslinking creates more organized or clumpy networks
based on the changes in conductivity after adding spacers to the conjugates.
As shown in Figure 5a, dehydrated samples of DNA–SWNT conjugates and DNA–
SWNT hydrogel both showed non-linear behaviors. With the same applied voltage pulses,
the measured current increased greatly in the hydrogel samples with spacers compared to
DNA–SWNT conjugates—over a 650-fold increase. Since MWNTs are highly conductive,
they showed a wire-like behavior with a much higher conductivity compared to the SWNT
samples. In the MWNT case, adding DNA spacers also increased the conductivity of DNA–
MWNT conjugates by 45-fold—see Figure 5b. When testing with DNA–AuNP samples,
the current increased four-fold after adding spacers to the conjugates and forming gel-like
networks, as shown in Figure 5c.
These electrical measurements demonstrate that modification and organization of
nanomaterials using DNA strands can be used to control the electrical behavior of percolat-
ing networks and can change the conductivity of composites by using DNA self-assembly
to connect the nanomaterials.
Appl. Sci. 2021,11, 2245 10 of 15
Appl. Sci. 2021, 11, 2245 10 of 15
Figure 5. Current–voltage (IV) curves of dehydrated samples. (a) DNA–SWNT conjugates (left) vs.
DNA–SWNT hydrogel (right); (b) DNA–MWNT conjugates (left) vs. DNA–MWNT hydrogel
(right); (c) DNA–AuNP conjugates (left) vs. DNA–AuNP hydrogel (right). All measured across gold
electrodes with 200 μm spacings. Legend shows pulse number.
4. Discussion
4.1. Characterizations
We first characterized citrate-stabilized AuNPs with TEM, demonstrating that syn-
thesized nanoparticles were homogeneous and that their average diameter was 13.1 ± 1.8
nm (Figures 2a and S3). As-synthesized AuNPs appeared clustered in groups of two or
three, with no clear spaces between individual nanoparticles within the groups. However,
the images of DNA–AuNP conjugates showed that DNA-attached AuNPs have a clear
and much more uniform spacing between neighboring particles, which was measured to
have an average of 0.78 nm (Figure 2b). By comparing the morphologies of AuNP cluster-
ing in Figures 2a,b, we showed that DNA strands modified the surfaces of AuNPs. We
further characterized DNA–AuNP conjugates using gel electrophoresis. Non-DNA-at-
tached AuNPs aggregated in the well and failed to enter the gel. DNA-modified AuNPs
did not aggregate and were able to migrate into the gel (as shown in Figure S4). The TEM
images agreed with the gel electrophoresis results and demonstrated that we successfully
decorated AuNPs with ssDNA. Next, we constructed DNA–AuNP hydrogel composites
Figure 5.
Current–voltage (IV) curves of dehydrated samples. (
a
) DNA–SWNT conjugates (left)
vs. DNA–SWNT hydrogel (right); (
b
) DNA–MWNT conjugates (left) vs. DNA–MWNT hydrogel
(right); (
c
) DNA–AuNP conjugates (left) vs. DNA–AuNP hydrogel (right). All measured across gold
electrodes with 200 µm spacings. Legend shows pulse number.
4. Discussion
4.1. Characterizations
We first characterized citrate-stabilized AuNPs with TEM, demonstrating that synthe-
sized nanoparticles were homogeneous and that their average diameter was 13.1
±
1.8 nm
(Figure 2a and Figure S3). As-synthesized AuNPs appeared clustered in groups of two or
three, with no clear spaces between individual nanoparticles within the groups. However,
the images of DNA–AuNP conjugates showed that DNA-attached AuNPs have a clear and
much more uniform spacing between neighboring particles, which was measured to have
an average of 0.78 nm (Figure 2b). By comparing the morphologies of AuNP clustering
in Figure 2a,b, we showed that DNA strands modified the surfaces of AuNPs. We fur-
ther characterized DNA–AuNP conjugates using gel electrophoresis. Non-DNA-attached
AuNPs aggregated in the well and failed to enter the gel. DNA-modified AuNPs did
not aggregate and were able to migrate into the gel (as shown in Figure S4). The TEM
images agreed with the gel electrophoresis results and demonstrated that we successfully
decorated AuNPs with ssDNA. Next, we constructed DNA–AuNP hydrogel composites
by combining DNA–AuNP conjugates and long spacers (Sl). We further confirmed the
Appl. Sci. 2021,11, 2245 11 of 15
formation of hydrogel composites by characterization of a hydrogel sample using TEM
(Figure 2c). The TEM images showed multiple layers of AuNPs on top of each other that
appeared to be held together during sample collapse from drying. Comparing that with
Figure 2b, it is apparent that DNA spacers had linked AuNPs together in a 3D structure.
Although differences in clustering and morphology between DNA–SWNT in conju-
gates versus hydrogel are not entirely distinctive using TEM characterization (
Figure 2d,e
),
the differences were apparent in the MWNT samples (
Figure 2f,g
). DNA–MWNT conju-
gates appeared to gather on the lacy carbon film on the copper grids and did not appear
to fill in most holes in the film. On the other hand, the dehydrated hydrogel made of
DNA–MWNT conjugates with long spacers (Sl) covered the entire lacy film (including
holes in the film) with its own networks formed by the nanotubes. DNA spacers helped
to connect MWNTs together into a web-like structure over a large area. When taking a
closer look at individual MWNTs, we observed regions of coating over the nanotubes,
with an average thickness of 1.51 nm. Figure 2h clearly shows that the middle area of the
MWNT was not wrapped by DNA, while the areas close to MWNT junctions were coated.
Figure 2h
also shows a thicker coating around MWNT junctions, indicating the location
of spacers. These images represent a direct observation of DNA acting as a “smart” glue
to bind and connect MWNTs together. Overall, we successfully constructed pure DNA
hydrogels and DNA hydrogel composites with nanomaterials.
4.2. Rheological Results
We would like to construct hydrogels of reasonable mechanical strength where the
crosslinked networks can effectively prevent diffusion of the nanomaterials and, thus, to
achieve hydrogels with confined architecture for further applications. Having a higher
storage modulus than loss modulus from oscillation frequency tests indicated that we
indeed made hydrogels with solid-like properties. When using the same DNA tiles as
crosslinkers, we observed that shorter spacers constructed hydrogels of higher mechanical
strengths as they are able to build a denser network. The situation is reversed and longer
spacers constructed more solid-like hydrogels when using DNA/nanomaterial conjugates
as crosslinkers. This is because the CNTs and AuNPs we used are much larger in scale
compared to DNA molecules; thus, having longer spacers helped to build more and more
stable bridges between nanomaterial/crosslinker components. Therefore, we used Sl for
all electrical studies and structural characterization analysis.
CNTs are well known for their mechanical reinforcement applications. Consistent with
previous observations in other composites, we observed a huge increase in storage modulus
with DNA–CNT hydrogel composites compared to pure DNA hydrogels. When using
one type of crosslinker, DNA–SWNT conjugates constructed the most solid-like hydrogels,
followed by using DNA–MWNT conjugates, DNA–AuNP conjugates, X-shaped DNA tiles,
and, finally, Y-shaped DNA tiles. We believe that we observed a higher storage modulus
from hydrogels constructed with DNA–SWNT conjugates than hydrogels constructed with
DNA–MWNT conjugates because DNA wraps around SWNTs more tightly with more
turns [
51
]. The TEM images (Figure 2) also showed that DNA coats on SWNTs much better
than on MWNTs. As the average diameter of CNTs increases from SWNTs (0.78 nm) to
MWNTs (~9 nm), (GT)
20
loses its strong binding around the nanotubes, which resulted in a
decrease in mechanical performance of hydrogels made with these conjugates. This result
shows the importance of crosslinked DNA as the binding material to connect nanomaterials
and to build networks. Furthermore, hydrogels constructed with both types of DNA–CNT
conjugate crosslinkers showed higher mechanical strength than hydrogels constructed
with DNA–AuNP conjugates because the high aspect ratios of CNTs are inherently in favor
of providing reinforcement to composites compared to sphere-shaped materials.
We further investigated and rationally improved the mechanical properties of hybrid
hydrogels by combining DNA/nanomaterial conjugates and DNA tiles as crosslinkers.
Since CNTs and AuNPs are in much larger scales than DNA molecules, there are available
spaces between individual nanotubes and nanoparticles in the hydrogels, where there is
Appl. Sci. 2021,11, 2245 12 of 15
no DNA filling besides the DNA network associated with binding strands and spacers.
These hydrogels have the capacity to integrate more materials that can fill in the spaces.
Therefore, we used different compositions of DNA/nanomaterial conjugates and DNA
tiles to construct hydrogels in which crosslinkers come in different scales. When we used
DNA tiles to make up to 25% of crosslinkers and DNA–CNT conjugates for the rest, the
resulted hydrogels were even more solid-like than when using 100% DNA–CNT conjugates
as crosslinkers. This is because substituting some of the DNA–CNT conjugates with a much
smaller type of crosslinkers helped to fill in the open spaces between CNTs and, thus, made
a denser hydrogel. However, DNA molecules are significantly weaker than CNTs, so we
observed a decrease in mechanical strength when substituting more DNA–CNT conjugates
with DNA tiles. In summary, we demonstrated adjustment of the mechanical properties of
hybrid hydrogels by combining different compositions of crosslinkers and achieved the
most solid-like hydrogel when using DNA–CNT conjugates as 75% of crosslinkers and
X-shaped DNA tiles for the remaining 25% of crosslinkers.
4.3. Electrical Studies
Studies on the conductivity of DNA mostly agree that DNA is not a good conductor
and does not contribute to conductivity in composites when conductive nanomaterials are
present [
51
]. However, DNA has been seen as a good candidate to self-organize nanocircuits
into a complex system. Therefore, besides mechanical reinforcement, another objective of
integrating nanomaterials into the hydrogel composites is to modify the electrical behavior
and add functionality to the hydrogels. For this reason, we used semiconducting SWNTs
when making DNA–SWNT conjugates. In a previous electrical study of a single SWNT
on parallel gold electrodes [
52
], the IV curves showed a saturation of conductance at high
voltages. We did not observe such a saturation from IV characterization of SWNT networks
from conjugates and hydrogels (Figure 5a).
Circuits built with AuNPs have a lower conductivity compared to the ones created
with CNTs, since the nanosphere structure does not have the advantage in reaching long-
range percolation as nanotubes of much greater aspect ratios do. For the same reason,
adding spacers to DNA–AuNP conjugates also does not result in as much of an enhance-
ment in conductivity. This result showed that the shape of nanomaterials needs to be
considered when designing hydrogels to ensure that the assembled circuits have the de-
sired performance. Increased control of the architectural characteristics of percolation paths
formed by CNTs and AuNPs (i.e., length scale, clumpiness, subcircuit structures, etc.) will
encourage further investigation of these embedded networks for potential applications in
neuromorphic and error-tolerant computing.
5. Conclusions
We designed and built pure DNA hydrogels as well as composites using DNA/nano-
material conjugate crosslinkers, DNA tile crosslinkers, and linear DNA spacers. We charac-
terized the nanomaterial networks in the composites and examined their mechanical and
electrical behaviors. We found that shorter spacers form more solid-like hydrogels when
combined with pure DNA crosslinkers, while longer spacers construct more solid-like
hydrogels when assembled with DNA/nanomaterial crosslinkers. We obtained hydro-
gel composites with significantly higher mechanical strength by combining DNA–CNT
conjugates and up to 25% DNA tiles as crosslinkers. In addition, dried networks from
both DNA–SWNT and DNA–AuNP conjugates and hydrogels show non-linear electrical
behaviors. By comparing the conductivities of dehydrated networks from conjugates and
hydrogels, we showed the ability of DNA self-assembly to integrate and connect percolating
networks with nanotubes and nanoparticles. These initial examples of biomolecular func-
tionality by design suggest that the basic concepts of DNA self-assembly can effectively be
used to create more complicated materials. Potentially, crosslinkers such as DNA-wrapped
CNTs can be used to create more sophisticated conjugates and nanostructures. We can
design DNA to realize control in nanoelectronics morphology through connection and
Appl. Sci. 2021,11, 2245 13 of 15
arrangement of nanomaterials. These materials have potential for applications in 3D in-
tegrated circuits and hardware with shorter production time, lower cost, lower power
consumption, and higher energy efficiency. Eventually, electronic hardware utilizing 3D
integration and assembled using DNA nanotechnology may achieve computing capabili-
ties in certain operations beyond the performance currently achieved by circuits fabricated
using traditional lithography techniques.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2076-341
7/11/5/2245/s1, Figure S1: Photos of (a) SWNT and (b) MWNT dispersions in deionized (DI) water
without (left) and with (right) DNA modification. SWNTs and MWNTs without DNA modification
settle at the bottom within 15 min after sonication; Figure S2: Photos of pure and DNA hydrogel
composites constructed by Sl with (from left to right) Y-shaped DNA tiles, X-shaped DNA tiles,
DNA–SWNT conjugates, DNA–MWNT conjugates, and DNA–AuNP conjugates; Figure S3: UV–Vis
spectrum of as-synthesized AuNPs. A peak
λmax
at 519 nm wavelength proves that these are well-
formed AuNPs with an average diameter of 13 nm. UV–Vis spectrum was recorded on a Thermo
Scientific NanoDrop 2000c Spectrophotometer (Waltham, USA); Figure S4: The band of pure AuNPs
(left) appears violet under visible light, while the band of DNA–AuNP conjugates (right) retains a red
color and shows a much better mobility; Figure S5: Gold electrodes of 200
µ
m spacing for electrical
characterization; Table S1: Sequences of ssDNA for the preparation of crosslinkers and spacers; Table
S2: Concentrations and ratios of crosslinkers and spacers to prepare for hydrogel.
Author Contributions:
Conceptualization, T.H.L., A.K., and M.G.; methodology, T.H.L., A.K., L.C.H.,
and M.G.; formal analysis, M.G., A.M., and A.K.; investigation, M.G., A.M., and A.K.; Writing—
Original draft preparation, M.G.; Writing—Review and editing, M.G., A.K., T.H.L., L.C.H., and A.M.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was partially funded by the US National Science Foundation through grant
NSF-CISE-CCF 1748459 to T.H.L.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Acknowledgments:
The authors thank E. Vetter, M. Hart, and C. Winkler for help with experiments.
This work was performed in part at the Analytical Instrumentation Facility (AIF) at North Car-
olina State University, which is supported by the State of North Carolina and the National Science
Foundation (award number ECCS-2025064). The AIF is a member of the North Carolina Research
Triangle Nanotechnology Network (RTNN), a site in the National Nanotechnology Coordinated
Infrastructure (NNCI).
Conflicts of Interest: The authors declare no conflict of interest.
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