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org Research Article

Near-Infrared-Active Copper Bismuth Oxide Electrodes for Targeted

Dissociation of Alzheimer’s β‑Amyloid Aggregates
Yerin Heo, Kayoung Kim, Jinhyun Kim, Jinhyeong Jang, and Chan Beum Park*
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ABSTRACT: The abnormal accumulation of β-amyloid (Aβ) aggregates in the brain is a major
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pathological hallmark of Alzheimer’s disease. We report a near-infrared (NIR)-active CuBi2O4-

based photocathodic platform that can target intact Aβ aggregates and dissociate them into
nontoxic species. Because of its relatively narrow band gap, CuBi2O4 exhibits strong absorption
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of NIR light, which allows for deeper tissue penetration and causes less photodamage to tissues
compared to visible light. Furthermore, its high stability in aqueous media, biocompatibility, and
robustness against photocorrosion make CuBi2O4 an ideal material for medical applications. For
the targeted clearance of Aβ aggregates, we have conjugated the KLVFF peptide which
specifically recognizes and captures Aβ aggregates on the surface of silver-doped CuBi2O4
(Ag:CuBi2O4). Upon illumination of NIR light under a cathodic bias, the KLVFF-immobilized
Ag:CuBi2O4 (KLVFF-Ag:CuBi2O4) effectively dissociated β-sheet-rich, long, and entangled Aβ
fibrillary aggregates into small fragmented, soluble species through photo-oxygenation. We also
verified that the KLVFF-Ag:CuBi2O4 photocathode is biocompatible and effective in reducing
Aβ aggregate-induced neurotoxicity. Our work demonstrates the potential of the KLVFF-
Ag:CuBi2O4 platform for the targeted disassembly of cytotoxic, robust Aβ aggregates with the aid of NIR energy and cathodic bias.
KEYWORDS: Alzheimer’s diseases, copper bismuth oxides, β-amyloids, peptide self-assembly, photocatalysis

■ INTRODUCTION
Alzheimer’s disease (AD) is the most pervasive neuro-
degenerative disorder affecting one in 10 people over the age
of 65, and the number of people suffering from the disorder
increases exponentially with aging.1 Despite the etiology of AD
being unclear, the abnormal accumulation of β-amyloid (Aβ)
aggregates in the brain is a key pathogenic hallmark in the
onset of AD.2 Increased levels of Aβ peptides by either
overproduction or failure of clearance promotes the self-
assembly of Aβ peptides into extracellular Aβ deposits such as
oligomers and fibrillary aggregates.3 Because Aβ assemblies
serve as a reservoir of toxic oligomers and accelerate the
formation of oligomers through secondary nucleation,4 many
attempts have been made to halt the progress of AD pathology
by clearing the Aβ aggregates.5−7 However, the dissociation of
Aβ aggregates is challenging because of the remarkable
structural stability of Aβ aggregates, which stems from their
closely packed cross β-sheet conformation. Recently, visible
(or UV) light-induced dissociation of mature Aβ aggregates
has been reported, which employed oxidative stress generated
by photosensitizing compounds or materials.8−10 For example,
Lee et al. demonstrated the breakage of preformed Aβ
aggregates by methylene blue under visible light illumination.8
In addition to molecular photosensitizers, photoactive
inorganic nanomaterials, such as titanium dioxide, were
investigated for the photocatalytic dissociation of Aβ
assemblies into nontoxic Aβ species under UV light
irradiation.9,11
Photoactive materials are an attractive option for light-
assisted therapy that enables local treatment through
spatiotemporal control of light.12−17 However, the use of
molecular or colloidal photosensitizers for treating brain
disorders poses potential issues, such as their delivery across
the blood−brain barrier to the hippocampus region and the
poor metabolism of light absorbers.18−20 Furthermore, high
absorbance of visible light in biological tissues leads to limited
tissue penetration below 2.5 mm, which restricts the
photoinduced therapies to superficial lesions of the human
body.21,22 In this regard, the photoelectrochemical approach
using near-infrared (NIR)-active electrodes is an attractive
strategy for the in situ light-assisted dissociation of Aβ
aggregates. Actually, electrode-based injectable devices are
already in clinical use for treating neurological disorders;23,24
deep brain stimulation delivers electrical impulses to a specific
area of the brain to mitigate the symptoms of Parkinson’s
disease, essential tremor, and dystonia. The safety of DBS
Received: February 7, 2020
Accepted: May 4, 2020
Published: May 4, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acsami.0c02349

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devices was approved by the Food and Drug Administration.
Furthermore, recent advances in optoelectronics allow for
direct photostimulation of deep brain regions via an implanted
NIR light source.25,26 For example, NIR light-emitting diode-
coupled fiber optics have delivered NIR light to a deeply
located lateral ventricle with minimal invasion.27 Compared to
visible (or UV) light, NIR light exhibits much deeper tissue
penetration and cause less damage to tissues by overheating; a
NIR light of 808 nm penetrates 10.9 mm of the brain
tissue.28−30 In addition, recent reports have revealed that the
injectable, NIR light-emitting fiber optics can be easily
combined with other inorganic materials,31 which hints at
the possibility of future development of the CuBi2O4-based
photoelectrode platform integrated with a NIR light source for
the localized dissociation of Aβ aggregates.
Herein, we report NIR light-triggered dissociation of Aβ
aggregates using a copper bismuth oxide (CuBi2O4) photo-
cathode, as depicted in Figure 1. Because of the relatively
Figure 1. Schematic illustration of disaggregation of Aβ assemblies by
the KLVFF-Ag:CuBi2O4 photocathode. Aβ aggregates are captured by
the cathode through the KLVFF moiety. When the cathodic bias was
applied to the KLVFF-conjugated Ag:CuBi2O4 under NIR light
illumination, reactive oxygen species (i.e. superoxide ions, hydrogen
peroxide, and so on) are generated to dissociate the Aβ aggregates
captured on the KLVFF-Ag:CuBi2O4 photocathode into nontoxic,
small species.
narrow band gap of 1.5−1.8 eV, p-type CuBi2O4 can absorb
NIR light, which causes minimal photodamage to the
surrounding cells along with high tissue penetration ability.32,33
Among various NIR-mediated photosensitizers, CuBi2O4 is
considered an ideal option because of its high stability in
aqueous media, biocompatibility, and robustness against
photocorrosion.34−36 The CuBi2O4-based photocathode ab-
sorbs NIR light, generates excited electron−hole pairs, and
drives redox reactions by modulating the energy level of the
electrons through the application of the cathodic bias. In the
current work, we have conjugated the KLVFF peptide on the
surface of CuBi2O4 for the targeted dissociation of Aβ
aggregates. KLVFF is a central recognition sequence (16−
20) of Aβ peptides, which binds to Aβ aggregates. Compared
to other recognition elements, such as antibodies (dissociation
constant, Kd, 10−8 to 10−12 M),37 the KLVFF peptide (Kd, 10−6
M) exhibits moderate binding affinity to Aβ aggregates.38
Nonetheless, the use of the KLVFF motif can avoid an
undesired immune response upon injection, the potential issue
of immunotherapy using monoclonal antibodies.39−43 By
conjugating the KLVFF peptide on the surface of the
CuBi2O4 photocathode, we demonstrate targeted disassembly
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of Aβ aggregates and alleviation of Aβ-associated toxicity with
the cathodic bias under NIR light illumination.
■ RESULTS AND DISCUSSION
We prepared silver-doped CuBi2O4 (Ag:CuBi2O4) photo-
cathodes via thermal oxidation of Ag-doped Cu/Bi films
electrodeposited on a fluorine-doped tin oxide (FTO)
substrate (Figure 2a). We doped the monovalent Ag ions to
CuBi2O4 to enhance the photocurrent intensity and the
photostability as well.44 Ag+ is an acceptor-type dopant that
substitutes the Bi sites of CuBi2O4 because of the similarity of
its ionic radius (115 nm) to that of Bi3+ (103 nm).44 The as-
synthesized Ag:CuBi2O4 film exhibited a porous morphology
with a thickness of 1 μm (Figures 2a and S1). According to our
analyses by energy-dispersive X-ray spectroscopy (EDX), Cu,
Bi, O, and Ag were successfully identified in the Ag:CuBi2O4
samples without any impurities (Figure S2 a−d). We carried
out X-ray diffraction (XRD) and transmission electron
microscopy (TEM) analyses to examine the crystallinity of
Ag:CuBi2O4. As shown in Figure S3, the XRD pattern was
consistent with the typical pattern of a kusachite structure
(PDF file no. 00-042-0334). The high-resolution TEM images
in Figure S2 illustrate the high crystallinity of as-synthesized
Ag:CuBi2O4 with highly exposed facets.36,45 To investigate the
changes in the oxidation states of Cu2+ and Bi3+ arising from
Ag doping, we conducted X-ray photoelectron spectroscopy
(XPS) analysis. As depicted in Figure S4, Cu peaks of Ag-
doped CuBi2O4 were identical to those of bare CuBi2O4 (Cu
2p1/2 at 950.2 eV and Cu 2p3/2 at 930.4 eV), which indicates
that monovalent Ag+ ion doping did not alter the oxidation
state of Cu. The Bi 4f peaks of Ag:CuBi2O4 were moved to a
higher binding energy by 0.1 eV compared to those of bare
CuBi2O4, implying that the substitution of Ag+ on the Bi3+ sites
of CuBi2O4 led to an increase in the oxidation state of Bi to
balance the charge neutrality.44 According to the UV/vis
spectrum (Figure 2b), Ag:CuBi2O4 absorbed light below 820
nm. The corresponding Tauc plot band gap indicated 1.6 eV,
showing the photo activity of Ag:CuBi2O4 in the NIR region.
We covalently immobilized KLVFF peptides on a N-
hydroxysuccinimide-activated Ag:CuBi2O4 surface, which was
confirmed by the attenuated total reflection Fourier transform
infrared spectroscopy (ATR−FTIR) analysis. Figure S5 shows
characteristic ATR−FTIR peaks that arise from the aromatic
C−H bending of phenylalanine at 846 and 738 cm−1,
indicating the presence of KLVFF on the Ag:CuBi2O4 film.
Our XPS analysis (Figure S6) further verified the immobiliza-
tion of the KLVFF moiety on the electrode; the content of
nitrogen for Ag:CuBi2O4 increased from 0.42 to 5.74% after
the conjugation with KLVFF. To investigate the dependency
of the immobilized KLVFF content on the capturing efficiency
of Aβ assemblies, we performed bicinchoninic acid assay and
quantitative wide-field fluorescence microscopy (WFM)
analysis; more Aβ aggregates were captured by KLVFF-
Ag:CuBi2O4 with increasing content of the immobilized
KLVFF and reached a plateau at 15.6 μM·cm−2 (Figures
S7−S9).
We investigated the capability of KLVFF-Ag:CuBi2O4 to
capture the preformed Aβ aggregates using quantitative WFM
analysis. For the experiment, we incubated bare Ag:CuBi2O4
and KLVFF-Ag:CuBi2O4 with mature Aβ aggregates for 1 h,
and then stained each electrode’s surface using thioflavin T
(ThT) dye that exhibits considerable fluorescence intensity at
485 nm upon its binding to β-sheet-rich Aβ aggregates.46−48 As
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Figure 2. (a) SEM image of Ag:CuBi2O4 (Inset: photograph of the as-synthesized Ag:CuBi2O4. (b) Absorbance spectrum of Ag:CuBi2O4 (Inset:
Tauc plot showing an energy gap of KLVFF-Ag:CuBi2O4, which is calculated from the absorbance spectrum). (c) WFM of bare Ag:CuBi2O4 and
KLVFF-Ag:CuBi2O4 after incubation with mature Aβ aggregates. Both photocathodes were stained with ThT. (d) Relative ThT fluorescence
intensities of the Aβ aggregate solution (20 μM) after incubation with the Ag:CuBi2O4 or KVFF-Ag:CuBi2O4 photocathode in the solution. Scale-
bar is 1 (a) and 20 μm (c).
shown in Figures 2c and S10, the fluorescence intensity of the
KLVFF-Ag:CuBi2O4 surface was approximately 92.8 times
stronger than that of bare Ag:CuBi2O4. According to our
analysis by ATR−FTIR (Figure S11), peaks at 1548.6 and
1633.4 cm−1 in the amide I and II bands newly appeared for
KLVFF-Ag:CuBi2O4, which is attributed to the β-sheet
structure of Aβ aggregates. The results suggest that Aβ
aggregates were efficiently captured by KLVFF-Ag:CuBi2O4.
The real-time monitoring of Aβ aggregates captured on
KLVFF-Ag:CuBi2O4 would be achieved by directly conjugating
ThT derivatives with flexible long alkyl chains on the KLVFF-
Ag:CuBi 2 O 4 electrode. To investigate the nonspecific
adsorption onto the Ag:CuBi2O4 photoelectrode, we exposed
fluorescence-labeled bovine serum albumin (FITC−BSA) and
fluorescence-labeled immunoglobulin G (FITC−IgG) on
Ag:CuBi2O4 and KLVFF-Ag:CuBi2O4. As depicted in Figure
S12, KLVFF-Ag:CuBi2O4 and bare Ag:CuBi2O4 exhibited
negligible fluorescence enhancement upon the exposure of
FITC−BSA and FITC−IgG. The result implies that the
physical adsorption of nonspecific proteins (such as BSA and
IgG) was negligible on both the Ag:CuBi2O4 and KLVFF-
Ag:CuBi2O4 surfaces despite the porous structure of CuBi2O4.
The Aβ aggregate solution exhibited a decreased ThT
fluorescence down to 30% after incubation with KLVFF-
Ag:CuBi2O4, whereas a negligible change in ThT fluorescence
was observed for the solution incubated with bare Ag:CuBi2O4
(Figure 2d), indicating KLVFF-Ag:CuBi2O4’s capability to
capture a substantial amount of Aβ aggregates in the solution.
We investigated the possible dissociation of Aβ assemblies
captured on the surface of KLVFF-Ag:CuBi2O4 under four
different conditions: (1) no bias in dark, (2) cathodic bias in
dark, (3) no bias under NIR light illumination, and (4)
cathodic bias under NIR light illumination. According to the
WFM results (Figures 3a,b), a drastic reduction (down to 5%)
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of ThT fluorescence was observed for KLVFF-Ag:CuBi2O4
incubated under condition (4), whereas negligible changes in
fluorescence intensities were observed for all the other
conditions (i.e., absence of either bias or light). We found
that the ATR−FTIR peak intensities at 1548.6 and 1633.4
cm−1 in amide I and II bands significantly decreased with the
cathodic bias under NIR light illumination (Figure S13). The
results show both the bias and light are required for the
elimination of Aβ aggregates from KLVFF-Ag:CuBi2O4. We
further examined the effect of illuminating time and optical
power density on the disassembly of Aβ assemblies. As shown
in Figure S14a, a gradual reduction (down to 4%) of ThT
fluorescence was observed for KLVFF-Ag:CuBi2O4 incubated
in accordance with NIR illuminating time (0 to 5 h) under a
cathodic bias. In addition, ThT fluorescence of Aβ aggregate’s
samples continuously decreased to approximately 4% with
increasing NIR light intensity (0−500 mWcm−2) and reached
a plateau at 500 mW·cm−2 of light intensity under the cathodic
bias (Figure S14b). Despite the low absorbance at 808 nm, the
KLVFF-Ag:CuBi2O4 photoelectrode exhibited high efficiency
in targeted disaggregation of Aβ assemblies under illumination
of NIR light and the application of cathodic bias. We attribute
these results to superior photoelectrocatalytic activities of the
Ag:CuBi2O4 photoelectrode under the application of a
cathodic bias of −0.4 V and NIR light illumination (Figure
S15).
We investigated the morphological and secondary structural
changes of Aβ samples dissociated from the photoactivated
KLVFF-Ag:CuBi2O4 under a cathodic bias by using ex situ
atomic force microscopy (AFM), dynamic light scattering
(DLS) analysis, and circular dichroism (CD) spectroscopy.
According to the results of AFM and DLS analyses (Figure
3c,e), highly dense and long fibrillary Aβ aggregates [hydro-
dynamic radius (Rh): 700−1200 nm] were fragmented into
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Figure 3. (a) False-colored WFM images of the surface of the KLVFF-Ag:CuBi2O4 photocathode incubated with Aβ aggregates under four
conditions: no-bias/dark, no-bias/light, bias/dark, and bias/light. (b) Quantitative fluorescence mean density of Aβ aggregates on the surface (n =
3). The fluorescence intensity was dramatically decreased upon the application of the cathodic bias with NIR illumination. Data were analyzed by
one-way ANOVA. ***p < 0.001 versus control. n.s.: not significant. The dissociation efficacy of Aβ aggregates by KLVFF-Ag:CuBi2O4 was analyzed
using (c) DLS and (d) CD analyses. For DLS analysis, the KLVFF-Ag:CuBi2O4 photocathode was immersed in the Aβ aggregate solution (20 μM)
and a cathodic bias of 0.4 V (vs Ag/AgCl) was applied for 5 h with NIR light illumination. The NIR-triggered dissociation of Aβ aggregates was
analyzed using (e) AFM (scale bar: 1 μm) and (f) BeStSel analyses. The Aβ samples were incubated with the KLVFF-Ag:CuBi2O4 photocathode
under NIR light illumination with the cathodic bias.

short strand-shaped or globular structures (Rh <180 nm).
Furthermore, the estimated β-sheet content of the detached Aβ
species (25.4%) was much lower than that of intact Aβ
aggregates (49.7%), which was evidenced by the results of CD
spectroscopy and β-sheet structure selection algorithm
(BeStSel) analysis (Figure 3d,f). The results imply that the
KLVFF-Ag:CuBi2O4 photocathode effectively breaks Aβ
aggregate’s β-sheet structure under cathodic bias and NIR
light illumination.
We hypothesized that the reactive oxygen species generated
by the KLVFF-Ag:CuBi2O4 photocathode accounts for the
dissociation of Aβ deposits. When the cathodic bias is applied
to the KLVFF-Ag:CuBi2O4 electrode under NIR light,
dissolved O2 in the solution is reduced to superoxide radicals
(O2•−) by receiving photoactivated electrons from the
electrode. At the same time, water is oxidized to hydrogen
peroxide (H2O2) or hydroxyl radicals (OH•) by accepting
photoexcited holes from the anodic compartment.49 To prove
the hypothesis, we examined the generation of reactive oxygen
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species (O2•−, H2O2, and OH·) by the KLVFF-Ag:CuBi2O4
photocathode with a cathodic bias under NIR light
illumination (λ = 808 nm) for 1 h, and their effects on the
disassembly of Aβ aggregates. We analyzed the formation of
O2•− using nitro blue tetrazolium (NBT) reduction assay; the
reduction of NBT by O2•− generates NBT formazan, which
can be monitored by the decrease of absorption at 259 nm
(Figure S16).50 As shown in Figure 4a, negligible O2•−
formation was observed under dark conditions (0.8 μM·h−1·
cm−2), whereas O2•‑ generation increased with increasing
cathodic bias (13.2 μM·h−1·cm−2 at −0.4 V vs Ag/AgCl) under
NIR light illumination (λ = 808 nm, P: 500 mW cm−2). The
amount of O 2•− generated by the KLVFF-Ag:CuBi2O 4
photocathode gradually increased in accordance with the
irradiation time under the cathodic bias (Figure S17). In
addition, with the increasing power density of NIR light, the
O2•− generation rate gradually increased and reached a plateau
of 6.9 μM·h−1·cm−2 at 500 mW·cm−2 (Figure S18). To
monitor H2O2 and OH• formation by KLVFF-Ag:CuBi2O4, we
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Figure 4. (a) The amount of O2•− generated by KLVFF-Ag:CuBi2O4 under various cathodic biases (from 0 to −0.4 V) in the presence or absence
of NIR light illumination for 1 h. The generation rate of O2•− was obtained by multiplying the concentration of NBT in aqueous solution (0.020
mM) and the percentage of reduced NBT, which can be monitored by the decrease of absorption at 259 nm. (b) The generation rate of H2O2 was
examined by oxidation of ABTS in the presence of hydrogen peroxidase. The amount of H2O2 produced by the KLVFF-Ag:CuBi2O4 photocathode
increased in accordance with the applied cathodic bias under illumination of NIR light. (c) ThT fluorescence intensities of Aβ samples incubated
with the KLVFF-Ag:CuBi2O4 photocathode with or without three different radical scavengers under a cathodic bias of 0.4 V and NIR irradiation.
(d) The DNPH assay to detect carbonyls in Aβ samples. The absorbance at 370 nm implies the relative amounts of carbonyl content. An increase
in the absorption intensity implies that Aβ aggregates were highly oxidized upon application of a cathodic bias of 0.4 V under NIR light
illumination.

performed 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic Aβ aggregates. To confirm the photooxidation of Aβ samples

acid) (ABTS) and terephthalic acid (TA) assays, respectively.
The hydrogen peroxidase-catalyzed oxidation of ABTS by
H2O2 forms ABTS+ that exhibits absorption at 420 nm,51
whereas the oxidation of TA by OH· yields fluorescence at 430
nm (λex: 315 nm).52,53 As shown in Figures 4b and S19, H2O2
and OH· generation increased in accordance with the applied
cathodic bias under illumination. In contrast, H2O2 and OH·
were negligibly produced under dark conditions. According to
the literature, the Met residues in Aβ peptides are readily
oxidized by reactive radicals, which leads to notable conforma-
tional changes in the peptides including two β-strands (Leu17-
Ala21 and Ile31-Val36).54 Because the hydrophobic interaction
between the strands is crucial for the aggregation of Aβ
peptides, the reactive radical-induced increase in the polarity of
Met residues destabilizes the self-assembled robust structure.55
To identify major radical species that caused the dissociation
of Aβ assemblies, we added radical scavengers [for example,
tert-butyl alcohol (TBA), sodium pyruvate (SP), or hydroxyl-
TEMPO (TEMPOL)] to the Aβ aggregate solution, which was
co-incubated with NIR-activated KLVFF-Ag:CuBi2O4 under a
cathodic bias of 0.4 V. According to the ThT assay (Figure 4c),
the addition of TEMPOL or SP led to the recovery of ThT
fluorescence, which was not affected by TBA. The results
suggest that O2•− and H2O2 mainly triggered the dissociation
of Aβ aggregates, whereas OH• did not engage in destroying
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by the KLVFF-Ag:CuBi2O4 photocathode, the oxidation of Aβ
samples was detected using 2,4-dinitrophenylhydrazine
(DNPH) assay; the reaction of DNPH with carbonyls in
proteins forms 2,4-dinitrophenylhydrazones, which can be
detected spectrophotometrically. As displayed in Figure 4d, a
considerable increase of the carbonyl content in Aβ aggregates
was observed after incubation with KLVFF-Ag:CuBi2O4 under
−0.4 V (vs Ag/AgCl) and NIR light.
To examine the photoelectrochemical stabilities of KLVFF-
Ag:CuBi2O4 and Ag:CuBi2O4, we performed the chronoam-
perometric analysis under NIR light illumination (500 mW·
cm−2) with a cathodic bias of −0.4 V (vs Ag/AgCl) for 18 h.
According to Figure S20, the changes in current densities were
under 10%, implying that the NIR light-driven cathodic
reactions can continue without significant loss of the
photoelectrochemical activities of KLVFF-Ag:CuBi2O4 and
Ag:CuBi2O4 over an extended period. We further investigated
the cyclic stability of the KLVFF moiety conjugated on
Ag:CuBi2O4 by measuring the amount of Aβ aggregates
captured on KLVFF-Ag:CuBi2O4 using WFM analysis. As
shown in Figure S21, the content of Aβ aggregates captured by
KLVFF-Ag:CuBi2O4 gradually decreased with increasing cycle
number, which is attributed to the inevitable damage of the
KLVFF moiety by the reactive radicals generated by the
KLVFF-Ag:CuBi2O4 electrode under NIR light illumination
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Figure 5. (a) Aβ-induced cytotoxicity of N2a cells incubated with or without the KLVFF-Ag:CuBi2O4 photocathode under dark/light with/without
a cathodic bias of −0.4 V (vs Ag/AgCl). Data were analyzed by one-way ANOVA (n ≥ 4). ***p < 0.001 vs control. n. s.: not significant. (b) Cell
viability assay of N2a cells cultured with or without bare, Ag-doped KLVFF immobilized on Ag-doped CuBi2O4.
and an applied cathodic bias. The stability issue would be
alleviated by introducing the long-length spacers (such as
polyethylene glycol) to the KLVFF moiety, which can limit the
access of reactive radicals toward the KLVFF moiety.
To investigate the possibility of alleviating Aβ aggregate-
induced toxicity by KLVFF-Ag:CuBi2O4, we examined cell
viability using mouse neuroblastoma (N2a) as a model cell line
that retains the ability of neuronal differentiation.56,57 N2a cells
were incubated with untreated or KLVFF-Ag:CuBi2O4-treated
Aβ aggregates, and the cell viability was assessed by alamarBlue
assay, in which resazurin (or alamarBlue) is reduced by viable
cells and produces highly fluorescent resorufin at 590 nm (λex:
540 nm).58 As shown in Figure 5a, a significant decrease of cell
viability (48%) by Aβ aggregates was observed, implying high
toxicity of Aβ assemblies to N2a cells. Similarly, Aβ samples
exhibited high cytotoxicity after co-incubation with KLVFF-
Ag:CuBi2O4 in the absence of either external bias or
illumination (≈49%). In contrast, KLVFF-Ag:CuBi2O4 low-
ered the toxicity of Aβ samples under −0.4 V bias and NIR
light illumination, increasing the cell viability up to 92%. We
also investigated the cell toxicity of bare and KLVFF-
conjugated Ag:CuBi2O4 themselves (in the absence of Aβ
aggregates), which exhibited nearly 100% of the cell viability
(Figure 5b). These results validate that the NIR light-assisted
KLVFF-Ag:CuBi2O4 electrode is effective for the mitigation of
Aβ-induced cytotoxicity through photocathodic dissociation of
Aβ aggregates into nontoxic ones.
This study broadens the horizon of the photoactive material-
based therapeutic application by demonstrating NIR-triggered,
targeted dissociation of neurotoxic Aβ aggregates by the
KLVFF-Ag:CuBi2O4 photocathode. Previously, a bismuth
vanadate (BiVO4)-based, n-type photoanode has been ex-
plored for the in situ light-assisted dissociation of Aβ
aggregates under an anodic bias and illumination; however,
the photo-induced disassembly of Aβ aggregates by the BiVO4-
based photoanode had several drawbacks such as the random
attack of reactive species and limited penetration depth (∼2.5
mm) of visible light in biological tissues. In the current work,
we employed deeply penetrating NIR light and p-type
CuBi2O4 for the photocathodic dissociation of Aβ aggregates.
The use of highly penetrating NIR light enabled the KLVFF-
CuBi2O4 electrode to effectively absorb the light energy by
minimizing the interference from the biological tissues; the
NIR light exhibited ca. 4.2 times higher transmittance than that
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of visible light in mouse brain tissues (Figure S22). Also, the
targeted dissociation of Aβ assemblies was achieved through
conjugation of the KLVFF peptide on the CuBi2O4-based
photocathode, which can minimize the side effect of damaging
normal cells. There are still several issues that should be
addressed for the practical application of the CuBi2O4-based
photocathode in the future. The clinical applicability of the
CuBi2O4-based photocathode needs to be assessed by
investigating in vivo studies using AD animal models.
Furthermore, the working electrode should be integrated
with the counter and reference electrodes in the form of a
multielectrode array (MEA). For incorporating KLVFF-
CuBi2O4 into the MEA platform, further studies are needed
to develop the microsized CuBi2O4 electrode.
■ CONCLUSIONS
We report the first example of a NIR-sensitized photoelectrode
platform that exhibits high efficacy in targeted disaggregation
of Aβ assemblies and the mitigation of cytotoxicity induced by
Aβ assemblies. For the targeted disassembly of mature Aβ
aggregates, the as-synthesized Ag:CuBi2O4 photocathode was
conjugated with KLVFF peptides that show strong binding
affinity to Aβ aggregates. The illumination on the KLVFF-
Ag:CuBi2O4 photocathode excited the electrons to the
conduction band, while the application of the cathodic bias
tuned the energy level of the electrons to drive redox reactions.
Upon the application of the cathodic bias with NIR light
illumination, O2 is reduced to superoxide radicals (O2•−) by
receiving the photoactivated electrons from the KLVFF-
Ag:CuBi2O4 electrode. According to our analysis, the
generated oxidative stress effectively disassembled toxic and
β-sheet-rich Aβ assemblies into nontoxic, small soluble species
by KLVFF-Ag:CuBi2O4 under NIR-light illumination with a
cathodic bias. Through the addition of radical scavengers in the
course of photocathodic dissociation of Aβ aggregates (e.g.,
TEMPOL, TBA, and SP), we verified that O2•− was the major
contributor to the dissociation of Aβ aggregates. Moreover, cell
viability tests demonstrated that the KLVFF-Ag:CuBi2O4
photocathode is nontoxic to cells, but effective in circum-
venting Aβ-induced cytotoxicity with a cathodic bias under
NIR light illumination. Our work shows the potential of the
CuBi2O4-based photocathode platform for NIR-triggered,
targeted dissociation of toxic Aβ assemblies into irreversible,
nontoxic, and small species.
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ACS Applied Materials & Interfaces
■
EXPERIMENTAL SECTION
Materials. Amyloid-beta and NH2-KLVFF-miniPEG were pur-
chased from AnaSpec, USA, and Peptron, Korea, respectively. For the
cytotoxicity test, fetal bovine serum (FBS) and penicillin were
purchased from Welgene, Inc., Korea. Dulbecco’s modified Eagle
medium (DMEM) was obtained from ThermoFisher, USA. All the
other chemicals were obtained from Sigma-Aldrich Chemical Co.,
USA.
Synthesis of Ag-Doped CuBi2O4 Electrodes. CuBi2O4 photo-
cathodes were synthesized according to the literature.44 To synthesize
the Cu/Bi layer, the precursor solution was prepared by dissolving
Cu(NO3)2.5 (30 mM), Bi(NO3)3 (100 mM), and silver nitrate (0.06
mM) in ethylene glycol (20 mL). The electrodeposition was
performed under a cathodic bias (−1.8 V vs Ag/AgCl) for 3 min
using an as-synthesized solution in the typical three-electrode cell
configuration with FTO glass (1 × 4 cm) as the working electrode,
Ag/AgCl (in 3 M NaCl) as the reference electrode, and platinum
mesh as the counterelectrode. Afterward, the Cu/Bi layer deposited
on FTO was washed with deionized (DI) and dried under N2 gas. For
the conversion of the Cu/Bi film into CuBi2O4, the samples were
annealed with a box furnace (Ajeon) at 500 °C for 3 h with a ramping
rate of 2.5 °C min−1. The top of the as-synthesized Ag-doped
CuBi2O4 photocathode was covered with the polyimide Kapton tape
to give a uniform exposed area of the Ag-doped CuBi 2 O 4
photocathode (1 × 2 cm).
Characterization of Electrodes. The morphology and the
crystal structure of CuBi2O4 photocathodes were observed using a
S-4800 scanning electron microscope (Hitachi Co., Japan) and an X-
ray diffractometer (Rigaku Co., Japan) with Cu Kα radiation. The
elemental composition analysis of the surface of the CuBi2O4
photocathode was examined by XPS (Thermo Scientific Inc., USA).
UV−vis absorbance of the electrode was determined using a V-650
spectrophotometer (Jasco Inc., Japan).
Preparation of Aβ42 Aggregates. Human Aβ42 (1 mg) was
dissolved in HFIP and incubated for 12 h at room temperature. The
solution was distributed in 16 equal aliquots, and the solvent was
evaporated using a vacuum desiccator. To make Aβ aggregates, the
Aβ42 peptide (1/16 mg) was dissolved in 30 μL of a buffer solution
composed of NaOH (8.5 10−3 M), CH3CN (144 × 10−6 M), and
Na2CO3 (144 × 10−6 M). After sonication of the solution for 30 min,
we diluted the samples with 300 μL of phosphate buffer solution (8.5
× 10−3 M), including NaCl (8.5 mM), Na2CO3 (14 μM), NaOH
(850 μM), and CH3CN (6.0%). The solution was incubated for
another 24 h at 37 °C to acquire mature Aβ aggregates.
Immobilization of KLVFF onto the Surface of the
Ag:CuBi2O4 Electrode. For attachment of the KLVFF peptide on
the Ag:CuBi2O4 electrode, the electrode was chemically activated.
The as-synthesized Ag:CuBi2O4 electrode was UV/ozone-treated for
2 h using an AC-6 UV ozone cleaner (Ahtech, Korea). Then, the
electrode was immersed in 3 vol % of APTES in ethanol/water (95:5
v/v) and kept in vacuum at room temperature in a vacuum desiccator.
After 1 h of modification, we rinsed the photocathode with 100%
ethanol and put it on the hot plate at 135 °C for an hour. For NHS
activation, the electrode was incubated in N,N-disuccinic carbonate
solution (20 mM) dissolved in sodium bicarbonate (50 mM) for 3 h.
After washing with DI and drying, NH2-KLVFF-miniPEG (200 μM)
dissolved in 10 μM PBS was covalently immobilized on the surface for
12 h at 4 °C.
Photocathodic Disaggregation of Aβ Assemblies. The fully
grown Aβ aggregates were incubated with KLVFF-immobilized
Ag:CuBi2O4 photocathode for 5 h at 37 °C under different
conditions; no-bias (dark/light) or cathodic bias (dark/light)
conditions. The photocathodic dissociation of Aβ aggregates was
performed in a conventional three-electrode configuration with the as-
synthesized KLVFF-Ag:CuBi2O4 photocathode as the working
electrode, an Ag/AgCl (in 3 M NaCl) electrode as the reference
electrode, and a graphite rod as the counterelectrode. For photo-
cathodic dissociation of Aβ aggregates, the CuBi2O4-based photo-
cathode was back-illuminated with NIR light (500 mW·cm−2) under
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an external bias of −0.4 V (vs Ag/AgCl), which exhibited moderate
temperature changes (<7 °C) (Figures S23 and S24), while other
control experiments were conducted in the absence of NIR light
illumination or cathodic bias.
CD Analysis. To examine the beta-sheet content of Aβ samples,
the far-UV CD spectra of Aβ samples were recorded using a J-815 CD
spectropolarimeter (Jasco Inc., Japan) at 20 °C. All the measurements
were obtained 3 times, and the conformational changes of the Aβ
aggregates were monitored by observing CD values at 215 and 195
nm.
ThT Assay. A total of 20 μL of Aβ samples (20 × 10−6 M) was
mixed with 480 μL of ThT (20 × 10−6 M). The fluorescence intensity
of the solution was measured using a JASCO FP6500 spectro-
fluorometer with excitation at 440 nm and emission at 485 nm. All the
measurements were replicated three times; all the reported values
imply the mean ± standard deviation (SD) (n = 3).
Atomic Force Microscopy (AFM). To acquire AFM images of
the samples, 10 μL of Aβ solution was spread on a mica for 15 min
and washed several times with DI water. The mica was dried with air
and observed using a Nanoscope V atomic force microscope (Veeco
Instruments, USA) in the tapping mode with an NCHR silicon
cantilever (Nanosensors Inc., Switzerland). The acquired AFM image
was analyzed using Image J software. The area of Aβ samples was
calculated by analyzing the mean pixel value of the images.
DLS Analysis. For the dynamic light scattering (DLS) analysis, Aβ
samples (20 μM) were sonicated for 30 min and diluted with DI. The
size distribution profiles of Aβ samples were analyzed using a
Zetasizer NanoZS (Malvern Instruments, UK). All the measurements
were replicated three times.
H2O2 Quantification. To detect the generation of H2O2 from the
KLVFF-Ag:CuBi2O4 photocathode, the KLVFF-Ag:CuBi2O4 elec-
trode was immersed in 8.5 mM phosphate buffer solution (pH 7.8)
composed of NaCl (8.5 mM), Na2CO3 (14 μM), NaOH (850 μM),
and CH3CN (6.0%). The cathodic bias was applied to the
photocathode for 1 h in the dark or with illumination of NIR light
(λ = 808 nm, power intensity: 500 mW/cm2). To eliminate the effect
from the anode compartment, the counterelectrode was separated by
a porous glass frit. The H2O2 concentration from the cathode
compartment was analyzed by the oxidation reaction of 2,2′-azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) catalyzed by
horseradish peroxidase. We prepared a reagent by dissolving 1 mM
ABTS and 2.5 U of horseradish peroxidase in 100 mM potassium
phosphate buffer (pH 5.0). Afterward, 50 μL of each Aβ sample was
added to 950 μL of the reagent. After vortex-mixing the sample
solution, the absorbance spectra of the sample solutions were
measured using a V-650 spectrophotometer (Jasco Inc., Japan) to
obtain the absorption intensity at 420 nm. The generation rate of
H2O2 was calculated using a calibration curve.
Nitroblue Tetrazolium (NBT) Reduction Assay. To analyze the
formation of O2•− by the photocathodes, the KLVFF-Ag:CuBi2O4
photocathode was immersed in the NBT solution (0.02 mM)
incubated under a cathodic bias in the dark or under illumination or
NIR light for 1 h. The relative amounts of generated O2•− of
photocathodes were obtained by analyzing the absorption intensity at
259 nm. The absorbance was measured using a V-650 spectropho-
tometer (JASCO Inc., Japan).
Dinitrophenylhydrazine (DNPH) Assay. Aβ samples (20 μM)
were mixed with trichloroacetic acid (TCA, 20% final concentration)
solution and kept in an ice bath for 30 min. After centrifugation of the
solution at 15,000 rpm for 10 min, the supernatant solution was
replaced with DNPH (10 mM) dissolved in HCl (2 M). After re-
precipitating the protein pellet with 20% TCA solution, the samples
were washed with an ethanol−ethyl acetate (1:1, v/v) solution three
times. A 6 M guanidine hydrochloride solution dissolved in 20 mM
potassium phosphate (pH 2.3) was added to each sample. After
incubation of the samples at 37 °C for 20−30 min, the absorption
spectra of final solutions were examined using a V-650 spectropho-
tometer (Jasco lnc., Japan).
Cell Viability Test for Neuroblastoma Cells. To evaluate the
cytotoxicity of photocathodes, the mouse neuroblastoma cell line
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ACS Appl. Mater. Interfaces 2020, 12, 23667−23676
ACS Applied Materials & Interfaces
(N2a) was obtained from KTCT (Daejeon, Korea). A density of 2 ×
105 cells per 1 mL was seeded in a 12-well plate in DMEM medium
composed of 10% FBS, 5% HS, and 1% antibiotics (ATG Korea,
Korea) under a humidified atmosphere with 5% CO2 at 37 °C.
Afterward, bare, Ag-doped, and KLVFF-conjugated CuBi2O4 photo-
cathodes (0.5 × 0.5 cm) were cleaned with 70% ethanol and sterilized
with irradiation of UV light. Each of the sterilized photocathodes was
put in each well and incubated with N2a cells for 60 h. Then, the
medium containing Aβ samples was removed and 500 μL of fresh
growth medium containing 10% alamarBlue solution was added. After
another 4 h of incubation, the fluorescence intensity at 595 nm was
measured (with λex = 540 nm) using a Victor 3 microplate reader
(PerkinElmer Inc., USA). To evaluate the Aβ-aggregate-induced
cytotoxicity, a density of 2 × 105 cells per 1 mL was seeded into a 96-
well plate and incubated for 24 h. Then, the pre-incubated Aβ samples
were mixed with the DMEM medium to a final concentration of 3
μM, and the cell medium was replaced with the mixture (100 μL).
After incubation for 60 h, the medium containing Aβ samples was
removed and 100 μL of fresh growth medium containing 10%
alamarBlue solution was added. After another 4 h of incubation, the
fluorescence intensity was measured in the same way as described
above.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsami.0c02349.
SEM image, EDX spectra, HRTEM images, ATR−FTIR
spectra, XPS spectra, XRD spectra, BCA assay, ThT
assay, NBT assay, hydroxyl radical production rate, DLS
results, fluorescence intensity on the surface of KLVFF-
Ag:CuBi2O4, chronoamperometry results, voltammetric
analysis, and WFM analysis results (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Chan Beum Park − Department of Materials Science and
Engineering, Korea Advanced Institute of Science and
Technology (KAIST), Daejeon 305-701, Republic of Korea;
orcid.org/0000-0002-0767-8629; Phone: +82-42-350-
3340; Email: parkcb@kaist.ac.kr; Fax: +82-42-350-3310
Authors
Yerin Heo − Department of Materials Science and Engineering,
Korea Advanced Institute of Science and Technology (KAIST),
Daejeon 305-701, Republic of Korea
Kayoung Kim − Department of Materials Science and
Engineering, Korea Advanced Institute of Science and
Technology (KAIST), Daejeon 305-701, Republic of Korea;
orcid.org/0000-0002-1746-9441
Jinhyun Kim − Department of Materials Science and
Engineering, Korea Advanced Institute of Science and
Technology (KAIST), Daejeon 305-701, Republic of Korea;
orcid.org/0000-0002-1616-1891
Jinhyeong Jang − Department of Materials Science and
Engineering, Korea Advanced Institute of Science and
Technology (KAIST), Daejeon 305-701, Republic of Korea
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.0c02349
Author Contributions
Y.H. and K.K. contributed equally. Y.H., K.K., and C.B.P.
conceived the research. Y.H. performed the experiments. Y.H.
and K.K. analyzed the data and wrote the manuscript. C.B.P.
23674
www.acsami.org Research Article
supervised the research. J.K. provided a discussion on
photoelectrochemistry and amyloid dissociation. J.J. contrib-
uted to the neurotoxicity analysis. All the authors discussed the
results and commented on the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This study was supported by the National Research
Foundation (NRF) via the Creative Research Initiative Center
(NRF-2015 R1A3A2066191), Republic of Korea.
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