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Diselenide-Bridged Carbon-Dot-Mediated
Self-Healing, Conductive, and Adhesive
Wireless Hydrogel Sensors for Label-Free
Breast Cancer Detection
Hyun Jeong Won, Benny Ryplida, Seul Gi Kim, Gibaek Lee,* Ji Hyun Ryu,* and Sung Young Park*
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sı Supporting Information

ABSTRACT: Recently, a great deal of research has focused on the study of

self-healing hydrogels possessing electronic conductivity due to their wide
applicability for use in biosensors, bioelectronics, and energy storage. The low
solubility, poor biocompatibility, and lack of effective stimuli-responsive
properties of their sp2 carbon-rich hybrid organic polymers, however, have
proven challenging for their use in electroconductive self-healing hydrogel
fabrication. In this study, we developed stimuli-responsive electrochemical
wireless hydrogel biosensors using ureidopyriminone-conjugated gelatin (Gel-
UPy) hydrogels that incorporate diselenide-containing carbon dots (dsCD)
for cancer detection. The cleavage of diselenide groups of the dsCD within
the hydrogels by glutathione (GSH) or reactive oxygen species (ROS)
initiates the formation of hydrogen bonds that affect the self-healing ability, conductivity, and adhesiveness of the Gel-UPy/
dsCD hydrogels. The Gel-UPy/dsCD hydrogels demonstrate more rapid healing under tumor conditions (MDA-MB-231)
compared to that observed under physiological conditions (MDCK). Additionally, the cleavage of diselenide bonds affects the
electrochemical signals due to the degradation of dsCD. The hydrogels also exhibit excellent adhesiveness and in vivo cancer
detection ability after exposure to a high concentration of GSH or ROS, and this is comparable to results observed in a low
concentration environment. Based on the combined self-healing, conductivity, and adhesiveness properties of the Gel-UPy/
dsCD, this hydrogel exhibits promise for use in biomedical applications, particularly those that involve cancer detection, due
to its selectivity and sensitivity under tumor conditions.
KEYWORDS: diselenide, carbon dot, self-healing, hydrogel sensor, stimuli-responsive

H ydrogels are emerging materials that possess applic-

ability for use in various biosensing applications,
particularly those that involve cancer detection. The
most prominent example of this is their ability to measure
changes in physicochemical properties of hydrogels, including
concentration of GSH is 4-fold, whereas the ROS is 10-fold
higher than that of the normal cell in a tumor environment.10−13
Moreover, adjusting the hydrogel matrices with redox-
responsive functional groups could possibly target the cancer,
allowing the detection of the diseases through the monitoring of
swelling behaviors in response to biological interactions and the biochemical reactions using optical and/or electrochemical
detection of biological events by bioreceptor-immobilized analysis with the redox reaction.14−16 Even though a current
hydrogels.1−5 The function of these hydrogels is also dependent detection system, such as positron emission tomography/
upon chemical and physical interactions between bioreceptors computed tomography (PET/CT), has been recognized, it still
and targeted biomolecules derived from the physiological contains several drawbacks such as intravenous injection of
environments.4,6 For instance, the changes in swelling properties
of various hydrogels, including ionic, pH-sensitive, redox-
responsive, and temperature-sensitive hydrogels, can be Received: March 24, 2020
detected in response to specific physiological conditions.7−9 Accepted: June 10, 2020
Redox-responsive hydrogels for cancer detection has been Published: June 10, 2020
widely investigated owing to the distinguish glutathione (GSH)
and reactive oxygen species (ROS) concentration between
normal and tumor conditions. It has been reported that the

© 2020 American Chemical Society https://dx.doi.org/10.1021/acsnano.0c02517

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Figure 1. Synthesis and characterization of dsCD. (a) Synthesis and chemical structures of dsCD. (b) Particle size distributions of dsCD after
treatments with phosphate-buffered saline (PBS), GSH, and H2O2 as measured by dynamic light scattering. Transmission electron microscopy
images of dsCD after treatments with (c) pH 7.4 PBS, (d) GSH, and (e) H2O2. (f) Photoluminescence intensity spectra of dsCD after treatments
with PBS, GSH, and H2O2. Fluorescence microscopy images of dsCD after (g) PBS, (h) GSH, and (i) H2O2 treatments. GSH and H2O2
concentrations are 10 mM and 100 μM, respectively. Scale bars indicate 100 μm.

radiopharmaceutical compounds, radiation exposure, and the developed.24 Poly(3,4-ethylenedioxythiophene):poly(4-styre-

need for expertise to interpret the data.17 The information from
PET/CT imaging is needed to identify the location for initial
assessment, and practically during surgery, a simple method for
the preliminary test is still required. Thus, it is highly desirable to
develop a self-healing and electronic signal-responsive biocom-
patible hydrogel for cancer sensors such as locating or placing a
patch at the tumor site and getting an immediate result,
diagnosis, or response under intracellular conditions during
tumor surgery.
One of the interesting aspects of the currently developed
hydrogels is self-healing via physical/chemical interaction by
modifying the polymer backbones.18,19 The reversibility of bond
formation and dissociation by physical cross-linking achieved by
hydrogen bonding, van der Waals interactions, π−π interactions,
π−cation interactions, and ionic bonds affects the re-formation
of polymeric networks after the hydrogels are destroyed.20−23
The ability of hydrogels to exhibit different self-healing
behaviors in the context of normal and targeted physiological
conditions could provide an excellent indicator for the ability of
these hydrogels to detect diseases and problems that cannot be
evaluated by current detection systems. Additionally, the self-
healing properties of electroconductive hydrogels could be
electrochemically detected by the attachment of electrodes to
the hydrogels.
To allow for the detection of electrochemical signals,
conductive polymer hydrogels and/or polymer hydrogels
incorporating electrically conductive materials have been
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nesulfonate) (PEDOT:PSS) composite hydrogels prepared
using thermal treatments represent one type of conductive
polymer;25 however, PEDOT:PSS hydrogels remain challenging
for use in medical applications due to their insolubility and
toxicity that limits their applicability, particularly within the
tumor microenvironment.26 Conductive nanocarbon sources,
such as carbon nanotubes, graphene, and carbon dots, can be
used for the enhancement of conductivity by the addition of
carbon sources into the hydrogels.27,28 The incorporation of
nanocarbon sources significantly improves hydrogel conductiv-
ity that can be detected by electrochemical devices and reduces
toxicity issues. For example, carbon dots (CDs) prepared using a
carbonization of polymers exhibit fluorescence and electro-
conductive properties and possess excellent water dispersibility,
mechanical properties, and low toxicity.29−32
In this study, we developed a redox-responsive self-healing
hydrogel that can detect the cancer environment and can
distinguish among various electronic signals that arise during
treatment in a redox setting. The main idea of this system is
utilizing the different concentrations of GSH and ROS between
normal and tumor cells. Therefore, the hydrogel that gives a
response to the mentioned stimuli was fabricated using
diselenide-containing carbon dots (dsCD) that incorporate
into ureidopyriminone-conjugated gelatin (Gel-UPy) hydrogels
achieving fluorescent, self-healing, and electronic materials. The
presence of GSH and H2O2 was essential to the cleavage of the
diselenide bonds of dsCD, which further control the self-healing,
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Figure 2. (a) Synthesis and chemical structures of Gel-UPy/dsCD hydrogels. (b) Photographic images of Gel-UPy and Gel-UPy/dsCD
hydrogels after cutting and attaching. (c) Photographs of GSH-treated and H2O2-treated Gel-UPy/dsCD hydrogels after cutting and attaching.
Proposed mechanisms of (d) unhealed Gel-UPy/dsCD hydrogels in pH 7.4 PBS and (e) self-healed Gel-UPy/dsCD hydrogels after GSH and
H2O2 treatments that resulted from hydrogen bonds. Resistance changes of Gel-UPy/dsCD hydrogels (f) in PBS, (g) with GSH, and (h) with
H2O2 before cutting, after cutting, and after self-healing. The resistance changes (i) in PBS, (j) with GSH, and (k) with H2O2 monitored by a
cellular phone under the same conditions described above. GSH and H2O2 concentrations are 10 mM and 100 μM, respectively.

adhesiveness, and the electrochemical properties of hydrogels.
The in vitro and in vivo experiments revealed that the hydrogel
biosensors could detect cancer cells with a high degree of
sensitivity and selectivity, suggesting wide applicability for the
visual and electrochemical detection of cancer.
RESULTS AND DISCUSSION
Synthesis and Characterization of Catechol-Contain-
ing Diselenide-Bridged Carbon Dots. The halogenated
catechol-containing alkanes (DOPA-Br) were initially synthe-
sized to form carbon−selenium (C−Se) bonds (Figure 1a) that
8411
are used in the final reaction step as the precursor of diselenide-
bridged dsCD. After the synthesis of DOPA-Br, the selenium
ions were introduced into the catechol-containing alkanes via
nucleophilic substitutions to obtain DOPA-Se.33,34 Additionally,
diselenide bonds (Se−Se) were spontaneously formed to
promote the formation of DOPA−Se−Se−DOPA, which will
be described as Di(DOPA-Se). To promote the self-oxidation of
catechol groups and their subsequent polymerization, Di-
(DOPA-Se) was incubated in pH 8.5 Tris-buffered solutions
for 24 h. During this process, the catechol group of DOPA loses
two protons and two electrons under basic reaction conditions,
ultimately leading to self-polymerization through the formation
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of dicatechol adducts.35 After the polymerization of Di(DOPA-
Se), the synthesized nanoparticles were carbonized in dimethyl
sulfoxide at 60 °C for 8 h to obtain the dsCD using the
hydrothermal method, and the structure was confirmed by 1H
NMR (Figure S1).
The particle size of Di(DOPA-Se) was approximately 118.29
nm as measured by dynamic light scattering (DLS), and this was
likely due to the physical interactions between catechol groups,
as shown in Figure 1b (i.e., hydrogen bonds and π−π
stacking).35,36 The obtained Di(DOPA-Se) was further analyzed
using MALDI-TOF to investigate the molecular weight of the
nanoparticle, which was determined to be 648 g/mol.
Additionally, the dsCD particle size was increased to 456 nm,
and the molecular weight of the nanoparticle was increased to
1938 g/mol, indicating that the Di(DOPA-Se) were covalently
cross-linked during the self-oxidative polymerization reac-
tions.37 As is well-known, the concentration of GSH in tumor
cells is 4-fold greater and the ROS in the intracellular condition
is 10-fold greater than those in normal cells, which makes it
easier to distinguish the cells. Additionally, due to the redox
reaction between GSH and ROS to diselenide, a redox-
responsive system consists of multiple diselenide bonds that
become a solution to fabricate a cancer-sensitive sensor. Due to
these facts, we verify the cleavage of diselenide bonds within the
dsCD during the redox reaction using 10 mM and 100 μM
concentrations of GSH and hydrogen peroxide (H2O2),
respectively, to mimic the intracellular condition of the tumor
cells.13,38,39 A decrease in particle size to 65.29 nm was observed
after GSH treatment, and the cleavage by GSH reduced the
molecular weight to 308 g/mol, further explaining the redox
reaction between dsCD and GSH. The addition of H2O2 could
also affect the decrease in dsCD size (∼68.42 nm), resulting
from the cleavage of diselenide bonds. Further confirmation of
the cleavage of diselenide bonds with dsCD was observed based
on the transmission electron microscopy images (Figure 1c).
After GSH and H2O2 treatments, the particle sizes were
significantly decreased, and this was consistent with DLS results
(Figure 1d,e). Moreover, the zeta-potential of dsCD before and
after treatment with GSH and H2O2 was obtained, as displayed
in Figure S2. Before treatment with either GSH or H2O2, the
zeta-potential of dsCD was identified to be −14.49 eV, which
further decreases after treatment with GSH and H2O2, reaching
−26.57 and −24.39 eV. The decrease indicated that the
diselenide bridge was disintegrated by GSH or H2O2, thus
oxidized −Se−Se− increased the negative charge owing to the
presence of oxygen of the nanoparticle surface.40 Carbon dots
are well-known for their tunable fluorescence, and we therefore
analyzed the fluorescence emission of the dsCD nanoparticles
before and after treatment with GSH and H2O2 using
photoluminescence (PL) spectrophotometry, as shown in
Figure 1f. A peak at the 482 nm wavelength was detected in
both GSH- and H2O2-treated dsCD, whereas no peak was found
in the untreated dsCD solution. It is noteworthy that the dsCD
fluorescence was quenched by the physical interactions of dsCD,
and this fluorescence was recovered after the cleavage of the
diselenide bonds. Additionally, Figure 1g reveals no fluorescence
of dsCD due to the quenching, which is in contrast to that
observed after treatments with GSH (Figure 1h) and H2O2
(Figure 1i) that resulted in the oxidation of diselenide groups in
dsCD.
Preparation and Characterization of Gel-UPy/dsCD
Hydrogels. The hydrogels were prepared by reacting the
gelatin with ureidopyrimidinone (UPy) via isocyanate−amine
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Article
group reactions that resulted in self-fabrication due to the
presence of UPy groups that can strongly bind to each other
through the utilization of multiple hydrogen bonds (Figure
2a).41−43 Additionally, the dsCD nanoparticle was incorporated
into the Gel-UPy hydrogels to obtain dsCD-embedded Gel-UPy
gels. The dsCD within the hydrogels exhibited a peak at the 481
nm wavelength after GSH or H2O2 treatments, whereas no peak
was observed to be produced by dsCD alone (Figure S3). This
indicated that the incorporation of dsCD into the hydrogels
could not significantly affect the fluorescence properties of
dsCD.
The visual photographs and the self-healing ability of the
fabricated hydrogel are displayed in Figure 2b, highlighted by
the distinctive shades that are observed between Gel-UPy and
Gel-UPy/dsCD hydrogels. The Gel-UPy hydrogels were
opaque and possessed a yellowish shade, whereas the Gel-
UPy/dsCD hydrogels were black due to the addition of dsCD.
In regard to the self-healing ability, the Gel-UPy hydrogels were
directly self-healed under all conditions including in phosphate-
buffered saline (PBS) solutions (Figure 2b) or after treatment
with GSH or H2O2 (Figure S4). This observation was also
supported by the rheological and the tensile strength analysis of
the Gel-UPy in Figure S5. The Gel-UPy/dsCD hydrogels
exhibited different behaviors, however, that included no self-
healing properties after treatment with a PBS solution due to the
presence of disturbances (dsCD) that prevent the intermolec-
ular interaction between UPy and gelatin.44 Additionally, the
Gel-UPy/dsCD hydrogels possessed excellent self-healing
properties after GSH or H2O2 treatments (Figure 2c) that
exhibited clear differences in the presence or absence of redox
potentials. For comparison, the self-healing performance of Gel/
dsCD was conducted to study the effect of dsCD hydrogen
bonding after treatment to hydrogel matrices. Figure S6 showed
that Gel/dsCD has no self-healing ability in PBS, but it has
preferable self-healing after treatment, which is similar to Gel-
UPy/dsCD. Furthermore, Fourier transform infrared (FT-IR)
of the Gel/dsCD (Figure S7) showed shifting in −O−H, −N−
H, and −C−O peaks, which indicate the effect of hydrogen
bonding promoted by dsCD after the cleavage by H2O2/GSH
treatment. To improve the clarity, the self-healing mechanism of
Gel-UPy/dsCD hydrogels is displayed in the schematic
illustrations (Figure 2d,e). The hydrogels exhibited no self-
healing behaviors in the absence of treatment, likely due to low
intermolecular interactions within the hydrogel;45 however, the
hydrogels were self-healed after treatment with GSH or H2O2
due to newly generated hydrogen bonds that resulted from the
cleavage of the diselenide bonds of dsCD. To confirm the
formation of hydrogen bonding of the Gel-UPy/dsCD after
GSH and ROS treatment, small-angle X-ray scattering (SAXS)
was performed. According to Figure S8, the increase of the q
peak to a higher value of Gel-UPy/dsCD during conjugation
with GSH/H2 O 2 treatment indicates the reduction of
interdomain spacing of Gel-UPy. Furthermore, the intensity of
Gel-UPy/dsCD after treatment increases owing to the change in
crystallinity of the hydrogel due to the influence of hydrogen
bonding to the matrices.46,47 Moreover, FT-IR showed that
there is a shift in the vibration of −O−H peaks from 3550 to
3300 cm−1 after UPy is introduced to the hydrogel matrices,
which is the effect of the hydrogen bond. Additionally, the peak
of −O−H changes to 3413 cm−1, determining the diminishing
of the hydrogen bond after dsCD loading. In a similar manner,
the −N−H peak at 1515 cm−1 of Gel-UPy/dsCD shifted to
1553 cm−1, and the appearance of the vibration peaks of −C−O
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Figure 3. Cancer-cell-responsive self-healing properties of Gel-UPy/dsCD hydrogels. (a) Photographic images of MDCK-cell-embedded
hydrogels with cutting and attaching. No self-healing occurred. Photographs of (b) MDCK-cell-embedded and (c) MDA-MB-231-cell-
embedded hydrogels with self-healing properties and the resistance changes of hydrogels treated with MDCK (b-1) and MDA-MB-231 (c-1)
cells after cutting and attaching. The monitored resistance of (d) MDCK- and (e) MDA-MB-231-cell-embedded hydrogels collected using a
cellular phone. Module changes for self-healing ability of the postwashed (f) MDCK- and (g) MDA-MB-231-treated hydrogels as determined by
the continuous step strain rheology test. The MDCK and MDA-MB-231 cell concentrations for each measurement are 101 cells/mL.

at 1081 cm−1 after GSH and H2O2 treatment demonstrate the
effect of hydrogen bonding to hydrogel matrices (Figure
S10).44,48 The presence of dsCD in the hydrogel matrices
blocked intermolecular hydrogen bonding between Gel-UPy.
However, after the cleavage by H2O2 or GSH, the intermolecular
bonding was present, which suddenly enhanced the self-healing
capabilities of the Gel-UPy/dsCD hydrogel. Thermogravimetric
analysis (TGA) was performed to investigate the thermal
stability, degradation, and hydrogen bond dissociation of the
hydrogel. According to TGA results in Figure S11, the Gel-UPy
demonstrated faster decomposition below 200 °C compared to
only gelatin gel owing to the breakage of intermolecular
hydrogen bonds. Other faster decomposition was also observed
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from Gel-UPy/dsCD after GSH and H2O2 treatment, due to the
effect of hydrogen bond dissociation.48
Rheology tests of the self-healing hydrogel during the course
of different treatments were conducted to analyze the elastic
response when the hydrogel was subjected to alternatively
changing amplitudes of oscillatory force (0.1−100%), as shown
in Figure S12. In Figure S12a, the PBS-treated hydrogel
exhibited poor recovery after high strain was applied due to
no cross-linked network recovery, and this was in contrast to
observations after GSH and H2O2 treatments (Figure S12b,c)
that quickly recovered to the initial value. This highlighted the
capacity for excellent self-healing that resulted from the
incremental hydrogen bonds within the matrices. To further
confirm the self-healing ability of the hydrogel, the electrical
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Figure 4. Electrochemical analysis of Gel-UPy/dsCD hydrogels embedded with MDCK and MDA-MB-231 cells. EIS analysis of the (a) MDCK-
and (b) MDA-MB-231-cell-embedded hydrogels. EIS measurements for hydrogels after elimination of (c) MDCK and (d) MDA-MB-231 cells.
(e) Resistance changes of hydrogels embedded with MDCK and MDA-MB-231 cells and after removing cells as a function of cell
concentrations. XPS analysis of (f) MDCK- and (g) MDA-MB-231-cell-embedded hydrogels after elimination of cells.

resistances of Gel-UPy/dsCD hydrogels with or without
treatments of GSH and H2O2 were measured after cutting and
healing. Initially, the fabricated hydrogels exhibited electronic
resistance around 825.9 Ω, and this was increased after the
hydrogel was cut due to no current passing through the system
(Figure 2f).49 In contrast, the resistance of Gel-UPy/dsCD
hydrogels after GSH (Figure 2g) or H2O2 (Figure 2h)
treatments recovered after being cut and self-healed, high-
lighting the reversibility of these hydrogel electronic properties.
It was noteworthy that the cleaved diselenide groups of dsCD
could significantly promote self-healing by allowing for the
formation of hydrogen bonds of individual dsCD, resulting in
resistance differences that could be detected by a commercially
available smart phone used as an electronic device (Figure 2i−
k). Moreover, the hydrogel exhibited self-healing reversibility
mechanically and electrically when cut and self-healed
repeatedly for 10 cycles, as shown in Figure S13. The Gel-UPy
hydrogel demonstrated preferable stability until 180 h compared
to that with gelatin hydrogel (90 h) owing to hydrogen bonding
that maintained the hydrogel structure (Figure S14).48
Electrochemical Studies Examining Cancer-Cell-Re-
sponsive Self-Healing Gel-UPy/dsCD Hydrogels. Prior to
cancer-cell-responsive analysis, the biocompatibility of the Gel-
UPy/dsCD hydrogel was identified with live and dead assays
using confocal laser scanning microscopy. As shown in Figure
S15, the hydrogel exhibited excellent biocompatibility as no
8414
dead cells were observed, which is comparable with the control.
Moreover, to verify the cancer-cell-responsive self-healing
properties of Gel-UPy/dsCD hydrogels, MDCK (normal) and
MDA-MB-231 (cancer) cells were each embedded into the Gel-
UPy/dsCD hydrogels. The Gel-UPy/dsCD hydrogels contain-
ing MDCK cells exhibited no self-healing properties due to the
low concentration of GSH (Figure 3b). In contrast, the MDA-
MB-231-embedded Gel-UPy/dsCD hydrogels (101 cells)
demonstrated self-healing ability after being cut and attached
(Figure 3c). Further analysis using high concentrations of
MDCK and MDA-MB-231 cells were then conducted. Higher
concentrations of MDCK cells (∼105 cells) could not initiate
healing due to an absence of reduction−oxidation by GSH and
H2O2, as shown in Figure S16. Additionally, there is no self-
healing observed when hydrogel was embedded with MCF10A
cells, which is a normal breast cell (Figure S17). In contrast,
hydrogels treated with higher concentrations of MDA-MB-231
(∼105 cells) exhibited rapid self-healing behaviors that were
likely due to higher redox potential levels that elevated the
hydrogen bonds within the hydrogel to help reconstruct the
hydrogels (Figure S18). It should be noted that the GSH
production in cancer cells could promote the self-healing of
hydrogels by facilitating the cleavage of diselenide bonds and the
subsequent formation of hydrogen bonds between the catechol
groups of dsCD.50,51 Additionally, we analyzed the resistances of
cut and healed cell-embedded Gel-UPy/dsCD hydrogels
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Figure 5. Ex vivo adhesion and in vivo cancer detection using Gel-UPy/dsCD hydrogels. (a) Schematic illustrations of measurements for ex vivo
adhesive properties. (b) Maximum detachment forces of hydrogels embedded with MDCK and MDA-MB-231 (cell concentration = 101 cells/
mL) cells as a function of cell concentration. (c) Schematic illustration of in vivo self-healing hydrogels in response to cancer cells. Photographic
images of hydrogels after 1 h implantation in (d) normal and (e) cancer tissues. (f) In vivo remaining weight of hydrogels as a function of time.
Histological assessments for hydrogels after (g) 5 and (h) 10 days.

according to a previously described method. Initially, the
resistance of MDCK-treated hydrogels was measured at 1825.9
Ω, and this further increased after cutting due to the absence of
self-healing and electron movement between the two cut
hydrogels (Figure 3b-1). In contrast, the MDA-MB-231-
embedded Gel-UPy/dsCD hydrogels exhibited repetitive self-
healing behaviors that included the recovery of resistance from
1826.1 Ω at initial conditions (before cut), unmeasured
resistance due to unconnected circuit condition after cutting,
and a return to 820.8 Ω after self-healing (Figure 3c-1). To
account for differences in the systems used to measure
resistance, MDCK- and MDA-MB-231-embedded Gel-UPy/
dsCD hydrogels were further analyzed using real-time measure-
ment connected to a smartphone via Bluetooth modules. For the
benchmark, the electrical signals from MDCK-cell-embedded
Gel-UPy/dsCD hydrogels prior to cutting were obtained. The
resistances of MDCK-treated hydrogels further increase due to
the absence of electrons passing through the system
(unconnected circuit), and this was decreased after the two
pieces of hydrogel were attached (before washing). Additionally,
to eliminate cells on the surface of the hydrogel, we washed the
hydrogel immediately and observed the signal that displays the
unmeasured signal (Figure 3d). The MDA-MB-231-embedded
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hydrogels did, however, exhibit a significant reduction in
resistance after attachment as a result of the self-healing
phenomena and a prolonged decrease in resistance after being
washed due to detachment of cells from the hydrogel (Figure
3e). The self-healing ability of the hydrogel during treatment
with MDCK and MDA-MB-231 cells before (Figure S19) and
after washing was also analyzed using a rheology recovery test on
different strains. The MDCK-treated hydrogel (Figure 3f)
exhibited no cross-link network recovery before and after
washing when subjected to oscillatory shear strain. These
phenomena indicated that the hydrogel network was broken
when high strain (100%) was applied to the hydrogel as the
recovery rate continued to decrease during the four oscillations.
Rapid recovery was observed, however, when the MDA-MB-
231-embedded Gel-UPy/dsCD hydrogel was subjected to 0.1
and 100% oscillation strains (Figure 3g). The MDA-MB-231-
treated Gel-UPy/dsCD hydrogel revealed that the G′
immediately recovered after the breaking strain was removed,
regardless of loading period. This further highlighted the
excellent cross-linked network recovery and self-healing
ability.37
To verify the effects of cancer-cell-dependent electrochemical
analyses, electrochemical impedance spectroscopy (EIS) was
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conducted. A Nyquist plot illustrating different hydrogel
treatments was fitted using a simple Randle equivalent circuit,
and the diameter of the circle in the plot corresponds to charge
transfer resistance between electrodes. Prior to EIS analysis, the
state of MDCK and MDA-MB-231 cells on the Gel-UPy/dsCD
hydrogel was observed using scanning electron microscopy
imaging. As shown in Figure S20a, the cells were attached to the
hydrogels that were embedded with either MDCK or MDA-
MB-231 cells; however, after the system was washed with PBS
for 1 h, almost all of the cells were removed, as shown in Figure
S20b. Additionally, the EIS results in Figure 4a,b revealed that a
higher number of cells embedded into the hydrogel increased
the resistance of the system, and this occurred regardless of cell
type. After the hydrogel was treated with cells for 1 h and then
washed with PBS to remove unattached cells from the surface,
the EIS experiments were then repeated. As shown in Figure 4c,
the MDCK-embedded Gel-UPy/dsCD hydrogels exhibited
decreased resistance, whereas MDA-MB-231-treated hydrogels
exhibited a much higher decrease that was due to the high
amount of GSH and H2O2 that cleaved the diselenide bonds to
create smaller particles that enhanced the electronic con-
ductivity of the hydrogel (Figure 4d).22,52 Additionally, the
cleaved diselenide bonds caused by a simultaneous redox
process of −Se−Se− to form −Se−O that is facilitated by GSH
and H2O2 promoted the proton−electron transfer due to
additional charge transfer interaction and hydrogen bond
formation between two organic π-electron components.53
These phenomena indicated that the hydrogel-based biosensors
could significantly detect cancer cells through electrochemical
measurements. Additionally, the resistances of Gel-UPy/dsCD
hydrogels correlate with the concentration MDCK and MDA-
MB-231 cells, and these data are presented in Figure 4e. The
resistances were almost constant either before or after removing
MDCK cells (101 cells/mL, 1907.0 Ω; 102 cells/mL, 2651.7 Ω;
104 cells/mL, 5485.6 Ω; and 105 cells/mL, 9713.4 Ω); however,
the resistances were significantly decreased in response to
various concentrations of MDA-MB-231 cells. Specifically, these
values were 1940.6 (101 cells), 3256.7 (102 cells), 6525.8 (104
cells), and 10670.8 Ω (105 cells) before removal to 1540.6 (101
cells), 1360.3 (102 cells), 837.6 (104 cells), and 350.8 Ω (105
cells) after removal. To confirm that the diselenide was cleaved
by the cells, the X-ray photoelectron spectroscopy (XPS) spectra
of Gel-UPy/dsCD hydrogels after the elimination of MDCK
and MDA-MB-231 cells were determined. In Figure 4f, the
peaks at 56.4 eV that could be assigned as the diselenide (Se−
Se) peaks were still present in the XPS spectrum of MDCK
groups;49 however, the peak for Se−Se was absent, and the new
Se−O was presented in the MDA-MB-231 (Figure 4g) groups,
indicating the cleavage of diselenide in the cancer cell
microenvironments. Thus, these hydrogel-based biosensors
may be used to detect cancer cells based on the diselenide-
mediated, self-healing of hydrogels in response to the generation
of GSH or ROS by cancer cells.
In Vivo Cancer-Cell-Specific Self-Healing Based on
Adhesiveness of Gel-UPy/dsCD Hydrogels. We hypothe-
sized that the Gel-UPy/dsCD hydrogels could be selectively
adhered to cancer tissues by cleavage of diselenide groups and
subsequent adhesion of remnant dsCD catechol groups.
Additionally, strong adhesiveness of hydrogels may result in
the successful detection of cancers. To evaluate the tissue
adhesion properties of Gel-UPy/dsCD hydrogels, we measured
the tensile strengths of Gel-UPy/dsCD hydrogels and
performed lap-shear adhesion testing using a universal testing
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machine (UTM).54,55 Figure 5a shows the schematic illustration
of adhesion measurements for the hydrogels. The porcine skins
(1 × 1 cm2) were attached to adherents, and the hydrogels were
placed between two porcine skins. As presented in Figure S21,
the standard force-displacement (F−D) curves of Gel-UPy/
dsCD hydrogels using different cell lines and concentrations
clearly revealed the cancer-cell-mediated self-healing and
adhesion properties of the hydrogels. The adhesion forces
were low in the MDCK groups, and the differences were not
significant as a function of cell concentration, indicating that the
normal cells could not affect the adhesion properties of Gel-
UPy/dsCD hydrogels. In contrast, Gel-UPy/dsCD hydrogels
using MDA-MB-231 cells exhibited a significant increase in
adhesion forces, as shown in Figure 5b (0.11 N for control,
0.4045 N for 101 cells, 0.8988 N for 102 cells, 1.8505 N for 104
cells, and 2.5013 N for 105 cells). As the main driving force of the
adhesion between the substrate and the hydrogel is the
attachment based on hydrogen bonds, and the cleavage of
diselenide bonds by GSH and H2O2 produced by cancer cells
enhanced the amount of hydrogen bonds and simultaneously
increased the adhesive behavior of the system. These
phenomena are also supported by the observation that the
surface area of nanoparticles within the hydrogel matrix was
increased due to a reduction in size after cleavage, thus
increasing the amount of contact area available during
attachment.
The Gel-UPy/dsCD hydrogels were further utilized as in vivo
cancer biosensors based on their self-healing properties. As
shown in Figure 5c, the MDCK and MDA-MB-231 cells (106
cells/20 μL) were injected into the subcutaneous regions. After
2 weeks, five pieces of the Gel-UPy/dsCD hydrogels were
implanted into the solid tissues of mice. The self-healing
behaviors of hydrogels were monitored for cancer detections
after 1 h. The Gel-UPy/dsCD hydrogels were separated on the
normal tissue (Figure 5d). Additionally, five pieces of the Gel-
UPy/dsCD hydrogels appeared as single hydrogels, but these
could readily be dissociated. In contrast, the pieces of Gel-UPy/
dsCD hydrogels on the cancer tissue formed a single hydrogel
that could not be separated (Figure 5e). The injected Gel-UPy/
dsCD hydrogels also maintained 57% of their original dry weight
after 5 days in vivo (Figure 5f). Although inflammatory
responses were observed after 5 days of hydrogel implantations
(Figure 5g), immune responses mostly disappeared by day 10
(Figure 5h). Thus, these hydrogel-based biosensors possess
enormous potential for use in biomedical applications as
implantable and cancer-detectable materials.
CONCLUSION
In this study, we report a wireless electrochemical self-healing
hydrogel biosensor that is based on the cleavage of diselenide in
response to exposure to glutathione and/or reactive oxygen
species in cancer cells. This Gel-UPy/dsCD hydrogel exhibited
cancer-cell-dependent self-healing properties with excellent
selectivity due to the different conditions that exist between
tumors and normal conditions that affect both electrochemical
signals and adhesive behavior. The hydrogel demonstrated fast
self-healing under tumor conditions simulated by the use of
MDA-MB-231 cells, and it remained unhealed under normal
conditions (MDCK cells) due to increased hydrogen bonds that
resulted from cleavage of the diselenide bonds of dsCD.
Additionally, different concentrations of cells embedded in the
hydrogels also allowed us to distinguish among electronic
responses, where the higher gap changes were detected under
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tumor conditions. Interestingly, the adhesiveness of hydrogels
was comparable during incubation with either MDCK or MDA-
MB-231 cells due to differences in intermolecular interactions
such as hydrogen bond and particle size after treatment with
GSH and H2O2 to enhance the attachment of the Gel-UPy/
dsCD hydrogels. The in vivo analysis also revealed no trace of
inflammation, confirming the excellent biocompatibility of these
hydrogels. This study provides a possibility involving the use of a
hydrogel biosensor particularly for cancer cell detection that
widens the opportunity for various biomedical applications such
as cancer-specific drug delivery and tissue engineering.
EXPERIMENTAL SECTION
Materials. Dopamine hydrochloride, triethylamine, selenium,
gelatin, 2-amino-4-hydroxy-6-methylpyrimidine, hexamethylene diiso-
cyanate, sodium borohydride (NaBH4), ethanol, n-pentane, diethyl
ether, dimethyl sulfoxide (DMSO), anhydrous N,N-dimethylforma-
mide (DMF), tetrahydrofuran (THF), and glutathione (GSH) were
purchased from Sigma-Aldrich, Korea. The 4-bromobutyryl chloride
was purchased from TCI. All other chemicals were of analytical grade.
DOPA-Br34 and 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-
4[1H]-pyrimidinone (UPy-NCO)41−43 were synthesized as previously
reported.
Synthesis of Diselenide-Bridged Carbon Dots. Initially, the
sodium hydrogen selenide was prepared by mixing the dry powders of
selenium (157 mg, 1.9 mmol) and NaBH4 (152 mg, 3.98 mmol) in
ethanol (98%, 0.7 mL). Anhydrous DMF (4 mL) was added into the
mixtures, and the color was observed to change from white to red-
brown. Additionally, more ethanol (80%, 0.45 mL) was added to the
mixture, and this was followed by a second addition of selenium powder
dissolved in DMF (5 mL). Dopa-Br (895 mg, 3.98 mmol) was added
into the selenide solution, and the reaction was allowed to continue for
24 h. After the reaction, double distilled water (DDW) (12 mL) was
added dropwise into the reaction solution to finalize the reactions. The
product was purified by precipitation using diethyl ether, dried in a
vacuum oven, and then labeled as Di(Dopa-Se) [EI-MS (m/z): 648 g/
mol, yield: 89.4%].
The Di(DOPA-Se) was allowed to polymerize by dissolving 1 g of
Di(DOPA-Se) into DMSO (90 mL) and a pH 8.5 Tris-buffered
solution (10 mL). The reaction time was 24 h, and the pH was adjusted
to 8.5 during the reaction. The obtained mixture was purified by
membrane dialysis (MWCO: 1 kDa), freeze-dried, and then labeled as
cross-linked Dopa-Se (yield: 36.4%). Finally, the cross-linked Dopa-Se
(1 g) was dissolved into DMSO (50 mL) and reacted for 8 h at 60 °C to
produce dsCD through a hydrothermal process. The dsCD was further
dialyzed for 3 days using a membrane (MWCO: 1 kDa) against water
and then lyophilized [EI-MS (m/z): 1938 g/mol, yield: 51.3%].
Synthesis of Ureidopyrimidinone-Conjugated Gelatin. Gel-
atin (4 g) was dissolved into DMSO (90 mL) at 60 °C. After gelatin was
homogeneously dissolved in DMSO, UPy-NCO (0.2 g) in DMSO (10
mL) was added dropwise into the gelatin solution using a dropping
funnel and reacted for 24 h. The Gel-UPy product was precipitated by
THF, further purified by dialysis against water (MWCO: 3.5 kDa
membrane) for 3 days, and then lyophilized (yield: 57.96%).
Fabrication of Gel-UPy/dsCD Hydrogels. To prepare the Gel-
UPy/dsCD hydrogels, Gel-UPy (100 mg) was dissolved into 1 mL of
DDW, and dsCD (5 mg) was added into the Gel-UPy solution. After
the Gel-UPy and dsCD were homogeneously mixed, the mixture
solution was poured into the molds. The final Gel-UPy/dsCD hydrogel
sponges were obtained using a simple freeze-drying method involving a
freezing and thawing process. To perform all of the hydrogel
experiments, the Gel-UPy/dsCD hydrogel sponges were swollen in
pH 7.4 PBS solutions for 24 h.
Glutathione- and H2O2-Treated Hydrogels. The 1 × 1 cm2
hydrogels were dipped into 1 mL of prepared solution (PBS 7.4, 10 mM
GSH in PBS 7.4, 100 μM H2O2 in PBS 7.4) and allowed to swell for 1 h
in the 37 °C incubator. The surface of the hydrogel was wiped to
remove excess solution before further analysis.
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Preparation of Cell-Embedded Gel-UPy/dsCD Hydrogels. To
prepare the cell-embedded Gel-UPy/dsCD hydrogels, the hydrogel
with dimensional structure at 1 × 1 cm2 was soaked in cell solution with
different concentrations (101, 102, 104, 105 cells/mL). The cell-
embedded hydrogel was then incubated for 24 h at 37 °C in a 5%
humidified CO2 atmosphere. For detachment of cells from hydrogel
matrices, the hydrogel was soaked in PBS 7.4 solution for 1 h and
shaken gently.
UV−Vis and Fluorescence Spectroscopic Studies. To perform
the UV−vis and fluorescence spectroscopic studies, the DOPA−Se−
Se−DOPA and dsCD were both dissolved into DDW at a
concentration of 1 mg/mL. For the treatments of GSH, 1 mL of
GSH (10 mM) was added into the dsCD-dispersed solution. To verify
the effectiveness of H2O2 treatments, 1 mL of H2O2 (100 μM) was
added into the dsCD solution. The final concentration of dsCD was
fixed to 1 mg/mL.
Electrochemical Studies for the Detection of Cancer Cells. To
measure the electrochemical signal, the resistances of Gel-UPy/dsCD
hydrogels were measured using a Keithley 2450 sourcemeter. After the
resistance of Gel-UPy/sdCD hydrogels was measured, the hydrogels
were treated with GSH (10 mM) or H2O2 (100 μM). The resistances of
GSH- or H2O2-treated hydrogels were measured after 60 min reactions.
Additionally, the resistances were monitored before cutting, after
cutting, and after attaching. For the cell experiments, the MDCK,
MCF10A, and MDA-MB-231 cells were seeded into the hydrogels as a
function of cell concentration (1 to 105 cells/single hydrogel) and then
incubated for 24 h at 37 °C in a humidified atmosphere under 5% CO2.
The resistances were monitored as described above. The EIS signals
were obtained using methods similar to those described above. The
MDCK, MCF10A, and MDA-MB-231 cells were seeded into the
hydrogels at predetermined cell concentrations (101, 102, 104, and 105
cells/one hydrogel) and cultured for 24 h at 37 °C in a humidified
atmosphere under 5% CO2. The EIS signals were obtained before and
after removing MDCK, MCF10A, and MDA-MB-231 cells. The
frequency was varied from 0.1 to 10 kHz, and the Ag/AgCl electrode
was used as a reference.
Ex Vivo Tissue Adhesive Forces of Gel-UPy/dsCD Hydrogels.
The tissue adhesive forces were measured using a lap-shear adhesion
testing method that employed a UTM. Briefly, the porcine skin tissues
were cut into 1 × 1 cm2 and attached to commercially available
polyethylene terephthalate OHP films. The Gel-UPy/dsCD hydrogels
with same dimensional size were placed and sandwiched to the porcine
skin tissue attached PET OHP film. The adhesiveness was measured
and collected by clipping the edges of PET OHP to a stationary edge
and another edge to move the grip of UTM with displacement rate of 1
mm/min. For the measurements of adhesive forces for MDCK- or
MDA-MB-231-containing Gel-UPy/dsCD hydrogels, the cells were
seeded into the hydrogels. The final concentrations of cells varied from
101 to 105 cells/single hydrogel.
In Vitro Biocompatibility of Gel-UPy/dsCD Hydrogel by Live
and Dead Assay. The cellular live and dead assays were conducted by
culturing 0.1 mL of 106 cells/mL of MDCK and MDA-MB-231 in a
Petri dish (control) and Gel-UPy/dsCD hydrogel and incubation at 37
°C in a humidified atmosphere under 5% CO2. After 24 h, the control
and hydrogel were washed with PBS and dyed with Calcein-AM for 15
min and propidium iodide for 1 min. After that, they were washed with
PBS for the final step. The cells on the control and hydrogel were
observed using confocal laser scanning microscopy.
In Vivo Self-Healing Properties of Gel-UPy/dsCD Hydrogels.
All animal experiments were performed in the Carbon Nano-
BioMaterials Laboratory of Wonkwang University and were approved
by the Animal Review Committee of Wonkwang University (WKU19-
54). Ten male BALB/c nude mice (16−20 g, 4−6 weeks old, Oriental
Bio. Co. Ltd., Korea) were used for cancer-specific self-healing
examinations of hydrogels. In brief, the MDA-MB-231 cells (106
cells/20 μL) were injected into the subcutaneous regions. After 2
weeks, a local growth of cancers was found on the back of the mouse.
The mouse was anesthetized using Zoletil/rumpun and immobilized
onto a surgical corkboard. Each mouse underwent surgery through a 1
cm long incision near the tumor tissue. Five pieces of the Gel-UPy/
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ACS Nano www.acsnano.org
dsCD hydrogels were prepared and implanted onto the tumor tissue.
After 1 h, the incision of each mouse was reopened to monitor the
hydrogels in a blind fashion. The MDCK cells were used as a control.
Histological Assessment for Gel-UPy/dsCD Hydrogels. The
Gel-UPy/dsCD hydrogels were subcutaneously implanted on the left
and right dorsal sides of mice (BALB/c, two mice). The tissues
surrounding the Gel-UPy/dsCD hydrogels were excised and collected
on postoperative days (5 and 10 days). The tissues were fixed in 10%
neutral buffered formalin for 24 h, embedded in paraffin, and then
sectioned at 20 μm following standard histological procedures. The
histological sections were stained with hematoxylin and eosin (H&E)
to observe the cell morphologies and distributions.
Real-Time Analysis of Hydrogel Self-Healing Properties via
Bluetooth-Connected Smartphone. Data acquisition was per-
formed by connecting self-prepared wireless equipment to the hydrogel
and transmitting the reading to the smartphone via Bluetooth
connection. The system configuration consists of a self-prepared
electronic circuit, microcontroller (Arduino Uno), and Bluetooth
module (AppGosu). The acquired self-healing performance was
displayed as a graph as an active communication between the
smartphone and the microcontroller via wireless connection.
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsnano.0c02517.
Characterization and equipment; 1H NMR data of
nanoparticles and hydrogel; zeta-potential of nano-
particles; self-healing of Gel-UPy treated with GSH and
ROS; SAXS, FT-IR, and TGA of hydrogel; hydrogel self-
healing reversibility and long-term stability; live and dead
assay; wireless resistance change with different concen-
tration of cells; scanning electron microscopy images of
cells containing hydrogel; standard F−D (force−displace-
ment) curves of hydrogels with various MDCK and
MDA-MB-231 cell concentrations (PDF)
AUTHOR INFORMATION
Corresponding Authors
Gibaek Lee − Department of Chemical & Biological Engineering,
Korea National University of Transportation, Chungju 380-702,
Republic of Korea; Email: glee@ut.ac.kr
Ji Hyun Ryu − Department of Carbon Convergence Engineering,
Wonkwang University, Iksan, Jeonbuk 54538, Republic of
Korea; orcid.org/0000-0002-0279-7477;
Email: jhryu4816@wku.ac.kr
Sung Young Park − Department of Chemical & Biological
Engineering, Department of Green Bio Engineering, and
Department of IT Convergence Engineering, Korea National
University of Transportation, Chungju 380-702, Republic of
Korea; orcid.org/0000-0002-0358-6946;
Email: parkchem@ut.ac.kr
Authors
Hyun Jeong Won − Department of Chemical & Biological
Engineering, Korea National University of Transportation,
Chungju 380-702, Republic of Korea
Benny Ryplida − Department of Green Bio Engineering, Korea
National University of Transportation, Chungju 380-702,
Republic of Korea; orcid.org/0000-0003-2272-3659
Seul Gi Kim − Department of Chemical & Biological Engineering,
Korea National University of Transportation, Chungju 380-702,
Republic of Korea
Complete contact information is available at:
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Article
https://pubs.acs.org/10.1021/acsnano.0c02517
Author Contributions
The experiments were designed and conceived by H.J.W. and
S.Y.P. The nanoparticle synthesis, characterization, and hydro-
gel fabrication were conducted by H.J.W. Mechanical properties
were analyzed by H.J.W., B.R., and G.L. The electrochemical,
electronic properties, and wireless communication were set up
by H.J.W. with the input from G.L. In vitro analysis was
conducted by H.J.W. and S.G.K. In vivo experiment was
performed by J.H.R. The manuscript was written by B.R.
J.H.R., and S.Y.P. and revised by H.J.W., B.R., and S.Y.P.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education (NRF-
2020R1A2B5B02001500 and 2018R1A6A1A03023788). We
thank A. Moon at Duksung Women’s University for providing
MCF10A cells
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