TBHP/NH4I-Mediated Direct N-H Phosphorylation of Imines and Imidates

Article abstract of DOI:10.1021/acs.joc.9b02301

A direct and practical metal-free N-H phosphorylation has been achieved via the TBHP/NH₄I-mediated cross-dehydrogenative coupling (CDC) reactions between imines/imidates and P(O)H compounds. This transformation provides an efficient synthetic r

Full text of DOI:10.1021/acs.joc.9b02301

Research Article
www.acsami.org
Cite This: ACS Appl. Mater. Interfaces 2019, 11, 8461−8469
Exploring the Antibacteria Performance of Multicolor Ag, Au, and
Cu Nanoclusters
Shanshan Wang,† Yingyi Wang,† Yi Peng, and Xiaoming Yang*
College of Pharmaceutical Sciences, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Ministry of Education),
Southwest University, Chongqing 400715, China
S
* Supporting Information
ABSTRACT: Integrating kinds of bactericides as one entirety shows prospect for improving antibacterial
efficiency, thus a new type of nanoclusters based on bactericide-directed herein emerged as a combination for
achieving a higher antibacterial effect. Specifically, an easily operated approach for preparing bacitracin-
directed silver, gold, and copper nanoclusters (AgNCs, AuNCs, and CuNCs, respectively) was proposed.
Meanwhile, AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin emitted cyan, yellow, and red
colors of fluorescence, respectively, and different sizes and nitrogen contents of these nanoclusters played a
critical role for their fluorescence alterations. Significantly, AgNCs@Bacitracin exhibited robust bacteria-killing
efficiency than other nanoclusters, owing to its marked damage on the bacterial membrane. Additionally,
propidium iodide (PI) staining indicated the bacterial membrane damage of 72.3% to the bacteria population
followed by the treatment with AgNCs@Bacitracin, and the introduction of AgNCs@Bacitracin lead to the
higher reactive oxygen species (ROS) production, facilitating their attractive antibacterial activity. Moreover,
this strategy of developing new nanoclusters broadened the avenues for designing the improved antibacterial
materials.
KEYWORDS: Ag, Au, and Cu Nanoclusters, multicolor, bacitracin-directed, improved antibacterial activity
designing various bioimaging probes on the basis of their
satisfactory photostability, applicable size, and lower toxic-
ity.21,22 Silver, as one kind of well-known antimicrobial agent,
may combine with other antibacterial agents to form silver
nanoclusters, thus producing a better antibacterial effect.23,24
Recently, considerable attention has been directed to the
attractive fluorescence performance and diverse values of Ag
(Au or Cu) nanoclusters; scarce antibacterial properties of the
nanoclusters have been applied.
Hereby, we developed three types of new water-soluble
nanoclusters via facile procedures, in which bacitracin served as
templates for encapsulating Ag, Au, or Cu atom (Figure 1).
Meanwhile, the as-prepared Ag, Au, and Cu NCs@Bacitracin
exhibited striking fluorescence such as cyan, yellow, and red,
suggesting their potential applications for bio-labeling and
beyond. Importantly, we explored the newly developed
nanoclusters toward their antibacterial functions. Through the
strategy of delivering Ag (Au or Cu) species and bacitracin into a
single package, which allowed an efficient and harmonious
utilization of nanoclusters and bacitracin, we obtained the
enhanced antibacterial effect of both agents, and AgNCs@
Bacitracin showed the strongest activity for killing Staphylococcus
aureus compared with that of other antibacterial agents. We did
not only demonstrate the antibacterial performance of the as-
prepared nanoclusters but also clarify their mechanism of
achieving the improved activity for killing bacteria.
1. INTRODUCTION
Bacterial infections have been considered as one of the critical
healthcare challenges, and various bacteria exhibit increasing
resistance to the existed antibiotics. For this case, developing
alternative antibacterial strategies is urgently required.1−3
Among the alternatives, employing antimicrobial nanomaterials
or nanotechnology has been considered as an effective strategy
whose rapid advances have indeed provided the opportunities
during the past decade.4,5 Currently, various antibacterial ways
based on nanomaterials have been proposed by the virtue of
their bactericidal and labeling properties.6−11
Extracted from organisms of B. subtilis, bacitracin is a type of
peptide antibiotic mainly against the Gram-positive bacteria in
vitro, which is proved to be water soluble, low toxic, neutral, and
stable against heat.12 It shows the laboratory level activity for the
mice infected by the hemolytic streptococcus and the pigs
infected by the gas gangrene in vivo.13,14 Furthermore, utilizing
bacitracin for hemolytic streptococcal and staphylococcal
infections of human being in clinical has provided the
encouraging results.
Defined as isolated particles, metal nanoclusters (MNCs) are
composed of a few to tens of atoms and usually exist less than 3
nm.15,16 Basically, these size particles are generally discrete and
exhibit tunable electronic transitions by sizes, leading to their
fascinating molecule-like properties such as obvious fluores-
cence.17−20 Compared with the routine fluorophores including
the organic dyes and semiconductor quantum dots (QDs), their
potential applications were possibly restricted because of the
weak photostability and toxicity issues, whereas the fluorescent
nanoclusters are considered as the promising candidates for
Received: December 25, 2018
Accepted: February 4, 2019
Published: February 4, 2019
© 2019 American Chemical Society
8461
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
(Chengdu, China), and a DF-101s thermostatic water bath was bought
from Gongyi Instrument Co., Ltd (Gongyi, China). Besides, the
solution blending was accomplished by vortex mixer QL-901 (Haimen,
China), and three-nanocluster powder was produced through
lyophilization by PiloFD8-4.3V (Charlotte, USA).
2.3. Synthesis and Purification of AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin. The bacitracin-
directed AgNCs, AuNCs, and CuNCs were innovatively synthesized
here. Generally, the glassware were first rinsed with aqua regia and
further washed by distilled water before use. Also, the magnetic stirring
bars were subjected to similar wash steps. To synthesize AgNCs@
Bacitracin, 1 mL of bacitracin (13 mg·mL−1) was introduced to 1.6 mL
of AgNO3 (17 mM). Subsequently, 200 μL of NaOH (1 M) and 20 μL
of NaBH4 (1 mM) were successively added. Then, the previous mixture
was incubated for 3.5 h at 37 °C. Similar for AuNCs@Bacitracin, 0.1 g
of bacitracin powder dissolved in 3 mL of ultrapure water followed by
an addition of 100 μL of HAuCl4 (100 mM). After 3 min, 1 mL of
NaOH (1 M) solution was introduced, and the mixture was incubated
at 4 °C for 12 h. Again for CuNCs@Bacitracin, 1 mL of bacitracin
solution (13 mg·mL−1) and 0.2 mL of CuSO4 (20 mM) were mixed
together with stirring for 10 min at 37 °C in a flask. Then, 20 μL of
NaBH4 (1 mM) and 60 μL of N2H4·H2O were added into the reaction
and incubated for 3.5 h with continuous stirring.
Toward purification, AgNCs, AuNCs, and CuNCs aqueous solutions
were initially centrifuged at 10000 rpm for 15 min, and the supernatant
was filtered by a membrane size of 0.22 μm to remove larger particles.
Then, the nanoclusters were obtained through a dialysis membrane
(300 MWCO) in deionized water for 24 h. Furthermore, the powder of
three nanoclusters was acquired with lyophilization, and their related
concentrations of 10 mg·mL−1 were obtained by dissolving in ultrapure
water for further applications.
2.4. Bacterial Cell Culture. S. aureus was initially recovered and
cultured in a trypticase soy broth (TSB) medium in a shaker of 37 °C
with 180 rpm, and its maintenance was achieved through the growth on
TSB agar (15% w/v agar in TSB medium). For each experiment, a
single colony of S. aureus was selected from the fresh TSB agar plate and
subjected to the culture at 37 °C and 180 rpm for 14 h. Finally, the
cultured bacteria were introduced for further experiments.
2.5. Nanoclusters Introduced into Bacteria. To identify the
antibacterial activity of three nanoclusters against S. aureus, the agar
diffusion test was performed. In particular, S. aureus were spread on agar
plates of Luria−Bertani. Then, five aseptic filter papers with AgNCs@
Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, bacitracin, and
ampicinlin (10 mg·mL−1) were separately placed on the culture plates.
Subsequently, these plates were incubated for 16 h with a temperature
of 37 °C. Again, we used a Vernier caliper to determine the inhibition
diameters, and all the experiments were conducted for three times with
a parallel format. Hence, we calculated average values of the inhibition
diameters. Next, AgNCs@Papain (10 mg·mL−1), AuNCs@Papain (10
mg·mL−1), CuNCs@Papain (10 mg·mL−1), AgNCs@BSA (10 mg·
mL−1), AuNCs@BSA (10 mg·mL−1), CuNCs@BSA (10 mg·mL−1),
Ag+ plus bacitracin (30 μM metal ions and 10 mg·mL−1 bacitracin),
Au3+ plus bacitracin (30 μM metal ions and 10 mg·mL−1 bacitracin),
and Cu2+ plus bacitracin (30 μM metal ions and 10 mg·mL−1
bacitracin) were treated similarly.
Growth curves of S. aureus with different treatments were plotted.
Initially, the cultured S. aureus was added into the fresh TSB medium to
obtain a concentration of 0.03 with OD600. Next, the previous
solutions were separately introduced into five round-bottom tubes with
1000 μL each, and 5 μL of AgNCs@Bacitracin, AuNCs@Bacitracin,
CuNCs@Bacitracin, Bacitracin (10 mg·mL−1), or water were added
into these tubes. Then, the mixtures were incubated at 37 °C with 180
rpm for 9 h, and their related absorbance at 600 nm was recorded for
each hour.
2.6. Membrane Damage of Bacteria. Propidium iodide (PI) was
cell impermeable and indicated a high uptake by the dead cells with the
damaged membrane, thus leaving the healthy cell unstained. Thereby,
PI was employed to trace the membrane damage of the bacterial cell.
Additionally, we introduced Hoechst 33342 for staining both the alive
and dead population of the bacteria. Specifically, the bacterial cells were
Figure 1. (A) Scheme of preparing AgNCs@Bacitracin, AuNCs@
Bacitracin, and CuNCs@Bacitracin. (B) Bacteria treated with
AgNCs@Bacitracin, AuNCs@Bacitracin, or CuNCs@Bacitracin.
2. EXPERIMENTAL SECTION
2.1. Chemicals and Materials. Bacitracin and N-acetyl-L-cysteine
(NAC) were obtained from Aladdin Co., Ltd. (Shanghai, China).
Copper sulphate (CuSO4), hydrogen tetrachloroaurate trihydrate
(HAuCl4), silver nitrate (AgNO3), propidium iodide (PI), sodium
borohydride (98%), and paraformaldehyde (PFA) were purchased
from Sigma-Aldrich (Milwaukee, USA). The acids and alkalis that
mainly include boric acid (H3BO3), phosphoric acid (H3PO4), sodium
hydroxide (NaOH), hydrazine hydrate (N2H4·H2O), and sodium
chloride (NaCl) were acquired from Dingguo Changsheng Biotechnol-
ogy Co., Ltd. (Beijing, China). 2′,7′-Dichlorofluorescein diacetate
(DCFH-DA) was bought from Nanjing Jiancheng Bioengineering
Institute. Methicillin-resistant S. aureus (ATCC 6538) were obtained
from China General Microbiological Culture Collection Center.
Besides, ultrapure water of 18.25 MΩ·cm, produced by an Aquapro
AWL-0502-P ultrapure water system (Chongqing, China), was utilized
during the whole experimental process.
2.2. Instrumentation. The fluorescent detections were achieved
by a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan)
with an excitation wavelength of 5 nm and an emission of 10 nm, while
the quartz cell is 1 × 1 cm. Next, a Shimadzu UV-2450
spectrophotometer (Tokyo, Japan) was used to obtain absorption
spectra. Toward the size distributions, a Zetasizer Nano Dynamic light
scattering (DLS) instrument (Malvern, England) was employed. Also,
Images of high-resolution transmission electron microscopy (HR-
TEM) were taken by a TECNAI G2F20 microscope (Portland,
America) at 200 kV. Meanwhile, a ESCALAB 250 X-ray photoelectron
spectrometer (XPS) and a Fourier transform infrared (FTIR)
spectroscopy (Tokyo, Japan) were applied to explore the elements
and chemical groups. The fluorescence quantum yields were
determined using Absolute PL quantum yield spectrometer C11347
(Hamamatsu, Japan). Images of the bacterial cells were obtained by a
Nikon A1+ confocal microscope (Tokyo, Japan). All pH values of the
solutions were detected using a Fangzhong pHS-3C digital pH meter
8462
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
Figure 2. Fluorescence spectra of (A) AgNCs@Bacitracin, (B) AuNCs@Bacitracin, and (C) CuNCs@Bacitracin. (D) UV−vis absorption spectra of
AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin. (E) FTIR analyses of bacitracin, AgNCs@Bacitracin, AuNCs@Bacitracin, and
CuNCs@Bacitracin. (F) XPS surveys of AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin. (G−I) Binding energy spectra of Ag 3d
(AgNCs@Bacitracin), Au 4f (AuNCs@Bacitracin), and Cu 2p (CuNCs@Bacitracin).
first incubated with Ag+, bacitracin, Ag+ plus bacitracin, and AgNCs@
Bacitracin solutions for 2 h, and then 30 μg·mL−1 PI was added into
each bacterial solution and incubated in the shakers at 37 °C and 180
rpm for 30 min. Later, the bacterial cells were subjected to
centrifugation at 5000 rpm for 2 min followed by washing with PBS
for three times and resuspended in the PBS as 1 mL. Finally, the
bacterial cells were fixed on the slides by 4% paraformaldehyde in PBS
for 15 min, and then, 1 μg·mL−1 Hoechst 33342 solution (Nuclear dye
reagent) was added onto the slides and fixed for 30 min. The bacterial
cells were observed by laser scanning confocal microscopy, and cell
numbers of PI- and Hoechst 33342-stained bacteria were counted.
Eventually, the percentage of PI-stained bacteria was calculated through
the number of PI stained bacteria divided by that of Hoechst 33342-
stained bacteria.23
was further treated with AgNCs@Bacitracin, AuNCs@Bacitracin, and
CuNCs@Bacitracin.25
2.8. Injury Model of Mice. Injury models of mice were introduced
to further investigate the antibacterial activities of AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin. Particularly, 12 female
Kunming mice were divided into four groups (each was3 weeks of age
with 18−22 g of weight) and infected with S. aureus through a measured
bored wound of 36 mm2. Then, saline AgNCs@Bacitracin (10 mg·
mL−1), AuNCs@Bacitracin (10 mg·mL−1), and CuNCs@Bacitracin
(10 mg·mL−1) were daubed on the blank band-aids. Again, we
monitored the four groups of mice treated with the various band-aids,
which were consecutively renewed for 24 h interval. Eventually, the
results were photographed during 3 days.
2.7. Reactive Oxygen Species Generation. To investigate the
intracellular reactive oxygen species (ROS) concentrations, the
bacterial cells were separately treated with 5 μL of AgNCs@Bacitracin,
AuNCs@Bacitracin, CuNCs@Bacitracin (10 mg·mL−1 for each) for 2
h, and followed by introducing 1 μM DCFH-DA for each, and they
were incubated at 37 °C and 150 rpm for 15 min. Subsequently, the
bacterial cell suspensions were centrifuged at 5000 rpm for 3 min,
washed with PBS for three times, and resuspended in PBS as 1 mL.
Then, the fluorescence of the DCF as-produced was recorded with an
excitation wavelength of 485 nm, and the relative ROS production level
was calculated by normalizing the ROS level of the treated group with
that of water treated. As the control, 25 mM N-acetyl-L-cysteine (NAC)
was separately introduced into three aliquots of bacterial cells, and each
3. RESULTS AND DISCUSSION
3.1. Optimizing Conditions of Synthesis. To obtain
optimal conditions of synthesized AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin, more detailed
experiments were conducted. As shown in Figures S1−S3, the
fluorescence intensities of three nanocluters were varied along
different temperatures (Figure S1A), incubation times (Figure
S1B), and amounts of AgNO3 (Figure S1C) and bacitracin
(Figure S1D) added, suggesting that the preparation of
AgNCs@Bacitracin was dependent on the optimization
parameters. Finally, at 37 °C for 3.5 h with 20 mM AgNO3
8463
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
and, 9 mM bacitracin were determined as the optimum
conditions for synthetizing AgNCs@Bacitracin, whereas at
room temperature for 1 h with 100 mM HAuCl4 and 80 mM of
bacitracin for AuNCs@Bacitracin (Figure S2A−D). Similarly, at
37 °C for 3.5 h with 20 mM CuSO4, and 9 mM bacitracin were
selected as the applicable conditions for synthesizing CuNCs@
Bacitracin (Figure S3A−D).
2E,F and Figure S5D−F) and CuNCs@Bacitracin (Figure 2E,F
and Figure S5G−I) were accordingly investigated by FTIR and
XPS, and surface groups of the two clusters were from bacitracin.
Additionally, binding energies of Au 4f (84.7 and 88.0 eV) and
Ag 3d (368.8 and 374.8 eV) of the two nanoclusters were shown
to be approximate to that of the gold (84.1 and 87.6 eV) and
silver atoms (368.0 and 374.0 eV) (Figure 2G,H), demonstrat-
ing their complete reductions.29 For Cu 2p spectrum, the
absence of the peak at 942 eV clearly excluded the presence of
Cu (II) species. Also, binding energies of Cu 2p (932.3 and
952.5 eV) exhibited a valence state that lied at 0 or +1 (Figure
2I), illustrating an acceptable agreement with the previous
reports.30
To picture these proposed nanoclusters, AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin were visualized by
HR-TEM, showing parallel morphology. In detail, most of
AgNCs@Bacitracin displayed well-dispersed particles (Figure
3A,B), and their corresponding sizes were measured as 3.2 0.6
nm with a polydispersity index (PDI) of 0.42 (Figure 3C) by
DLS, proving a favorable agreement with the data of HR-TEM.
Similarly, the majority of AuNCs@Bacitracin were of uniform
dispersity with sizes of 4.6 0.8 nm as PDI = 0.57 (Figure 3D−
F), and the size of CuNCs@Bacitracin were within a size of 5.5
1.1 nm with a PDI of 0.50 (Figure 3G−I).31 Thereby, the
quantum size effect was responsible for regulating fluorescence
emissions of three clusters.32,33 Through detailed character-
ization (Table 1), only the particle sizes and nitrogen contents
(6.94, 11.19, and 16.68% for AgNCs@Bacitracin, AuNCs@
Bacitracin, and CuNCs@Bacitracin, respectively) of three
clusters were proved to be distinctly different. These variations
were believed to be responsible for the different fluorescence of
three nanoclusters.28,34 Moreover, the current three nano-
clusters showed less toxicity for mammalian cells, even though
they were incubated with a high dosage amount of 50 μg·mL−1
for 24 h, clarifying their satisfactory biocompatibility toward the
potential applications in vivo (Figure S6A−C).
3.3. Stability of AgNCs@Bacitracin, AuNCs@Bacitracin,
and CuNCs@Bacitracin. To investigate fluorescent stabilities
of AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@
Bacitracin, the related experiments were conducted. First, the
fluorescent signal of AgNCs@Bacitracin hardly showed any
variation for different reaction times (10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, and 120 min) under UV light, indicating their
favorable stability (Figure S7A). Also, reaction temperatures of
30, 40, 50, 60, 70, 80, and 90 °C were examined, and in a range of
30−50 °C was the optimal temperature toward the favorable
stability (Figure S7B). Besides, fluorescent intensities of
AgNCs@Bacitracin exhibited less change along with different
concentrations of NaCl (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
and 1.0 M), describing their acceptable tolerance in the
hypersaline environment (Figure S7C). Also, proposed
AgNCs@Bacitracin was stable upon addition to different
kinds of organic solvents, revealing that the fluorescence of
AgNCs@Bacitracin was barely affected by the regular organic
solvents (Figure S7D).
In a similar manner, the stabilities of AuNCs@Bacitracin and
CuNCs@Bacitracin were showed as being satisfactory (Figures
S8A and S9A) even under 30−50 °C (Figures S8B and S9B) or
in the hypersaline environment (Figures S8C and S9C), and the
organic solvents (Figures S8D and S9D) were hardly affected
the stability of both AuNCs@Bacitracin and CuNCs@
Bacitracin, suggesting that all these three clusters showed
favorable stability toward various conditions.
3.2. Characterization of AgNCs@Bacitracin, AuNCs@
Bacitracin, and CuNCs@Bacitracin. According to the
particular design, the three metal NCs were prepared in an
aqueous solution. Initially, fluorescence spectroscopy was
employed to identify their related fluorescent properties. Briefly,
the emission peak of AgNCs@Bacitracin emerged at 452 nm
with cyan fluorescence under UV light (Figure 2A), whereas
AuNCs@Bacitracin showed yellow fluorescent emission at 563
nm (Figure 2B). Likewise, CuNCs@Bacitracin emitted red
fluorescence at 632 nm (Figure 2C). Meanwhile, their
corresponding excitation spectra for these nanoclusters were
separately gained. For AgNCs@Bacitracin, the excitation peak
was at 370 nm, and AuNCs@Bacitracin was excitated at 410 nm,
whereas CuNCs@Bacitracin was at 508 nm. Again, the absolute
PL quantum yields (QY) were separately obtained as 8.3, 5.8,
and 3.2% for AgNCs@Bacitracin, AuNCs@Bacitracin, and
CuNCs@Bacitracin, respectively, and the lifespan of CuNC@
bacitracin was longer than that of both AuNCs@Bacitracin and
AgNC@bacitracin (Figure S4). Besides, UV−vis absorption
spectra of all three clusters exhibited peaks of 285 nm, which
were resulted from the n → π* transition by CO (Figure 2D).
Functional groups and components of three clusters were
obtained and analyzed by FTIR and XPS. First, surface groups of
AgNCs@Bacitracin were investigated with FTIR (Figure 2E).
More precisely, a characteristic absorption of 3438 cm−1
indicated the O−H stretching vibration and 1708 cm−1 for
CO stretching vibration, describing there existed carboxylic
groups. Again, the peak at 3412 cm−1 proved N−H stretching
vibration.26,27 To identify element components of AgNCs@
Bacitracin, three major peaks for C, O, and N were obviously
appeared in the XPS survey (Figure 2F), demonstrating that the
as-prepared were mainly composed of C (51.86%), O (6.94%),
and N (40.01%) as shown in Table 1. Meanwhile, the C 1s band
Table 1. Elementary Composition and Corresponding Ratios
(C, N, and O) of AgNCs@Bacitracin, AuNCs@Bacitracin,
and CuNCs@Bacitracin
elementary composition
C (%)
N (%)
O (%)
AgNCs@Bacitracin
AuNCs@Bacitracin
CuNCs@Bacitracin
51.86
57.64
59.59
6.94
11.19
16.68
40.01
29.98
21.47
of C 1s spectrum could be divided into four peaks (Figure S5A),
referring to sp2 carbons (CC, 284.5 eV), sp3 carbons (C−O/
C−N, 285.1 eV), carbonyl carbons (CO, 287.7 eV), and
carboxyl carbons (COOH, 289.4 eV). Also, the N 1s band was
divided into three peaks at 398.8, 399.4, and 400.5 eV represent
pyridinic-N, amino-N, and pyrrolic-N (Figure S5B), respec-
tively, and the O 1s band contains three peaks at 530.9, 531.6,
and 532.7 eV for CO, C−O−C, and C−OH (Figure S5C),
respectively.28 Altogether, FTIR and XPS data provided the
evidence for AgNCs@Bacitracin consisting of both the
oxidation and π-conjugated domains. Besides, high polarity
and hydrophilicity of AgNCs@Bacitracin were derived from the
multiple hydroxyl groups. Similarly, AuNCs@Bacitracin (Figure
8464
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
Figure 3. HR-TEM images and DLS analyses of (A−C) AgNCs@Bacitracin, (D−F) AuNCs@Bacitracin, and (G−I) CuNCs@Bacitracin.
3.4. Antibacterial Activity of AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin. To further
identify functions and applications of AgNCs@Bacitracin,
AuNCs@Bacitracin, and CuNCs@Bacitracin, their antibacterial
activities against S. aureus were subsequently explored.
Particularly, all zones of inhibition treated by AgNCs@
Bacitracin displayed a much higher antibacterial activity than
that by AuNCs@Bacitracin, CuNCs@Bacitracin, or bacitracin
(Figure 4A). Meanwhile, their average inhibition diameters and
minimum inhibitory concentrations (MICs) toward S. aureus
were calculated (Figure 4D and Table S1), and the diameter and
MIC of S. aureus treated with AgNCs@Bacitracin were 0.82 cm
and 6.25 μg·mL−1, which was obviously longer than that of
AuNCs@Bacitracin (0.54 cm, 200 μg·mL−1), CuNCs@
Bacitracin (0.62 cm, 50 μg·mL−1), or bacitracin (0.50 cm, 200
μg·mL−1).
To identify interactions of the metal atoms with bacitracin or
other proteins, several nanoclusters (AgNCs@Bacitracin,
AuNCs@Bacitracin, CuNCs@Bacitracin, AgNCs@Papain,
AuNCs@Papain, CuNCs@Papain, AgNCs@BSA, AuNCs@
BSA, and CuNCs@BSA) were subjected to the antibacterial
experiments. The inhibition diameters and MIC of S. aureus
treated by three current NCs@Bacitracin showed much higher
antibacterial activity than that by three clusters derived from
Papain (0 cm, 0 μg·mL−1) and three clusters based on BSA (0
cm, 0 μg·mL−1), suggesting their antibacterial functions were
mainly derived from the cooperation between bacitracin and the
metal atoms (Figure 4B−E). Furthermore, AgNCs@Bacitracin
(0.82 cm) displayed a higher antibacterial activity than Ag+ plus
bacitracin (0.68 cm), whereas AuNCs@Bacitracin (0.54 cm)
was higher than Au3+ plus bacitracin (0.51 cm) and CuNCs@
Bacitracin (0.62 cm) higher than Cu2+ plus bacitracin (0.59 cm)
(Figure 4C−F). To be specific, bacitracin displays the
antibacterial activity mainly against the Gram-positive bacteria,
whereas BSA and papain indicate a nonantibacterial effect.
Thereby, the antibacterial activity of bacitracin was retained
while bacitracin cooperated with Ag atoms to form nanoclusters.
Taken together, these data demonstrated that silver clusters
show the best performance.
3.5. Principle of Antibacterial Activity. To explore the
enhanced-antibacterial activity of AgNCs@Bacitracin, growth
curves of S. aureus treated with AgNCs@Bacitracin, AuNCs@
Bacitracin, or CuNCs@Bacitracin (10 μg·mL−1) were built up.
As shown in Figure 5A, a longer lag phase (6 h) was observed for
AgNC-treated S. aureus than that of AuNC-treated or CuNC-
treated S. aureus (4 h). Obviously, AgNCs exhibited the
enhanced-antibacterial activity compared with AuNCs or
CuNCs. Meanwhile, both AuNC- and CuNC-treated S. aureus
reached the stationary phase over 8 h of growth, and their
maximum cell densities showed similarity. These findings
suggest that AgNCs shows stronger inhibition for the growth
of S. aureus.
8465
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
Figure 4. (A) Zone inhibitions of AgNCs@Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, bacitracin, and ampicinlin against S. aureus. (B) Zone
inhibitions of AgNCs@Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, AgNCs@Papain, AuNCs@Papain, CuNCs@Papain, AgNCs@BSA,
AuNCs@BSA, and CuNCs@BSA against S. aureus. (C) Zone inhibitions of AgNCs@Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, Ag+ plus
bacitracin, Au3+ plus bacitracin, and Cu2+ plus bacitracin. (D−F) Related average calculated diameters. Data are mean SD, n = 3, student t test, p <
0.05, asterisk (*) means significant against bacitracin, whereas pound sign (#) as being significant against AgNCs@Bacitracin, p < 0.05.
To further illuminate the improved antibacterial activity of
AgNCs@Bacitracin, the more detailed investigations for the
bacterial membrane damage were performed. Basically, the
group treated with water barely damaged the bacterial
membrane (Figure 5B). In contrast, the groups with AuNCs@
Bacitracin, CuNCs@Bacitracin, or bacitracin treatment sepa-
rately exhibited 26.6, 30.5, and 33.7% of the bacteria with the
damaged membrane. Again, the group treated by Ag+ or Ag+ plus
bacitracin led to a ratio of 24.3% and 42.1% (Figure S10). In
striking contrast, the group of AgNCs@Bacitracin-treated
showed a ratio of 72.3% for the membrane-damaged bacteria,
suggesting its most powerful activity for antibacteria (Figure
5C). These findings demonstrated that AgNCs@Bacitracin
shows the most efficient damage on the bacterial membrane.
Besides, ROSs, which mainly including hydroxyl radicals
Taking the above explorations together, AgNCs@Bacitracin
could lead to more broken membranes of bacteria and higher
production of ROS, thus facilitating the maximum amount of
bacteria cell death. Consequently, AgNCs@Bacitracin shows
the highest antibacterial efficiency. Additionally, bacitracin
served as the template of the nanoclusters here, formed a
package by cooperating with Ag atoms, and also improved the
antibacterial activity.36
3.6. Band-Aid of Three Nanoclusters Applied for
Wounded Mice. To evaluate the antibacterial efficiency of in
vivo three metal nanoclusters, Kunming mice wound on their
backs were employed as the model.37 Specifically, the mice were
divided to four groups, and each group was treated with band-
aids of AgNCs@Bacitracin, AuNCs@Bacitracin, CuNCs@
Bacitracin, and Saline. Also, concentrations of three clusters
introduced were with 10 mg·mL−1 each, which was much lower
than that of antibiotics commonly used for the wound
disinfection. Accordingly, photos of the back-wounded mice of
four groups were obtained, and each band-aid was changed every
12 h of interval. During the therapeutic process, recovering
situations of the wounds was different, although no change for
the body weight was observed. Simultaneously, wounds of mice
treated with three band-aids of AgNCs@Bacitracin, AuNCs@
Bacitracin, and CuNCs@Bacitracin did not appear with any
erythema and edema (Figure 6). Overall, these results suggested
that three nanoclusters show the potentiality of serving as the
effective antimicrobial nanomaterial, although AgNCs@Baci-
tracin showed the most powerful activity for killing bacterial
cells.
(·OH), superoxide anion radicals (O2 ), and hydrogen peroxide
·
(H2O2), were generally refered to the reactive oxygen species of
living organisms. As well-known, the excessive production of
ROS can cause the damage to the membrane, DNA, and protein
of bacteria, thus leading to the cell death. To elucidate the
enhanced-antibacterial efficiency of AgNCs@Bacitracin more
clearly, we asked whether their antimicrobial efficiency was
related to produce ROS. Thereby, we have identified their ROS
amounts while S. aureus treated with different nanoclusters
through a DCFH-DA dye. As shown in Figure 5D, AgNCs@
Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin in-
creased the intracellular ROS production. Importantly,
AgNCs@Bacitracin induced the maximum increase of ROS,
thus inducing the increased cell death for S. aureus.25,35
8466
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
Figure 5. (A) Growth curve of S. aureus treated with AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin. (B) Fluorescent photos for
the bacteria cells with different treatments (scale bar: 25 μm). (C) Percentage of the bacteria cells stained with PI by introducing water, AgNCs@
Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, or bacitracin. n = 3, asterisk (*) means significance against the bacitracin-treated group, p < 0.05.
(D) Relative ROS levels of bacteria treated with AgNCs@Bacitracin, AuNCs@Bacitracin, and CuNCs@Bacitracin, whereas ROS values for two
groups treated by water or water with NAC defined as 1, n = 3, asterisk (*) means significance against the group treated with water, p < 0.05; circumflex
accent (^) means significance against non-NAC-treated group, p < 0.05.
nanoclusters are proposed originated from differences of the
particle sizes and nitrogen contents. Significantly, the as-
designed AgNCs@Bacitracin exhibited dramatic bacteria-killing
efficiency, which was owing to the hybrid structure of Ag atoms
integrated with bacitracin that could effectively damage the
bacterial membrane. Besides, the further study suggested that
AgNCs@Bacitracin could lead to the higher ROS-production of
the bacteria, facilitating their attractive activity over bacteria.
More importantly, this strategy broadened the potential for us to
design more efficient ways toward the antibacterial substances.
ASSOCIATED CONTENT
■
Figure 6. Images of the wounded mice treated with AgNCs@
Bacitracin, AuNCs@Bacitracin, CuNCs@Bacitracin, or saline for 0,
24, 48, and 72 h.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.8b22143.
4. CONCLUSIONS
Optimization of syntheses conditions, fluorescence and
spectral analyses and images, effects of different organic
substances at various conditions, and minimum inhibitory
concentration of bacitracin (PDF)
In summary, we here innovatively synthesized three antibacterial
nanoclusters including AgNCs@Bacitracin, AuNCs@Bacitra-
cin, and CuNCs@Bacitracin with the multicolor fluorescence
such as cyan, yellow, and red. Different emissions of the three
8467
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
(16) Lv, S.; Li, Y.; Zhang, K.; Lin, Z.; Tang, D. Carbon Dots/g-C3N4
Nanoheterostructures-Based Signal-Generation Tags for Photoelec-
trochemical Immunoassay of Cancer Biomarkers Coupling with
Copper Nanoclusters. ACS Appl. Mater. Interfaces 2017, 9, 38336−
38343.
(17) Zhang, L.; Han, F. Protein Coated Gold Nanoparticles as
Template for the Directed Synthesis of Highly Fluorescent Gold
Nanoclusters. Nanotechnology 2018, 29, 165702.
(18) Halawa, M. I.; Gao, W.; Saqib, M.; Kitte, S. A.; Wu, F.; Xu, G.
Sensitive Detection of Alkaline Phosphatase by Switching on Gold
Nanoclusters Fluorescence Quenched by Pyridoxal Phosphate. Biosens.
Bioelectron. 2017, 95, 8−14.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ming4444@swu.edu.cn. Phone: 86-23-68251225. Fax:
86-23-68251225.
■
ORCID
Xiaoming Yang: 0000-0002-2514-8672
Author Contributions
†These authors contributed equally.
Notes
The authors declare no competing financial interest.
(19) Gao, G.; Zhang, M.; Gong, D.; Chen, R.; Hu, X.; Sun, T. The
Size-effect of Gold Nanoparticles and Nanoclusters in the Inhibition of
Amyloid-β Fibrillation. Nanoscale 2017, 9, 4107−4113.
(20) Xu, M.; Gao, Z.; Wei, Q.; Chen, G.; Tang, D. Label-free Hairpin
DNA-scaffolded Silver Nanoclusters for Fluorescent Detection of Hg2+
Using Exonuclease III-assisted Target Recycling Amplification. Biosens.
Bioelectron. 2016, 79, 411−415.
(21) Han, L.; Xia, J.-M.; Hai, X.; Shu, Y.; Chen, X.-W.; Wang, J.-H.
Protein-Stabilized Gadolinium Oxide-Gold Nanoclusters Hybrid for
Multimodal Imaging and Drug Delivery. ACS Appl. Mater. Interfaces
2017, 9, 6941−6949.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support by Fundamen-
tal Research Funds for the Central Universities
(XDJK2018AC004), Chongqing Research Project of Basic
Research and Frontier Exploration (cstc2018jcyjAX0197),
Fundamental Research Funds for Innovative Project on
Designing and Screening Drug Candidates of Chongqing
(cstc2015zdcy-ztzx120003), and Innovative Research Project
for Postgraduate Students of Chongqing (CYS18099).
(22) Xu, Y.; Liu, Q.; He, R.; Miao, X.; Ji, M. Imaging Laser-Triggered
Drug Release from Gold Nanocages with Transient Absorption
Lifetime Microscopy. ACS Appl. Mater. Interfaces 2017, 9, 19653−
19661.
(23) Zheng, K.; Setyawati, M. I.; Lim, T.-P.; Leong, D. T.; Xie, J.
Antimicrobial Cluster Bombs: Silver Nanoclusters Packed with
Daptomycin. ACS Nano 2016, 10, 7934−7942.
(24) Sangsuwan, A.; Kawasaki, H.; Matsumura, Y.; Iwasaki, Y.
Antimicrobial Silver Nanoclusters Bearing Biocompatible Phosphor-
ylcholine-Based Zwitterionic Protection. Bioconjugate Chem. 2016, 27,
2527−2533.
(25) Zheng, K.; Setyawati, M. I.; Leong, D. T.; Xie, J. Antimicrobial
Gold Nanoclusters. ACS Nano 2017, 11, 6904−6910.
(26) Yang, K.; Liu, M.; Wang, Y.; Wang, S.; Miao, H.; Yang, L.; Yang,
X. Carbon Dots Derived From Fungus for Sensing Hyaluronic Acid and
Hyaluronidase. Sens. Actuators, B 2017, 251, 503−508.
(27) Zeng, R.; Tang, Y.; Zhang, L.; Luo, Z.; Tang, D. Dual-readout
Aptasensing of Antibiotic Residues Based on Gold Nanocluster-
functionalized MnO2Nanosheets with Target-induced Etching Re-
action. J. Mater. Chem. B 2018, 6, 8071−8077.
(28) Ding, H.; Yu, S.-B.; Wei, J.-S.; Xiong, H.-M. Full-Color Light-
Emitting Carbon Dots with a Surface-State-Controlled Luminescence
Mechanism. ACS Nano 2016, 10, 484−491.
(29) Zhou, S.; Zhang, M.; Yang, F.; Wang, F.; Wang, C. Facile
Synthesis of Water Soluble Fluorescent Metal (Pt, Au, Ag and Cu)
Quantum Clusters for the Selective Detection of Fe3+ Ions as Both
Fluorescent and Colorimetric Probes. J. Mater. Chem. C 2017, 5, 2466−
2473.
(30) Yang, X.; Feng, Y.; Zhu, S.; Luo, Y.; Zhuo, Y.; Dou, Y. One-step
Synthesis and Applications of Fluorescent Cu Nanoclusters Stabilized
by L-cysteine in Aqueous Solution. Anal. Chim. Acta 2014, 847, 49−54.
(31) Wang, Y.; Wang, S.; Lu, C.; Yang, X. Three Kinds of DNA-
directed Nanoclusters Cooperating with Graphene Oxide for Assaying
Mucin 1, Carcinoembryonic Antigen and Cancer Antigen 125. Sens.
Actuators, B 2018, 262, 9−16.
(32) Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes
for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44,
8636−8663.
(33) Zhang, L.; Wang, E. Metal Nanoclusters: New Fluorescent
Probes for Sensors and Bioimaging. Nano Today 2014, 9, 132−157.
(34) Jiang, K.; Sun, S.; Zhang, L.; Lu, Y.; Wu, A.; Cai, C.; Lin, H. Red,
Green, and Blue Luminescence by Carbon Dots: Full-Color Emission
Tuning and Multicolor Cellular Imaging. Angew Chem., Int. Ed. 2015,
54, 5360−5363.
REFERENCES
■
(1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial
Biofilms: A Common Cause of Persistent Infections. Science 1999, 284,
1318−1322.
(2) Ebrahiminezhad, A.; Barzegar, Y.; Ghasemi, Y.; Berenjian, A.
Green Synthesis And Characterization Of Silver Nanoparticles Using
Alcea Rosea Flower Extract as a New Generation Of Antimicrobials.
Chem. Ind. Chem. Eng. Q. 2017, 23, 31−37.
(3) Rai, M.; Yadav, A.; Gade, A. Silver Nanoparticles as a New
Generation of Antimicrobials. Biotechnol. Adv. 2009, 27, 76−83.
(4) Stewart, P. S.; Costerton, J. W. Antibiotic Resistance of Bacteria in
Biofilms. Lancet 2001, 358, 135−138.
(5) Mah, T. F.; O’Toole, G. A. Mechanisms of Biofilm Resistance to
Antimicrobial Agents. Trends Microbiol. 2001, 9, 34−39.
(6) Hancock, R. E. W.; Sahl, H.-G. Antimicrobial and Host-defense
Peptides as New Anti-infective Therapeutic Strategies. Nat. Biotechnol.
2006, 24, 1551−1557.
(7) Lemire, J. A.; Harrison, J. J.; Turner, R. J. Antimicrobial Activity of
Metals: Mechanisms, Molecular Targets and Applications. Nat. Rev.
Microbiol. 2013, 11, 371−384.
(8) Steenbergen, J. N.; Alder, J.; Thorne, G. M.; Tally, F. P.
Daptomycin: a Lipopeptide Antibiotic for the Treatment of Serious
Gram-positive Infections. J. Antimicrob. Chemother. 2005, 55, 283−288.
(9) Muangsiri, W.; Kirsch, L. E. The Protein-binding and Drug
Release Properties of Macromolecular Conjugates Containing
Daptomycin and Dextran. Int. J. Pharm. 2006, 315, 30−43.
(10) Xiu, Z.-m.; Zhang, Q.-b.; Puppala, H. L.; Colvin, V. L.; Alvarez, P.
J. J. Negligible Particle-Specific Antibacterial Activity of Silver
Nanoparticles. Nano Lett. 2012, 12, 4271−4275.
(11) Ji, H.; Sun, H.; Qu, X. Antibacterial Applications of Graphene-
based Nanomaterials: Recent Achievements and Challenges. Adv. Drug
Delivery Rev. 2016, 105, 176−189.
(12) Godin, B.; Touitou, E. Mechanism of Bacitracin Permeation
Enhancement through the Skin and Cellular Membranes from an
Ethosomal Carrier. J. Controlled Release 2004, 94, 365−379.
(13) Engberg, R. M.; Hedemann, M. S.; Leser, T. D.; Jensen, B. B.
Effect of Zinc Bacitracin and Salinomycin on Intestinal Microflora and
Performance of Broilers. Poult. Sci. 2000, 79, 1311−1319.
(14) Mascher, T.; Margulis, N. G.; Wang, T.; Ye, R. W.; Helmann, J. D.
Cell Wall Stress Responses in Bacillus Subtilis: the Regulatory Network
of the Bacitracin Stimulon. Mol. Microbiol. 2003, 50, 1591−1604.
(15) Zhou, Q.; Lin, Y.; Xu, M.; Gao, Z.; Yang, H.; Tang, D. Facile
Synthesis of Enhanced Fluorescent Gold-Silver Bimetallic Nanocluster
and Its Application for Highly Sensitive Detection of Inorganic
Pyrophosphatase Activity. Anal. Chem. 2016, 88, 8886−8892.
8468
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469

ACS Applied Materials & Interfaces
Research Article
(35) Yuan, X.; Setyawati, M. I.; Leong, D. T.; Xie, J. Ultrasmall Ag+-
rich Nanoclusters as Highly Efficient Nanoreservoirs for Bacterial
Killing. Nano Res. 2014, 7, 301−307.
(36) Martinez-Castanon, G. A.; Nino-Martinez, N.; Martinez-
Gutierrez, F.; Martinez-Mendoza, J. R.; Ruiz, F. Synthesis and
Antibacterial Activity of Silver Nanoparticles with Different Sizes. J.
Nanopart. Res. 2008, 10, 1343−1348.
(37) Miao, H.; Zhong, D.; Zhou, Z.; Yang, X. Papain-Templated Cu
Nanoclusters: Assaying and Exhibiting Dramatic Antibacterial Activity
Cooperating with H2O2. Nanoscale 2015, 7, 19066−19072.
8469
DOI: 10.1021/acsami.8b22143
ACS Appl. Mater. Interfaces 2019, 11, 8461−8469