Photo-induced synthesis of molybdenum oxide quantum dots for surface-enhanced Raman scattering and photothermal therapy

Article abstract of DOI:10.1039/c9tb02102g

By means of a simple and photo-induced method, four colors of molybdenum oxide quantum dots (MoO_x QDs) have been synthesized, using Mo(CO)₆ as the structural guiding agent and molybdenum source. The as-prepared MoO_x QDs display diverse optical properties due to the different configurations of oxygen vacancies in various nanostructures. Among them, crystalline molybdenum dioxide (MoO₂) with a deep blue color shows the most intense localized surface plasmon resonance effect in the near-infrared (NIR) region. The strong NIR absorption endows MoO₂ QDs with a high photothermal conversion efficiency of 66.3%, enabling broad prospects as a photo-responsive nanoagent for photothermal therapy of cancer. Moreover, MoO₂ QDs can also serve as a novel semiconductor substrate for ultrasensitive surface-enhanced Raman scattering (SERS) analysis of aromatic molecules, amino acids and antibiotics, with SERS performance comparable to that of noble metal-based substrates. The therapeutic applications of MoO₂ QDs open up a new avenue for tumor nanomedicine.

Full text of DOI:10.1039/c9tb02102g

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Photo-induced synthesis of molybdenum oxide
quantum dots for surface-enhanced Raman
scattering and photothermal therapy†
Cite this:DOI: 10.1039/c9tb02102g
a
a
a
a
a
Haihong Yu,‡ Zhengfei Zhuang,‡ Dongling Li, Yanxian Guo, Yang Li,
a
b
a
a
Huiqing Zhong, Honglian Xiong, Zhiming Liu * and Zhouyi Guo
*
By means of a simple and photo-induced method, four colors of molybdenum oxide quantum dots
MoO QDs) have been synthesized, using Mo(CO) as the structural guiding agent and molybdenum
source. The as-prepared MoO QDs display diverse optical properties due to the different configurations of
oxygen vacancies in various nanostructures. Among them, crystalline molybdenum dioxide (MoO ) with a
deep blue color shows the most intense localized surface plasmon resonance effect in the near-infrared
NIR) region. The strong NIR absorption endows MoO QDs with a high photothermal conversion efficiency
of 66.3%, enabling broad prospects as a photo-responsive nanoagent for photothermal therapy of cancer.
Moreover, MoO QDs can also serve as a novel semiconductor substrate for ultrasensitive surface-enhanced
Raman scattering (SERS) analysis of aromatic molecules, amino acids and antibiotics, with SERS performance
(
x
6
x
2
(
2
Received 26th September 2019,
Accepted 4th December 2019
2
DOI: 10.1039/c9tb02102g
comparable to that of noble metal-based substrates. The therapeutic applications of MoO
up a new avenue for tumor nanomedicine.
2
QDs open
rsc.li/materials-b
As a star member of the transition metal oxides (TMOs),
molybdenum oxide (MoO ) provides a plasmonic resonance
Introduction
x
Flake-like two-dimensional (2D) nanomaterials have captured from visible to NIR wavelengths. The morphology and size of
substantial attention for their favorable light–matter interactions MoO nanostructures, including nanoparticles, nanodumbbels,
x
in a wide range from ultraviolet (UV) to near-infrared (NIR) nanoclusters, 2D nanosheets (NSs) and their 0D derivatives, and
regions since the discovery of graphene, black phosphorus (BP) quantum dots (QDs), can be easily modulated via intelligent
1
6–11
and their derivatives. The utilization of 2D nanostructures as synthesis strategies.
It is well known that biodegradability is
promising photo-responsive nanoprobes for optical imaging, one of the critical parameters for clinical translation of a
12
x
molecular sensing and phototherapy has been exploited pre- nanomaterial. MoO is biodegradable, which has been discussed
2
,3
7,13,14
valently in recent years.
light-responsive features of nanomaterials in the NIR region tion property. The nanosheets are relatively stable at acidic pH
also named as the ‘‘biological transparency window’’) are while being degradable at physiological pH, which leads to longer
advantageous, which however poses a dilemma to graphene- tumor retention of those nanosheets but little influence on normal
For biomedical applications, the in several papers.
x
MoO NSs show a pH-responsive degrada-
(
13
or BP-based nanostructures. More recently, plasmonic 2D tissues. More interestingly, the LSPR peak of MoO is tunable by
materials have received great interest because of their appreci- controlling the concentration of oxygen vacancy in the lattices,
able localized surface plasmon resonance (LSPR) effect derived making MoO an efficient NIR-responsive nanomaterial for diverse
from the collective oscillations of valence electrons in the optical applications.
nanostructures under illumination.
x
15
x
16–18
For example, Song et al. described a
4
,5
hydrothermal strategy for fabricating biodegradable MoO NSs
x
8
using ammonium molybdate as the Mo source. The 2D semi-
conductor showed good capabilities as a NIR nanoprobe for tumor
photoacoustic imaging and photothermal therapy (PTT) with
rapid body clearance. MoO QDs can also be prepared by the
hydrothermal method. In a study conducted by Liu et al.,
a
MOE Key Laboratory of Laser Life Science & SATCM Third Grade Laboratory of
Chinese Medicine and Photonics Technology, College of Biophotonics, South China
Normal University, Guangzhou, Guangdong 510631, P. R. China.
E-mail: liuzm021@126.com, ann@scnu.edu.cn
x
b
Department of Physics and Optoelectronic Engineering, Foshan University,
Guangdong, P. R. China
molybdenum powder was chosen as a precursor for the pre-
paration of MoO
x
QDs with size around 2.5 nm, which dis-
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
played strong NIR harvesting ability to convert NIR lasers into
hyperthermia for cancer theranostics.
c9tb02102g
These authors contributed equally to the work.
1
9
‡
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In addition, the LSPR effect will lead to an electromagnetic Hexacarbonylmolybdenum (Mo(CO)6) was purchased from Yuanye
2
0
enhancement in Raman scattering. In fact, many efforts have Biotechnology (Shanghai, China). N,N-Dimethylformamide was
been made to exploit novel surface-enhanced Raman scattering obtained from Aladdin (Shanghai, China). Dulbecco’s modified
(
SERS) substrates based on semiconductor materials in recent Eagle’s medium (DMEM) and fetal bovine serum (FBS) were
2
1
years, especially plasmonic TMOs. Compared to the tradi- purchased from GIBCO (Grand Island, NY, USA). The Annexin
tional SERS substrates composed of noble metals (e.g., Au and V-FITC-PI Apoptosis Detection Kit was purchased from Dalian
Ag), TMOs are cheaper, more versatile and have better biocom- Meilun (Liaoning, China). All reagents were used without further
patibility. Furthermore, the Raman enhancement induced by purification. Deionized water (Milli-Q System, Millipore, USA)
plasmonic TMOs has proven to be comparable to noble metals, was used in all experiments.
and the SERS effect is largely dependent on the density of d-orbital
22
free electrons in the oxygen vacancies. This is evident in Synthesis of molybdenum oxide quantum dots
dumbbell-like MoO nanostructures reported by Zhang et al., where
x
Preparation of reduced state molybdenum quantum dots: a
general fabrication process is as follows: first, 1 mM Mo(CO)6
was dispersed into dimethyl formamide and sonicated for
the MoO nanodumbbells without a LSPR band showed almost no
2
SERS effect on R6G molecules, while remarkable Raman enhance-
ment was achieved using their plasmonic counterparts as the SERS
30 min. In the presence of a flow of nitrogen, the mixed solution
9
substrates. The Raman enhancement factor (EF) and the limit of
was added into a three necked flask and heated to 80 1C for 1 h
under magnetic stirring. During this process, the color of the
solution changed from colorless to yellowish-brown. When cooled
to room temperature, the yellow intermediate was moved into
an ultra-low temperature freeze dryer for 12 h. Excess ethanol
was added to wash the products and a dry yellow solid was
obtained by freeze drying.
6
detection (LOD) of plasmonic MoO
10
2
reached 3.75 Â 10 and
À7
2
M, respectively. Another striking example is 2D MoO NSs
À8
5
whose LOD and EF were observed to be 4 Â 10 M and 2.1 Â 10 ,
23
respectively. Li et al. also reported that amorphous MoO3 QDs
synthesized from MoS powder with the assistance of supercritical
2
2
CO presented a superior EF of methyl blue (MB) molecules of up
5
24
to 9.5 Â 10 . It should be noted that the Raman tests in the
current work were mostly carried out on dried samples, while
liquid-phase Raman analysis is rarely performed.
Synthesis of molybdenum oxide quantum dots with
different degrees of oxidation: the initial yellow intermediate
(0.4 mg) was dispersed into 50% ethanol solution (10 mL). After
There are many ways to synthesize molybdenum oxides, such
sonicating for 30 min, the mixed solution was put in sunlight
7
,8,25,26
27
as the hydrothermal method,
laser ablation, chemical
À2
(
B0.2 mW cm ) for 2 h to get a light green product (G). At 5 h,
2
8
24
vapor deposition, the supercritical CO assisted method, and
2
the colour changed to light blue, which meant the formation
of B. At 10 h, DB was acquired with the colour of the solution
turning dark blue. If the yellow mixed 50% ethanol aqueous
so on. Most of the synthetic methods require high temperature,
high pressure, femtosecond lasers, etchants, etc., which are
operationally hazardous, energy extravagant and environment-
unfriendly. Therefore, safe, low pollution, energy-saving and
controllable synthesis methods urgently need to be exploited.
Herein, a simple photo-induced method for preparing plasmonic
MoO QDs is proposed using molybdenum hexacarbonyl (Mo(CO) )
solution was put under a UV lamp for 5 h, G was synthesized
2
(green color).
Coating of calcium carbonate
CaCl O (20 mg) and MoO
Á2H
x
6
À1
2
2
2
(40 mL, 10 mg mL ) were
as the molybdenum source, by means of which four varieties of
dispersed in a beaker containing 10 mL of ethanol solution, which
was adjusted to pH 6.8 and sealed with a plastic wrap to form a
number of pores. The bottle was then placed in a desiccator with
two bottles of amino bicarbonate (NH4HCO3) at a temperature
of 45 1C. After 1 day of vapor diffusion reaction, the product was
centrifuged and collected at 12 000 rpm for 20 min.
MoO QDs are obtained. The plasmonic nanostructures and oxygen
x
vacancy defects in the MoO
irradiation dose. The optimal LSPR feature of the MoO
can be observed when the molybdenum precursor underwent
sunlight irradiation for 10 h, namely MoO QDs. The NIR-
responsive feature allows the MoO QDs to act as photothermal
x
QDs can be controlled by the light
x
QDs
2
2
nanoagents for high-performance cancer PTT. Liquid-phase
SERS analysis of dye molecules is also performed using MoO2
Characterization
QDs as ultrasensitive SERS substrates, wherein the LOD and the Transmission electron microscopy (TEM) images were obtained on a
À8
6
maximum EF of MB in solution are about 10 M and 7 Â 10 , 200 kV JEM-2100HR transmission electron microscope (JEOL, Japan)
respectively. Furthermore, the SERS detection of phenylalanine equipped with an EDX spectrometer. UV-vis-NIR absorption spectra
and amphotericin B is also achieved, indicating the promising were collected on a spectrophotometer (UV-6100S, MAPADA, China).
potential of MoO
2
QDs for bioanalysis.
The X-ray diffraction (XRD) pattern was obtained using a Bruker D8
focus X-ray diffractometer under CuKa radiation (l = 1.54051 Å).
Raman spectra were measured by a Renishaw inVia microspectro-
meter (Derbyshire, England) equipped with an excitation wavelength
of 785 nm. XPS spectra were measured by using a Thermo Fisher
Scientific K-Alpha photoelectron spectrometer (Shanghai, China).
Experimental
Materials
Methylene blue (MB), malachite green (MG), and calcium Apoptosis assay was performed via ACEA NovoCyteTM Flow
chloride (CaCl
2
)
were purchased from Sigma-Aldrich. Cytometry (San Diego, USA).
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Raman measurement
by DMSO, and the absorbance at 495 nm was measured using a
microplate reader.
Different kinds of aqueous dye solutions with concentrations
À3
À1
À8
Hep G2 cells were seeded into 6-well culture plates (about
varying from 10 to 10 M were mixed with equivalent MoO
x
4
5 Â 10 cells per well), followed by incubation with MoO QDs
2
QDs (1 mg mL ). Next, 4 mL of suspension was extracted and
dropped onto a cleaned silicon wafer for Raman scanning.
The Raman spectrum was obtained by using a high-resolution
confocal Raman spectrometer (Derbyshire, England) at the
excitation wavelength of 785 nm with the laser power of 0.5 mW.
A 20Â objective lens was used to focus the sample. All the samples
were measured at least six times and the EFs were calculated
as follows:
À1
(
100 mg mL ) or PBS for 8 h. Finally, the Hep G2 cells were
À2
irradiated with an 808 nm laser at the power density of 2 W cm
for 2.5 min. Subsequently, the cells were stained with calcein
AM/PI for 15 minutes, and imaged by a fluorescence micro-
scope (Calcein AM lex = 488 nm, lem = 515 nm; PI lex = 535 nm,
lem = 617 nm).
Apoptosis assay was performed using the Annexin-V-FITC-PI
apoptosis detection kit (Meilun, Dalian, China). Briefly, 5 mL of
annexin-V-FITC and 5 mL of PI solution were added to 100 mL of
EF = ISERS  CRaman/IRaman  CSERS
(1)
5
cell suspension (1 Â 10 ). Cells were incubated in the dark for
where ISERS represents the SERS signal caused by the concentration
of probe molecules (CSERS), and IRaman refers to the normal
Raman signal obtained due to the concentration of probe
molecules (CRaman).
15 minutes at 25 1C, and then supplemented with 400 mL of
binding buffer. Finally, a flow cytometer was used to analyze
the apoptosis of the cells.
To understand the subcellular localization of MoO
the fluorescence co-localization imaging experiment was per-
formed. MoO QDs were first mixed with an equivalent fluo-
rescent probe (RhB) to acquire MoO QDs/RhB. Then, the
2
QDs,
Photothermal properties of MoO
x
QDs
À1
2
The temperature changes of MoO QD solutions (0–100 mg mL
)
x
2
2
under 808 nm laser irradiation (0.33, 1 and 2 W cm )
were measured by an infrared thermal camera (Fluke Ti200,
FlukeCorp, Washington, USA). The temperature was recorded
every 30 s for 10 min. The photothermal conversion efficiency
Hep G2 cells pre-cultured in Petri dishes were incubated with
À1
200 mg mL
2
MoO QDs/RhB for 8 h. After washing with
PBS, the cells were co-stained with different organelle-specific
trackers (Lyso-Tracker Green, Mito-Tracker Green and ER-Tracker
Blue-White DPX). Finally, the cells were washed and fixed by 4%
paraformaldehyde for 10 min, and observed under a fluorescence
microscope.
4
7
(
Z) was estimated using the equation below:
Z = hs(Tmax À Tmax water)/I(1 À 10À )
hs = SmC /t
tS = Àt/ln y
y = (Tamb À T)/(Tamb À Tmax
A
,
(2)
(3)
(4)
(5)
p
S
Results and discussion
Synthesis and characterization of molybdenum oxide quantum
)
where h represents the heat conversion coefficient and s dots
denotes the surface area of the container. Here, t represents
the system time constant of the sample, and m and C represent
S
Molybdenum oxide nanomaterials were synthesized via a typi-
cal light-controlled strategy (Fig. 1a and b). Mo(CO) was first
p
6
the mass (1 g) and specific heat capacity of the solvent (Cp,water
=
heated in dimethyl formamide (DMF) at 80 1C for 1 h to get
the yellow intermediate (Y), which has been characterized as
À1
4.2 J g ), respectively. Tamb is the ambient temperature of the
surroundings, and Tmax and Tmax,water are the equilibrium
temperatures of the MoO2 solution and water, respectively.
À2
I is the laser power density (2 W cm ), and A represents the
absorbance of MoO solution at 808 nm (A = 0.294).
2
808
In vitro photothermal cancer therapy
Hep G2 cells were obtained from the Laboratory Animal Center
of Sun Yat-sen University (China) and cultured in DMEM (Gibco,
USA) supplemented with 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin at 37 1C in a humidified atmo-
sphere with 5% CO till the desired confluency was achieved.
2
The standard tetrazolium salt (MTT) assay was performed to
determine the cell viability. Briefly, Hep G2 cells were seeded in
4
9
6-well plates with a density of 10 cells per well and incubated
in a CO2 incubator for 12 h at 37 1C. Then, the medium
was displaced by fresh medium containing various concentra-
tions of the MoO
incubator for 24 h. Subsequently, MTT (5 mg mL ) was added
2
QDs. The cells were cultured in a CO
2
Fig. 1 Schematic illustration of the synthesis process of MoO
x
QDs.
À1
(
a) Synthesis of yellow reduced molybdenum. (b) Synthesis of three
to each well for 4 h at 37 1C. Then, the medium was replaced molybdenum oxides with different degrees of oxidation.
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Fig. 3 (a) XRD analysis of G, B, DB and G
to standard patterns of Mo 11 (JCPDS No. 05-0508), MoO
No. 35-0609) and MoO (JCPDS No. 32-0671), respectively. (b) Raman
analysis of G, B, DB, and G
2
. Black lines correspond
4
O
3
(JCPDS
2
2
Fig. 2 TEM images of (a) DB, (b) B, (c) G and (d) G , respectively. The
2
. (c) UV-Vis-NIR absorption spectra of MoO
x
upper insets show the corresponding selected area electron diffraction
SAED) patterns, and the lower insets display the HRTEM images of the
À1
QDs at the same Mo concentration of 0.5 mg mL
.
(
nanostructures.
and 64.9 degrees in the XRD pattern of DB, which matches
QDs in with MoO (JCPDS No. 32-0671). Similarly, B is MoO (JCPDS
elementary Mo (Fig. S1 and S2, ESI†). Then, MoO
x
2
3
different oxidation states were obtained under sunlight or No. 35-0609) and the others (G2 and G) are Mo O (JCPDS
4
11
ultraviolet (UV) irradiation. Significant color changes were No. 05-0037). The Raman spectra of MoO QDs are shown in
x
À1
x
observed under sunlight, and the color of the solution changed Fig. 3b, where the typical peaks of MoO QDs at 993 and 820 cm
from yellow to green (G), blue-green (B) and dark blue (DB), can be attributed to the stretching vibration of terminal oxygen
respectively. On the other hand, the yellow intermediate (MoQO) and double coordinated bringing oxygen (Mo QO),
And the intensities of these two peaks increase
gradually from DB to G , which means an increasing oxygen
2
31,32
became only green (G
2
) under UV irradiation, and the color respectively.
did not change as the radiation time increased.
2
Transmission electron microscopy (TEM) and high resolu- content of molybdenum oxide. The optical property was recorded
tion TEM (HRTEM) were used to evaluate the dimensions and by using UV-Vis-NIR absorption spectroscopy. As shown in Fig. 3c,
crystal structures of the samples. As shown in Fig. 2a–d, four G, B and DB reveal strong light absorption across visible and
kinds of molybdenum oxides form in the shapes of quantum NIR regions, which is in keeping with the earlier theories based
33,34
dots with the sizes of 3–5 nm. The HRTEM image in the lower on Mie-Gan’s calculations.
inset in Fig. 2a reveals that the lattice fringes of DB are 0.25 nm indicates the promising potential of MoO
and 0.34 nm, which can be indexed to the (111) and (200) facets thermal nanoagents for PTT.
This excellent optical property
x
QDs as potent photo-
2
9,30
of monoclinic MoO
2
, respectively.
The corresponding
In order to determine the chemical composition and valence
x
selected area electron diffraction (SAED) pattern presented in state in the MoO nanostructure, X-ray photoelectron spectro-
the upper inset also shows an obvious crystal structure. In scopy (XPS) was used. Fig. 4a shows the Mo, O, and C elements
addition, the lattice fringes of B and G are 0.15 nm and 0.1 nm, in the XPS spectra of the Mo-based samples, where the char-
respectively (Fig. 2b and c). G2 has no lattice fringes, which may acteristic peaks of MoO
x
can be observed, such as 231.1
be ascribed to the lattice structural destruction caused by UV (Mo 3d), 397.1 (Mo 3p3/2), 415.1 (Mo 3p1/2), 531.1 (O 1s), and
3
2
light. The amorphous structure of G2 can also be confirmed by 974.1 eV (Mo(A). The signals of the oxidation state of various
the SAED pattern (Fig. 2d). Mo species in the MoO QD samples are divided into six
The as-prepared molybdenum oxide nanomaterials were bands, namely Mo 3d5/2, Mo 3d3/2, Mo 3d5/2, Mo 3d3/2
x
4
+
4+
5+
5+
,
6
+
6+
further studied by X-ray diffraction (XRD) analysis. As shown Mo 3d5/2 and Mo 3d3/2, respectively. Fig. 4b shows the high-
in Fig. 3a, obvious sharp peaks can be observed in the XRD resolution XPS spectrum of G, where the binding energies at
patterns of DB and B, indicating that DB and B have good 231.13 and 234.23 eV are in accordance with Mo 3d5/2 and
5
+
5
+
34
crystal structures. The crystal structure of G can also be proved Mo 3d3/2 of MoO
by the peak (7.11) emerging in the XRD pattern. However, no 235.38 eV are separate traits of Mo 3d5/2 and Mo 3d3/2
peaks are observed in the XRD pattern of G . According to the Calculated on the basis of area ratios of Mo species derived
standard patterns, there are three typical peaks at 38.4, 44.5, from XPS spectra, the content is 35.25%, 26.20%, 24.34% and
x
, respectively. The peaks at 232.18 and
6
+
6+
36
.
2
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2 x
Fig. 4 (a) XPS spectra and (b–e) the high-resolution XPS spectra of Mo 3d in G, B, DB and G , respectively. (f) XPS spectra of O 2p in MoO QDs.
5
+
6+
5+
6+
1
5.80% for Mo 3d5/2, Mo 3d5/2, Mo 3d3/2 and Mo 3d3/2
,
intense, and it reaches a maximum at the DB stage (i.e. MoO ),
2
1
8
respectively, certifying that a large percentage of Mo element which is consistent with the data reported previously. The
existed in a reduction state. As displayed in Fig. 4c, the binding concentration-dependent SERS activity of MoO is further illu-
2
5
+
energies at 231.08 and 234.23 eV correspond separately to Mo
strated in Fig. 5b, where the SERS spectral pattern of MB excited
5
+
35
3
d
5/2 and Mo 3d3/2
.
The peaks at 232.18 and 235.38 eV are in by MoO
2
fits well with the Raman spectrum of pure MB; and four
6
+
6+
À1
accordance with Mo 3d5/2 and Mo 3d3/2. The atom contents characteristic Raman peaks of MB molecules, R
1
(1625 cm ),
4
(774 cm ), can clearly be
À1
À1
À1
were concluded to be 40.16%, 21.66%, 27.73% and 13.34%
R
2
(1192 cm ), R
detected on the MoO
Fig. 4d reveals that the initial 3d3/2 and data of the four typical SERS bands, where the changes in the
d5/2 double peak values at 230.1 and 233.2 eV are characteristic peaks obey a concentration-dependent manner in the concen-
3
(1181 cm ) and R
5
+
6+
5+
6+
for Mo 3d5/2, Mo 3d5/2, Mo 3d3/2 and 20Mo 3d3/2
,
2
substrate. Fig. 5c displays the quantitative
3
7,38
respectively.
3
4
+
39
À3
À8
of Mo oxidation of MoO2. The energy gap of 3.1 eV between tration range from 1 Â 10 M to 1 Â 10 M. The intensities of
4
0
the two doublets is in keeping with an earlier report. As
shown in Fig. 4e, the peaks at 230.78 and 234.33 eV are ascribed
5
+
5+
to Mo 3d5/2 and Mo 3d3/2, respectively. The binding energy
6
+
6+
at 232.23 and 235.43 eV can be assigned to Mo 3d5/2 and Mo
3/2, respectively. By estimating the area ratio of Mo species
from XPS spectra, the DB form represents MoO , while the
3d
2
others (B, G and G ) represent MoO
forms. The sunlight
3Àx
2
5
+
results in the increase of the Mo states, which transforms G
into B, further breaking the oxygen bond, forming DB (with
4
+
41
abundant Mo states). This change from MoO
promotes broad absorption in the Vis-NIR spectral region
Fig. 3c). Fig. 4f shows the XPS spectra of oxygen 2p peaks of
all four samples, which shows the O binding energies of G , B,
DB, and at 529.98, 530.23, 530.68, and 530.33 eV,
respectively.
x 2
to MoO also
(
2
G
4
2
SERS properties of molybdenum oxide quantum dots
To evaluate the SERS performance of molybdenum oxide
quantum dots, we chose the frequently-used methylene blue Fig. 5 SERS measurement of MB molecules deposited on MoO
x
sub-
6
strates. (a) SERS spectra of MB molecules (10À M) deposited on four
(
MB) molecule as a Raman probe. Fig. 5a exhibits the SERS
À6
MoO
molecules with different concentrations deposited on MoO
c) The SERS intensities of four typical Raman peaks extracted from the
panel. (d) The Raman enhancement factors of MB at different concentra-
substrates.
x
QDs with different oxygen defects. (b) Raman spectra of MB
spectra of MB at the concentration of 10 M induced by four
molybdenum oxide substrates. G displays a relatively low SERS
effect on MB (also see Fig. S3, ESI†). With the increase of oxygen
2
substrates.
(
defects, the SERS performance of the MoO
x
QDs becomes more tions deposited on various MoO
x
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Fig. 7 SERS spectra of (a) phenylalanine (5 mM) and (b) amphotericin B
(
2
0.5 mM) deposited on the MoO or silicon wafer substrate.
À1
in Fig. 7a, two obvious Raman bands at 1156 cm
1
and
À1
560 cm emerge in the SERS spectrum of amphotericin B,
Fig. 6 (a) Repeatability of SERS detection of MB on the same MoO
2
substrate at twenty different sample spots. (b) Uniform pseudo color which can be assigned to the C–C stretch, the C–C–H bend and
4
5,46
map of Raman spectra of MB molecules deposited on the MoO
2
substrate.
the symmetric CQC stretch, respectively.
The enhancement
À8
The MB concentration is 10 M. (c–e) The Raman intensities of the three
À1
factors of phenylalanine (1611 cm ) and amphotericin B
2
typical peaks collected from 20 random points on the MoO substrate.
À1
4
5
(
1559 cm ) induced by MoO
2
QDs are 1.26 Â 10 and 1.37 Â 10 ,
which are equivalent to that triggered by noble metal-based
À1
peak R1 (1625 cm , C–C ring vibration) are larger than the SERS substrates, indicating the promising potential of MoO2
other Raman signatures at all concentrations and are used for QDs as cheap alternatives for gold or silver for SERS detection.
EF calculation. As displayed in Fig. 5d, the enhancement To our knowledge, this is the first time that SERS analysis of
factors of MB molecules deposited on the three molybdenum biomolecules has been demonstrated using a MoO
oxide substrates increase with decreasing concentration. The semiconductor substrate.
Raman enhancement factors of MB molecules deposited on the
x
-based
DB substrate are the biggest among the three molybdenum oxides.
6
x
Photothermal ablation of cancer cells based on MoO QDs
The maximum EF for R can be up to 7 Â 10 at a concentration of
1
À8
10
M. Therefore, the DB colored MoO QDs (MoO ) were used For photothermal tumor therapeutic application, the photo-
x 2
for further investigation.
thermal characteristics of MoO QDs were investigated. Fig. 8a
x
For the repeatability test, 20 spectral lines randomly shows the temperature elevating curves of MoO
2
QD aqueous
À1
collected on the MoO
2
substrate are displayed in Fig. 6a, where solutions (100 mg mL ) under 808 nm laser with the power
À2
highly unified SERS spectral patterns can be observed. We can densities of 0.33, 1 and 2 W cm
for 600 s, respectively,
also notice the uniform color distribution in the corresponding indicating a good photothermal conversion performance of
pseudo-color map (Fig. 6b), indicating an excellent SERS repeat- MoO
2
QDs. A concentration-dependent photothermal heating
ability of the MoO QDs. Fig. 6c–e display the relative standard
2
À1
À1
deviations (RSDs) of Raman peaks at 1625 cm , 1192 cm
and 1181 cm , which are 3.04%, 6.40%, and 5.50%, respec-
tively. The good SERS repeatabilities of MB molecules on the
,
À1
MoO
Fig. S4 (ESI†). The SERS measurement of malachite green
molecules using the MoO substrate was also conducted, which
2
substrate at different concentrations can also be seen in
2
obtained satisfactory results (Fig. S5, ESI†). These results
strongly demonstrate that MoO QDs can be used as remark-
2
able SERS substrates with uniform SERS signals.
2
In addition, MoO was further employed for SERS detection
of phenylalanine and amphotericin B, two important bioactive
molecules. Phenylalanine is one of the essential amino acids in
man, while amphotericin B is a polyene antifungal drug whose
residues are harmful to human health. As shown in Fig. 7, the
Raman signals of phenylalanine and amphotericin B are
À1
Fig. 8 (a) Temperature heating curves of MoO
2
QD solutions (100 mg mL
)
under 808 nm laser at different power densities. (b) Temperature changes
À1
with gradient concentrations (10–100 mg mL ) of the MoO
2
QD aqueous
significantly enhanced by MoO QDs. There are only two peaks solutions under irradiation at a wavelength of 808 nm laser with a power
2
À2
density of 2 W cm for 600 s. (c) Infrared thermal images of MoO
2
QDs
in the normal Raman spectrum of phenylalanine (Fig. 7a).
However, more abundant fingerprint information is observed
in its SERS counterpart, such as 1024 cm (n12, ring breathing
(n9a, C–H bend), 1215 cm
ring-C stretch) and 1611 cm (n8a, C–Cstretch).
with different concentrations under NIR irradiation. (d) The temperature
variation of primary heating and cooling of MoO QDs. (e) The cooling
time vs. ln y after 808 nm laser irradiation. On the basis of the linear
À1
2
À1
À1
43,44
mode), 1157 cm
(symmetric
s 2
regression analysis, the t value (the slope of the plot) for MoO QDs is
À1
As shown determined to be 322 s.
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effect of MoO
maximal temperature of MoO
2
QDs was also noticed (Fig. 8a–c), where the
QD solution under NIR laser
2
irradiation can reach 82 1C, which far exceeds the tolerance of
cancer cells. The temperature induced by MoO QDs is much
2
higher than that induced by other kinds of molybdenum oxides
(
Fig. S6 and S7, ESI†). To quantitatively demonstrate the
photothermal feature of MoO QDs, the photothermal trans-
duction efficiency (Z) at 808 nm was calculated according to our
2
4
7
previous work. As shown in Fig. 8d and e, the sample system
time constant (t ) was determined to be 322 s, and Z is 66.3%,
S
which confirms the outstanding NIR photothermal perfor-
mance of the molybdenum oxide-based nanostructures. MoO2
QDs also exhibit good photostability after NIR irradiation,
which can be confirmed by the absorption spectrum and
TEM observation (Fig. S8, ESI†).
Fig. 10 Fluorescence co-localization analysis of MoO
lysosome and mitochondria trackers. Scale bar: 100 mm.
2
QDs with the ER,
Encouraged by the high photothermal conversion, we then
assessed the in vitro photothermal efficacy of MoO
order to improve the stability and biocompatibility of MoO
QDs in physiological environments, a layer of calcium carbo-
2
QDs. In
the co-localization coefficients are calculated to be 88.5%, 82.0%
and 26.6% for lysosomes, ER and mitochondria, respectively,
2
2
indicating that MoO QDs are mainly distributed in ER and
nate (CaCO ) was coated on the surface of the nanostructures
3
lysosomes (Fig. S11, ESI†).
(
the characterization of the CaCO coating is displayed in
3
3
0,48,49
For the in vitro photothermal ablation of cancer cells, Hep
Fig. S9, ESI†).
Hep G2 cells were co-cultured with MoO
2
G2 cells treated with MoO QDs were exposed to NIR irradiation
QDs with different concentrations for 12 h, and then the classic
MTT assay evaluated the cellular viability. As shown in Fig. 9a,
all the cellular viabilities of Hep G2 cells exceed 90%, suggesting
2
for 2.5 min. As shown in Fig. 9a, a MoO QD concentration-
2
dependent cellular viability was observed. The tumor inhibition
of MoO
2
QDs under NIR laser can achieve 61% when the
that MoO
2
QDs have no apparent toxic effect on Hep G2 cells in
À1
À1
concentration of nanostructures increases up to 200 mg mL
,
the concentration range from 0 to 200 mg mL . MoO QDs also
2
illustrating a good PTT effect. The photothermal tumor abla-
tion performance of MoO QDs was further investigated by
show little influence on the normal hepatocytes (Fig. S10, ESI†).
We also investigated the subcellular localization of MoO QDs.
2
2
calcein AM and propidium iodide (PI) double-staining assay.
Fig. 9b displays the fluorescence images of Hep G2 cells after
different treatments. Intense and obvious green fluorescence
signals can be observed in the cancer cells treated with PBS,
2
Hep G2 cells incubated with RhB-labelled MoO QDs were
co-stained with different organelle-specific trackers (lysosomes,
ER and mitochondria). The data are shown in Fig. 10, where the
2
fluorescence signals ascribable to MoO QDs do not match that
NIR and MoO
G2 cells treated with MoO
cence, indicating an effective anticancer effect of MoO
2
QDs, respectively. In contrast, most of the Hep
QDs + NIR laser emit red fluores-
QDs. In
of the mitochondria tracker but coincide with that of lysosome
and ER probes after 8 h of incubation. For accurate evaluation,
2
2
addition, the photothermal treated cancer cells were double-
stained with annexinV-FITC and PI for flow cytometry assay. As
shown in the four-quadrant flow scatter plots (Fig. 9c), abundant
early (5.23%) and late (38.71%) apoptotic cells are observed in
the MoO
the Control, MoO
.71%, respectively. The data described here strongly prove the
2
QDs + NIR laser group, while the apoptotic cells in
2
QD and NIR groups are 5.32%, 7.13%, and
5
2
effective anti-tumor effect in vitro using MoO QDs as a novel NIR
photothermal nanoagent.
Conclusion
x
In conclusion, four MoO QDs with different oxygen defects
have been prepared using a facile and photo-induced strategy.
The optical properties of MoO QDs are reliant on the oxygen
x
Fig. 9 (a) MTT assay of the cell viabilities of Hep G2 cells treated with
contents in the nanostructures. Among them, MoO QDs with
2
different concentrations of MoO
2
QDs under NIR laser irradiation for
4+
the Mo state exhibit optimal NIR optical absorption and SERS
2
.5 min. The data are represented as mean Æ SD of the three independent
activity. The SERS detection limit and maximum EF of MB in
experiments. (b) Calcein-AM/PI live/dead staining of Hep G2 cells after
treatment (scale bar: 100 mm). (c) Flow cytometry analysis of apoptosis in
Hep G2 cells after treatment.
À8
6
solution are 10
M and 7 Â 10 , respectively. MoO
2
QDs
can also serve as a cheap non-metal SERS substrate to analyze
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Journal of Materials Chemistry B
the fingerprint information of bioactive molecules with an 14 Y. Wang, W. Wang, X. Shen, B. Zhou, T. Chen, Z. Guo,
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4
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Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
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of China (11874021, 61675072, 61335011, 81601534 and 21505047),
the Science and Technology Project of Guangdong Province
of China (2017A020215059), and the Science and Technology
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