Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 350−360 www.acsami.org

One-Step in Situ Detection of miRNA-21 Expression in Single Cancer

Cells Based on Biofunctionalized MoS2 Nanosheets
Gerile Oudeng,†,‡ Manting Au,† Jingyu Shi,†,‡ Chunyi Wen,*,† and Mo Yang*,†,‡
†
Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong, P. R. China
‡
The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen 518057, P. R. China
*
S Supporting Information

ABSTRACT: Here, we report the one-step in situ detection

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of targeted miRNAs expression in single living cancer cells via

MoS2 nanosheet-based fluorescence on/off probes. The
strategy is based on the folic acid (FA)−poly(ethylene
glycol)-functionalized MoS2 nanosheets with adsorbed dye-
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labeled single-stranded DNA (ssDNA). Once the nanoprobes

are internalized into cancer cells, the hybridization between
the probes and target miRNA results in the detachment of
dye-labeled ssDNA from MoS2 nanosheets surface, leading to
the green fluorescence recovery. In this nanoprobe, MoS2
nanosheets offer advantages of high fluorescence quenching
efficiency and extremely low toxicity. The FA conjugation
could protect the probes and improve cancer cell transfection efficiency. The ability of this nanoprobe for endogenous miRNA
detection in single living cancer cells is demonstrated for two types of cancer cells with different miRNA-21 expressions (MCF-7
and Hela cells). This functionalized MoS2 nanosheet-based nanoprobes could provide a sensitive and real-time detection of
intracellular miRNA detection platform.
KEYWORDS: miRNAs, in situ detection, MoS2 nanosheets, fluorescence resonance energy transfer (FRET), cancer cells

1. INTRODUCTION Accordingly, reliable and sensitive detection of miRNAs in

living cells is needed for early diagnosis and therapy.14,15
MicroRNAs (miRNAs) are short noncoding sequences with
Fluorescence resonance energy transfer (FRET) is a
19−25 nucleotides.1 They can regulate the diverse gene
sensitive technique to detect biomolecules on the basis of
expression by inhibiting gene translation or enhancing the
the energy transfer from donor to acceptor.16−19 Recently,
mRNA degradation in the post-transcriptional level.2 miRNAs graphene and graphene-like two-dimensional nanomaterials,
work in a relatively early and upstream level in the molecular including graphene oxide (GO), graphitic carbon nitride (g-
pathways of cells, and many of them express specifically in C3N4) nanosheets, and transition-metal dichalcogenides, such
diverse normal cells, tumors, or viruses.3 miRNAs are also as molybdenum disulfide (MoS2), have been used as efficient
reported to be closely related to drug resistance of cancer fluorescence quenchers due to their large surface area and
cells.4,5 Thus, miRNA is becoming a potential “seeded player” unique optical properties.20−27 Particularly, MoS2 nanosheets
for molecular biomarker in early prediction and indication of have aroused a lot of interests for intracellular applications,
tumorigenesis or metastasis. For example, in a perspective such as intracellular adenosine triphosphate detection, gene
view, miRNA-21 was reported to have abnormally high delivery, cellular imaging, and therapy due to their low
expression in many kinds of cancer cells lines, involving breast cytotoxicity even compared to GO.28−33 However, the
cancer, cervical cancer, lung cancer, pancreatic cancer, and so possibility using MoS2-based probes for intracellular miRNA
on, and it was described closely related to the chemo- detection has not been explored yet.
therapeutic sensitivity of tumors.6−8 Herein, we report, for the first time, monitoring of
The current methods for miRNA detection are mainly based endogenous miRNA expression via MoS2 nanosheet-based
on end-point techniques, such as qualitative reverse tran- nanoprobe, which could realize one-step in situ miRNA
scription polymerase chain reaction (qRT-PCR),9 northern expression detection at single-cell level. For such purpose,
blotting,10,11 and microarray,12,13 which can sensitively folate-functionalized MoS2 nanosheets immobilized with
measure miRNAs. However, these methods suffer from the fluorescence-labeled ssDNA probes (ssDNA−MoS2−PEG−
need of a large amount of cell samples, time-consuming
process, and end-point checking feature. Due to the low-level Received: November 28, 2017
expression of miRNAs in cells, the rapid and sensitive Accepted: December 14, 2017
detection of miRNAs in living cells is still a challenge. Published: December 14, 2017

© 2017 American Chemical Society 350 DOI: 10.1021/acsami.7b18102

ACS Appl. Mater. Interfaces 2018, 10, 350−360
ACS Applied Materials & Interfaces Research Article

FA) were prepared by covalently immobilizing FA on MoS2 (USA origin), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

nanosheets via the lipoic acid−poly(ethylene glycol) ester bromide (MTT), and penicillin/streptomycin, were purchased from
(LA−PEG−NH2) and π−π stacking between the dye-labeled Life Technologies Inc.
ssDNA probe and MOS2 nanosheets. As shown in Scheme 1, Agarose gel electrophoresis retardation assay was purchased from
Biowest, Spain. The DNA ladder (M1100-50) was purchased from
Solarbio Science & Technology Co. Ltd., Beijing, China. The main
Scheme 1. Schematic of ssDNA−MoS2−PEG−FA Probe- reagents for Western blotting, including radio-immunoprecipitation
Based FRET Platform for Intracellular miRNA-21 Detection assay lysis buffer, protease inhibitor cocktail, β-actin, horseradish
peroxidase-labeled goat antimouse IgG, and bicinchoninic acid
protein assay kit, were purchased from Servicebio Technology Co.
Ltd., China. Annexin V-FITC/PI Apoptosis Detection Kit for flow
cytometry was purchased from NanJing KeyGen Biotech Co. Ltd.,
China. Molybdenum Standard for ICP TraceCERT was purchased
from Sigma-Aldrich.
2.2. Characterization. The morphology and size of MoS2
nanosheets were characterized using a JEOL-2100F transmission
electron microscope installed with an Oxford Instruments EDS
system (200 kV). The absorbance spectra of MoS2 nanosheets were
obtained with a UV−vis spectrophotometer (Ultrospec 2100 pro).
Size distribution and ζ potential of MoS2 nanosheets were determined
at neutral pH with a Zetasizer Nano Z system (Malvern Instruments
Ltd). Powder X-ray diffraction (XRD) pattern of MoS2 nanosheets
was obtained using an X-ray diffractometer (Rigaku SmartLab).
Fourier transform infrared (FTIR) spectra of MoS2 nanosheets and
this fluorescence turn-on sensor is established by quenching MoS2−PEG−FA were acquired with a Bruker Vertex-70 FTIR
the absorbed dye-labeled ssDNA probes on MoS2 nanosheets spectrometer. Thermogravimetric analysis (TGA) was performed
to an “off” state due to the fluorescence resonance energy with a thermogravimetric analyzer (Netzch STA 449C, Jupiter). X-ray
photoelectron spectra (XPS) for functionalization analysis were
transfer (FRET) effect. The conjugated folate via LA−PEG
determined on an AXIS ULTRA DLD X-ray photoelectron
linker could protect ssDNA probes and improve cancer cell spectrometer (Kratos, Tokyo, Japan)
targeting and internalization process. When ssDNA−MoS2− 2.3. MoS2 FRET Nanoprobe Establishment. Folic acid (FA)
PEG−FA nanoprobes are internalized by cancer cells, the was first conjugated with LA−PEG−NH2 to form LA−PEG−FA
higher binding force between target miRNA-21 and ssDNA following a previous protocol.30,34 For this purpose, 0.15 mmol folic
probes leads to the rapid fluorescence recovery due to the acid was dissolved in 2.5 mL of dimethyl sulfoxide (DMSO) for 8 h.
detachment of dye-labeled ssDNA probes from MoS2 nano- FA solution was then added with 0.17 mmol EDC and 0.33 mmol
sheets. With this simple strategy, we used this functionalized NHS for activation. LA−PEG−NH2 (0.15 mmol) was then added for
MoS2 nanosheet-based nanoprobes for miRNA-21 detection in reaction overnight. The final product was collected and dialyzed with
both cell-free system and inside living cancer cells with a 1 kDa dialysis bag for 4−5 days, and dialysis water was changed
every day to remove the organic solution and the excess reagent. The
different expressions of miRNA-21. This platform can be a final product LA−PEG−FA was lyophilized using a ScanVac cs55-4
potential platform for in situ detection of intracellular miRNA CoolSafe freeze dryer.
in early diagnostics and treatment applications. LA−PEG−FA was then conjugated on MoS2 nanosheets to form
MoS2−PEG−FA complex. LA−PEG−FA (65 μg) powder was added
2. EXPERIMENTAL SECTION into 0.6 mg of MoS2 nanosheets dispersed in 1.2 mL of water. The
2.1. Materials. MoS2 material was purchased from Muke Nano mixture was sonicated for 20 min and stirred for 5 h. Then, the
Science and Technology Ltd., Nanjing, China. After sonication, MoS2 samples were filtered by 100 kDa MWCO filters and centrifuged to
nanosheets dispersion solution was dialyzed with a filter membrane remove the extra LA−PEG−FA. The obtained MoS2−PEG−FA
with 3500D molecular weight cutoff (MWCO) for 1 day to eliminate complex was highly water-soluble and then stored in 4 °C for further
lithium hydroxide (LiOH). Then, the dispersion was centrifuged at usage.
2000 rpm for 5 min. All of the samples of miRNA-21, scrambled In quenching efficiency experiment, FAM-labeled miRNA-21 probe
DNA, miRNA-20a, and target miRNA-21 were synthesized and with a fixed concentration of 30 nM was incubated with MoS2−PEG−
purified by Sangon Biotech, Shanghai, China. The first three DNA FA nanosheets in a series of concentrations (5−120 μg/mL) for 30
sequences were modified with carboxyfluorescein (FAM) in the 5′ min at room temperature. Then, the fluorescence intensity was
terminal. The sequences are described as follows: scrambled DNA measured with 488 nm excitation wavelength. As control groups,
probe (FAM-5′-TGC GCT CCT GGA CGT AGC CTT-3′), FAM-labeled scRNA probes and FAM-labeled miRNA-20a probes
miRNA-20a (FAM-5′-FAM-TAA AGT GCT TAT AGT GCA were loaded with same amount under the same experimental
GGT AG-3′), miRNA-21 probe (FAM-5′-TCA ACA TCA GTC conditions.
TGA TAA GCT A-3′), and target miRNA-21 sequence (5′-UAG The stabilities of ssDNA carried by MoS2 and MoS2−PEG under
CUU AUC AGA CUG AUG UUG A-3′). All of the synthesized DNA DNase I treatment were evaluated through gel electrophoresis assay.
sequences were dissolved in ultrapure water as stock solution and kept Briefly, naked ssDNA, ssDNA−MoS2, and ssDNA−MoS2−PEG were
at −20 °C. Ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) incubated with DNase I (0.15 unit/μL) at 37 °C for 5 and 20 min,
and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from respectively. Naked ssDNA, ssDNA−MoS2, and ssDNA−MoS2−PEG
Sigma-Aldrich. Folic acid was also purchased from Sigma-Aldrich. without nuclease treatment were used as control groups. Samples of
Lipoic acid−PEG−NH2 (MW: 2000 Da) was purchased from each group were stirred and loaded into the prepared agarose gel
ToYongBio Ltd., Shanghai, China, and stored at −20 °C. Dialysis (3%) at 120 V for 30 min. After electrophoresis, the gel was stained
bags (MWCO: 3500D and 1000D) and tubes (MWCO: 1000D) with 0.5 μg/mL ethidium bromide (EtBr) and washed with water.
were purchased from GE Lifescience and stored at −4 °C. All of the The bands were visualized by Tanon 1600/1600R Gel Imaging
cell culture reagents, including Dulbecco’s modified Eagle’s medium System.
(DMEM) (high glucose), trypsin 0.25% ethylenediaminetetraacetic 2.4. In Vitro miRNA Detection. For target detection, ssDNA−
acid (EDTA), phosphate-buffered saline (PBS), fetal bovine serum MoS2−PEG−FA nanocomplex was incubated with a series of

351 DOI: 10.1021/acsami.7b18102

ACS Appl. Mater. Interfaces 2018, 10, 350−360
ACS Applied Materials & Interfaces Research Article

Figure 1. (a) Schematic of the preparation process of nanoprobes. (b) TEM image of synthesized MoS2 nanosheets. (c) TEM image of synthesized
MoS2−PEG−FA. (d) Size distribution of MoS2 nanosheets and MoS2−PEG−FA. (e) FTIR spectra of MoS2 nanosheets and MoS2−PEG−FA.

concentrations of miRNA-21 target sequences from 10 to 50 nM. The cell viability (%) was calculated as (Am/Acontrol) × 100, where Am
After 2 h incubation at 37 °C in the dark environment, the represents the absorbance at 570 nm of cells. The MCF-7 cells were
fluorescence signal of each sample was measured. To investigate the cultured in 6 well plates with a density of 4.0 × 104/well and then
specificity, FAM-labeled scRNA probe and miRNA-20a probe were incubated with MoS2 (200 μg/mL) and MoS2−PEG (200 μg/mL)
used with the same detection protocol. Fluorescence spectra of for 24 h, respectively. Afterward, MCF-7 cells were treated with
quenching and fluorescence recovery were recorded with an EDTA-free trypsin and washed with PBS. After further washing with
Edinburgh FLSP920 spectrophotometer equipped with a 450 W PBS at 4 °C, the MCF-7 cells were resuspended with 200 μL of
steady-state xenon lamp at room temperature. binding buffer and treated with 5 μL of Annexin V-FITC and 5 μL of
2.5. Cell Culture and Cytotoxicity Assay. MCF-7 cells and propidium iodide in the dark environment for 15 min. The apoptosis
Hela cells were cultured in a high-glucose medium containing 10%
of cells was analyzed with CytoFLEX (Beckman).
fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5%
2.6. Western Blotting Analysis. The expression levels of surface
CO2 atmosphere. For cell viability experiment, the cells were first
folate receptor (FR) in MCF-7, Hela, NIH3T3, and HepG2 cell lines
seeded on a 96 well plate and medium was changed each day. MoS2
nanosheets and the MoS2−PEG−FA complex with a series of were determined by Western blotting assay. The protein samples were
concentration from 0 to 200 μg/mL were added into the 96 well plate analyzed by 12% sodium dodecyl sulfate polyacrylamide gel
with a density of about 4.0 × 104 cells/well. After incubation with electrophoresis and transferred to a poly(vinylidene difluoride)
nanomaterials for 24 h, 10 μL of 12 mM MTT stock solution was membrane. After electroblotting of the gels, the filters were blocked
added into each well and the cells were incubated at 37 °C in 5% CO2 with 5% skimmed milk in Tris-buffered saline Tween-20 (TBST)
for 4 h. Then, the medium in each well was replaced with DMSO for buffer (0.1% Tween-20, 10 mM Tris−HCl, pH = 8) and then
another 10 min incubation in 37 °C. After sufficiently mixing, the incubated with anti-FR protein monoclonal antibody at 4 °C
absorbance of the samples was measured at 570 nm using a Tecan overnight. After washing, the blots were incubated with 1:3000
Infinite F200 microplate reader to evaluate the viability of the cells. diluted goat antimouse IgG monoclonal antibody in TBST buffer.

352 DOI: 10.1021/acsami.7b18102

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Figure 2. (a) TGA diagram of MoS2 nanosheets and MoS2−PEG−FA. (b) UV−vis spectra of MoS2, FA, and MoS2−PEG−FA. (c) Full XPS
images of synthesized MoS2 nanosheets and MoS2−PEG−FA. High-resolution XPS images of (d) Mo 3d, (e) S 2p, and (f) N 1s.

Finally, the protein bands were visualized with enhanced chem- inductively coupled plasma mass spectrometer (ICP-MS). The
iluminescence. intensity ratios of the molecular Mo4+ ions to the internal standard
2.7. Inductively Coupled Plasma Mass Spectrometry (ICP- Mo4+ ions were determined and plotted against molybdenum
MS) Quantitative Analysis of Cellular Uptake. To quantitatively standard solution to generate a calibration curve (Figure S1).
evaluate the effect of FA modification on cellular uptake of 2.8. Intracellular miRNA Monitoring. MCF-7 cells were first
nanoprobes, four different kinds of cell lines (MCF-7, Hela, cultured in 75 cm2 flask for 1 week with a cell density of 4.0 × 104
NIH3T3, and HepG2) were seeded with a density of 4.0 × 104/ cells/mL. To monitor the endogenous miRNA-21 expression in
well at 75 cm2 flask. The cells were incubated with MoS2−PEG and MCF-7 cells and Hela cells, the cells were seeded into confocal dishes
MoS2−PEG−FA (100 μg/mL) for 4 h. After incubation, the cells (35 mm) and cultured for 24 h at 37 °C in a 5% CO2 environment.
were washed with PBS three times and then digested with trypsin. Then, ssDNA−MoS2−PEG−FA complex of 100 μg/mL, which was
The cell suspension was fixed in 2.5% glutaraldehyde and dehydrated confirmed with 90% cell viability and 92% quenching efficiency, was
with a series of ethanol concentrations. The collected cells were then then loaded with FAM-labeled miRNA-21 probe and added into the
dried in a vacuum desiccator and weighed. The cell samples were then confocal dishes. After 4 h incubation at 37 °C and 5% CO2
dissolved by nitric acid solution and diluted by deionized water to 10 environment, the cells were washed with PBS gently for confocal
mL with indium (5 ppb) as the internal standard.35,36 The imaging with a Leica TCS SPE confocal microscope system. The
concentration of Mo4+ was determined by an Agilent 7500ce excitation laser was set at 488 nm, and the green fluorescence signal

353 DOI: 10.1021/acsami.7b18102

ACS Appl. Mater. Interfaces 2018, 10, 350−360
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Figure 3. (a) Photos of MoS2 nanosheets and MoS2−PEG−FA complex distributed in water, PBS, and cell medium after 4 h of standing. (b) Gel
electrophoresis of ssDNA, ssDNA−MoS2, and ssDNA−MoS2−PEG with and without DNase I treatment; lanes 1−3: ssDNA without DNase I,
ssDNA treated with DNase I for 5 and 20 min; lanes 4−6: ssDNA−MoS2 without DNase I, ssDNA−MoS2 treated with DNase I for 5 and 20 min;
lanes 7−9: ssDNA−MoS2−PEG−FA without DNase I, ssDNA−MoS2−PEG−FA treated with DNase I for 5 and 20 min. (c) Cell viability analysis
of MCF-7 cells incubated with MoS2 nanosheets and MoS2−PEG for 24 h by MTT assay. (d) Cell apoptosis analysis of MCF-7 cells incubated
with MoS2 nanosheets (left) and MoS2−PEG (right) for 24 h by flow cytometry.

was collected within the range of 505−540 nm under the same on cancer cells was conjugated via LA−PEG−NH2 linker
conditions. Cell images in bright field, fluorescence, and overlap of the through the EDC/NHS chemistry.30 Different character-
two fields were captured and analyzed. Single-cell fluorescence images izations were used to confirm the successful functionalization
were then analyzed with ImageJ software. Flow cytometry was used to
quantitatively analyze fluorescence recovery signals of multiple cells.
process. Figure 1b,c shows the TEM images of MoS2
The cell samples were first incubated with nanoprobes and then nanosheets and MoS2 nanosheets after PEG−FA modification.
collected with trypsin followed by washing with PBS several times. After FA−PEG modification process, the thickness of MoS2
The fluorescence recovery signals of multiple cells were then analyzed nanosheets obviously decreased and the average size deceased
with a CytoFLEX flow cytometer (Beckman) fitted with a 488 nm from 180 nm to about 142 nm by dynamic light scattering
excitation laser. (DLS) analysis (Figure 1d). The decrease of thickness and size
could be explained by the partial breakdown of MoS2
3. RESULTS AND DISCUSSION nanosheets and further exfoliation during PEGylation and
3.1. Synthesis of ssDNA−MoS2−PEG−FA Probes. The sonication. After FA−PEG modification, the ζ-potential of
proposed ssDNA−MoS2−PEG−FA nanoprobes provide a MoS2 nanosheets shifted from −40.7 to −30.8 mV (Figure
rapid and sensitive strategy for intracellular miRNA-21 S4), indicating the presence of the PEG−FA. The conjugation
detection in living cancer cells. As shown in Figure 1a, MoS2 of FA−PEG on MoS2 nanosheets was then analyzed by Fourier
nanosheets were first prepared by a simple sonication-assisted transform infrared (FTIR) spectroscopy (Figure 1e). The peak
exfoliation approach from bulk MoS2 powder. XRD pattern at 472 cm−1 was observed in both MoS2 and MoS2−PEG−FA,
was then used to characterize the MoS2 nanosheets. As shown which was attributed to the Mo−S bonding. The peaks at
in Figure S2, MoS2 nanosheets showed the characteristic peak 640−680 and 837 cm−1 were attributed to the S−S
of pristine MoS2 centered at 2θ = 14.50°, which matched with characteristic stretching.38,39 The absorption bands at 1000
the JCPDS data of MoS 2 . 37 The as-synthesized MoS 2 and 1114 cm−1 were attributed to the S−O stretching.40 The
nanosheets had an average size of about 180 nm with certain bending modes of O−H around 1635 and 3371 cm−1 were
thickness (Figure S3a) and the lattice fringe around 0.234 nm assigned to the water molecules. The characteristic vibration
with polycrystalline structure in selective area electron peaks within the range of 2800−3000 cm−1 and a weak peak at
diffraction (Figure S3b). 1338 cm−1 were attributed to the C−H stretching of PEG and
To improve the performance of MoS2 nanosheets, lipoic FA.30,35 These results demonstrated the successful conjugation
acid-modified PEG-amine (LA−PEG−NH2) is conjugated to PEG−FA on MoS2 nanosheets.
MoS2 surface at the defect sites. Folic acid (or folate), which is Thermogravimetric analysis (TGA) was also performed to
used for specific targeting of the folate receptor overexpressed show weight losses of 67.09 and 37.87% for MoS2 and MoS2−
354 DOI: 10.1021/acsami.7b18102
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ACS Applied Materials & Interfaces Research Article

PEG−FA at 890 °C, respectively (Figure 2a). The gradual performed for MCF-7 cells incubated with various concen-
weight loss of MoS2−PEG−FA between 300 and 800 °C is due trations of MoS2 and MoS2−PEG up to 24 h. As shown in
to the removal of stable oxygen functional groups adhered on Figure 3c, no obvious toxicity could be observed for both
MoS2 surface, indicating the attachment of PEG−FA on MoS2. MoS2 and MoS2−PEG with cell viabilities above 80% even at
As shown in Figure 2b, the UV−vis spectra of MoS 2 high concentrations of up to 200 μg/mL. And MoS2−PEG
nanosheets exhibited characteristic peaks around 215 and showed a slightly higher cell viability compared to MoS2
280 nm of FA, indicating the conjugation of FA on MoS2 nanosheets. The apoptosis degrees of MCF-7 cells incubated
nanosheets. Furthermore, X-ray photoelectron spectroscopy with MoS2 nanosheets and MoS2−PEG for 24 h were further
(XPS) measurement was performed to analyze the bonding quantitatively determined by flow cytometry. As shown in
energy information change during modification (Figure 2c). Figure 3d, MCF-7 cells incubated with MoS2−PEG showed a
The high-resolution XPS images of Mo 3d, S 2p, and N 1s are lower apoptosis ratio of 5.80% compared to 8.47% for MoS2,
shown in Figure 2d−f, respectively. The peaks of Mo 3d5/2 and which also exhibited the slightly improved biocompatibility.
Mo 3d3/2 in pristine MoS2 nanosheets were 232.2 and 235.4 Except for the cytocompatibility, hemocompatibility is another
eV, and the two peaks shifted to 230.9 and 234 eV for MoS2− major concern for an ideal material to be used for biological
PEG−FA, respectively. The S 2p5/2 and S 2p3/2 of pristine application. As shown in Figure S6, no visible hemolysis effect
MoS2 nanosheets were 163.2 and 167.8 eV and shifted to was observed in blood for MoS2−PEG over a concentration
162.8 and 165.9 eV for MoS2−PEG−FA, respectively. Both range of 10−100 μg/mL.
Mo 3d and S 2p peaks showed a shift to lower binding energy To demonstrate the specific targeting of MoS2−PEG−FA
due to PEG−FA conjugation. In addition, there was no N 1s nanoprobes on folate receptor overexpressed cancer cells, the
peak for MoS2 nanosheets, and the N 1s peak at 399.2 eV was cellular uptake experiments were performed with cancer cell
observed in MoS2−PEG−FA, which indicated the formation of lines of MCF-7 and Hela and normal cell lines of NIH3T3 and
CONH− between FA and PEG and the presence of N ring HepG2. The surface folate receptor (FR) expressions of MCF-
structure in FA.41,42 7 cells, Hela cells, NIH3T3 cells, and HepG2 cells were first
3.2. Stability and Biocompatibility Testing of PEG− measured by Western blotting. MCF-7 cells and Hela cells had
FA Conjugation. Stable dispersion of MoS2 nanosheets in obviously higher FR expression levels compared to NIH3T3
various physiological solutions is critical for their application in and HepG2 cells (Figures 4a and S7). For the cellular uptake
biosensing. Pristine MoS2 nanosheets might agglomerate due experiments, MoS2−PEG and MoS2−PEG−FA were incu-
to restacking, leading to poor stability in solution. The layer- bated with each kind of cell line for 4 h. The cellular uptake
by-layer stacking might hide probe gene sequences and lead to levels in different kinds of cell lines were then determined by
the failure of hybridization between probe and target.43 As measuring Mo4+ content using inductively coupled plasma
shown in Figure 3a, it can be clearly seen that the MoS2−
PEG−FA has a better stability compared to pure MoS2 for 4 h
standing in solutions including water, PBS, and DMEM, which
allowed further usage in biological and physiological environ-
ments. The size distributions of MoS2 nanosheets before and
after PEG−FA modification in the above solutions after 4 h
were also measured by a DLS size analyzer. It was shown that
the average size of MoS2−PEG−FA was kept within 120−140
nm and that the average size of MoS2 was in the micrometer
range due to the possible agglomeration (Figure S5).
In real physical environment, naked oligonucleotides are
easily cleaved by deoxyribonuclease I (DNase I), which
directly led to the break of oligonucleotide probes and false
fluorescence recovery signal.39,44 The attachment of oligonu-
cleotide probes on MoS2 nanosheet surface could increase the
probe stability and protect oligonucleotide probes from
enzymatic degradation by DNase I. To demonstrate this, gel
electrophoresis experiments with ssDNA, ssDNA−MoS2, and
ssDNA−MoS2−PEG−FA under DNase I treatment with
various time periods were performed. As shown in Figure 3b,
naked ssDNA (lanes 1−3) were digested quickly and no visible
signal was detected for both 5 min (lane 2) and 20 min (lane
3), which demonstrated the rapid degradation of ssDNA by
DNase I. For ssDNA loaded on the MoS2 nanosheets (lanes
4−6), ssDNA partially remained for 5 min (lane 5) and largely
disappeared for 20 min (lane 6). In contrast, there was no
obvious digestion in the presence of DNase I for ssDNA
loaded on MoS2−PEG−FA for both 5 min (lane 8) and 20
min (lane 9). This protection of ssDNA on MoS2−PEG−FA
may be attributed to the steric hindrance effect to prevent the Figure 4. (a) Western blot of FR expression in Hela, MCF-7, NIH-
enzymes absorption onto the nanocomposite surface.45 3T3, and HepG2 cells; β-actin was used as the loading control. (b)
To study the cytotoxicity of MoS2 nanosheets before and ICP-MS for cellular uptake amount measurement of MoS2−PEG and
after PEG coating, standard MTT cell viability assays were MoS2−PEG−FA in cells with different FR expression levels.

355 DOI: 10.1021/acsami.7b18102

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ACS Applied Materials & Interfaces Research Article

Figure 5. (a) Overlapping between adsorption spectrum of MoS2 nanosheets and excitation and emission spectra of FAM-labeled DNA. (b)
Photoluminescence spectra of FAM-labeled miRNA-21 probes incubated with MoS2 nanosheets with a series of concentrations. (c) Quenching
efficiency (%) and PL intensity of FAM-labeled miRNA-21 probe quenched by a series of concentrations of MoS2−PEG−FA nanosheets.

Figure 6. (a) Photoluminescence spectra of MoS2−PEG−FA-based FRET sensing platform with the addition of miRNA-21 target with increasing
concentration from 10 to 50 nM. (b) Intensity of recovered fluorescence signal with the addition of various concentrations of target miRNA-21
(inset: fitting logarithmic curve of the relative fluorescence intensity and the target miRNA-21 concentrations). (c) Comparison of relative
fluorescence recovery signal for scRNA, miRNA-20a, and miRNA-21 with the same concentration of 50 nM.

mass spectrometry (ICP-MS). As shown in Figure 4b, the quenching by MoS2 nanosheets. As shown in Figure 5c, the
cellular uptakes of MoS2−PEG for all of the four kinds of cell quenching efficiency reached 96.95 ± 3.01% with 120 μg/mL
lines are quite similar. FA conjugation on MoS2−PEG of MoS2 nanosheets, showing the high quenching efficiency of
significantly increased the cellular uptake of nanoprobes for MoS2 nanosheets. The quenching capabilities of MoS2 and
cancer cell lines of MCF-7 and Hela. The cellular uptakes of MoS2−PEG−FA were also compared to the same concen-
MoS2−PEG−FA showed 113 and 116% increase for MCF-7 tration. As shown in Figure S8a, there is no obvious difference
and Hela cells compared to MoS2−PEG, respectively. In for the quenching spectra of MoS2 and MoS2−PEG−FA on
contrast, there is only a slight cellular uptake change of FAM-labeled probes with the same concentration of 120 μg/
nanoprobes before and after FA conjugation for normal cell mL. Both nanomaterials could achieve almost complete
lines. The above experimental results demonstrated the quenching. The quenching capabilities of MoS2 and MoS2−
targeting functions of MoS2−PEG−FA on folate receptor PEG−FA under various concentrations also did not show
overexpressed cancer cells. much difference (Figure S8b). Both ssDNA−MoS2 and
3.3. In Vitro Sensing of miRNA-21. High quenching ssDNA−MoS2−PEG−FA complexes showed stable quenching
efficiency is the precondition for a “turn-on” FRET sensor efficiency of above 90% in solutions over 4 h standing (Figure
establishment. The good matching between emission spectra S8c).
of FAM-labeled probes and adsorption spectra of MoS2 makes The capability of the established FRET sensing platform was
it possible to establish an efficient FRET system (Figure 5a). then tested by incubating the established ssDNA−MoS2−
To achieve a high quenching efficiency, the ratio between the PEG−FA nanoprobes with synthetic miRNA-21 at a series of
donor and acceptor molecules should be optimized. Thus, concentrations from 10 to 50 nM. After incubation for 2 h at
FAM-labeled probe as the donor molecule with fixed 37 °C, the recovered fluorescence signal was measured. As
concentration (30 nM) was incubated with a series of shown in Figure 6a, the fluorescence signal gradually enhanced
concentrations of MoS2−PEG−FA nanosheets ranging from with increasing concentrations of the miRNA-21 target. The
5 to 120 μg/mL. The fluorescence intensity of FAM-labeled fluorescence intensity peak at 520 nm showed a linear
probe of 30 nM in the same volume of PBS was considered as relationship with logarithmic concentration of target as the
control signal. As shown in Figure 5b, with the increasing equation y = −20.328 + 7.396x, where y is the relatively
concentrations of MoS2−PEG−FA, the fluorescence emission recovered fluorescence signal (Fr − Fq)/Fq and x is the
peaks of FAM-labeled probe at 520 nm decreased gradually logarithmic concentration of miRNA-21 (Figure 6b). Fluo-
due to the strong affinity between the nanosheets and single- rescence stability of sensing against various ions, pHs, and
stranded probes. The quenching efficiency was calculated using amino acids was evaluated by PL fluorescence measurement
the equation Qe = (F0 − Fq)/F0, where F0 is the original (Figure S9), and it showed negligible change in different
fluorescence intensity of FAM-labeled probe and Fq represents physiological solutions. Furthermore, to investigate the
the fluorescence intensity of FAM-labeled probe after specificity of the MoS2 nanosheet-based FRET sensor for
356 DOI: 10.1021/acsami.7b18102
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ACS Applied Materials & Interfaces Research Article

Figure 7. (a) Confocal microscopy images of MCF-7 cells incubated with ssDNA−MoS2−PEG−FA nanoprobes for 0, 1, and 4 h. (b)
Representative single-cell fluorescence image signal analysis with various incubation periods. (c) Fluorescence intensity per unit area of single cells
with various incubation periods. (d) Representative histogram plots of flow cytometry showing the intracellular fluorescence recovery of MCF-7
cells upon incubation with nanoprobes. (e) Summary data of flow cytometry results (*P < 0.05, **P ≤ 0.01). The bar represents the mean
fluorescence intensity (MFI) ± standard error of the mean (SEM).

miRNA-21 detection, miRNA-20a and scrambled RNA h showed the highest signal, which was almost 2-fold (P <
(scRNA) were tested with same procedures. Under the same 0.05) higher than that at 1 h (Figure 7e).
concentration of 50 nM, miRNA-21 showed almost three times To examine the specificity of the MoS2-based probes for
of signal intensity compared to scRNA and miRNA-20a, which miRNA-21 detection, human epithelial cervix carcinoma (Hela
shows obvious discrimination for intracellular sensing (Figure cell) was chosen as control due to its low endogenous
6c). expression of miRNA-21.46 As demonstrated by qRT-PCR,
3.4. Intracellular Sensing of miRNA-21. For intracellular MCF-7 cells showed a much higher miRNA-21 expression
miRNA-21 monitoring, the breast cancer MCF-7 cell, which compared to Hela cells (Figure S10). In this experiment, both
overexpresses miRNA-21, was chosen as the cell model. In this MCF-7 cells and Hela cells were treated with 100 μg/mL
experiment, ssDNA−MoS2−PEG−FA probes were first nanoprobes for 4 h under the same experimental conditions.
incubated with MCF-7 cells for 0, 1, and 4 h. High-resolution Figure 8a shows the confocal fluorescence images of MCF-7
confocal fluorescence images were then captured to analyze the cells and Hela cells after 4 h incubation. MCF-7 cells showed
fluorescence signal recovery due to the binding with miRNA- higher fluorescence recovery signals compared to Hela cells. As
21 (Figure 7a). Single-cell fluorescence analysis was performed shown in Figure 4b, the cellular uptake of nanoprobes for Hela
using ImageJ software. A gradual increase of fluorescence signal cells was slightly higher than that of MCF-7 cells. This
across the cell body (x-axis) was observed with increasing indicated that the higher fluorescence recover signals of MCF-
incubation time at single-cell level (Figure 7b). The individual 7 cell should be mainly contributed by the higher endogenous
cell fluorescence intensity was also normalized to individual expression of miRNA-21 rather than the internalized amount
cell area, which showed that the fluorescence intensity per unit of nanoprobes. The fluorescence recovery signals from
area increased gradually with increasing incubation time multiple single cells of MCF-7 and Hela cells were then
(Figure 7c). This fluorescence signal recovery was due to the quantified and compared using flow cytometry. Figure 8b,c
release of FAM−ssDNA from MoS2 nanosheets by the shows the representative quantitative flow cytometry results of
stronger binding between FAM−ssDNA and endogenous ssDNA−MoS2−PEG−FA probes incubated with MCF-7 cells
miRNA-21. The previous approaches for in situ miRNA and Hela cells after 4 h incubation, respectively. The
fluorescence recovery signal of MCF-7 cells was 58.7% higher
detection need long-term incubation to achieve enough
than that of Hela cells after 4 h incubation (Figure 8d). The
internalization of nanoprobes (>10 h).25 The above exper-
above results demonstrated a potential screening approach
imental results demonstrated that our ssDNA−MoS2−PEG−
using the MoS2 nanosheet-based FRET probes for rapidly and
FA probes could enable rapid internalization into cancer cells
sensitively monitoring different endogenous expression levels
with overexpression of folate receptor for rapid in situ
of target miRNA at single-cell level.
detection. The fluorescence recovery signals from multiple
single cells of MCF-7 were then quantified using flow
cytometry. Figure 7d showed the representative quantitative 4. CONCLUSIONS
flow cytometry results of ssDNA−MoS2−PEG−FA probes In this work, MoS2 nanosheet-based FRET probes were
incubated with MCF-7 cells at various time points. As developed for measuring miRNA-21 expression in living cancer
expected, the control cells at t = 0 h showed a low level of cells. This miRNA sensing strategy is based on monitoring
autofluorescence signals. The fluorescence recovery signal at 4 fluorescence “off−on” change of internalized ssDNA−MoS2−
357 DOI: 10.1021/acsami.7b18102
ACS Appl. Mater. Interfaces 2018, 10, 350−360
ACS Applied Materials & Interfaces Research Article

Figure 8. (a) Confocal microscopy images of MCF-7 cells and Hela cells incubated with ssDNA−MoS2−PEG−FA nanoprobes for 4 h. (b, c)
Representative histogram plots of flow cytometry showing the intracellular fluorescence recovery upon incubation with nanoprobes for MCF-7 cells
and Hela cells, respectively. (d) Summary data of flow cytometry results for Hela cells and MCF-7 cells for 4 h incubation. The bar represents the
mean fluorescence intensity (MFI) ± SEM.

PEG−FA probes due to the hybridization of endogenous

miRNA with FAM−ssDNA. PEG-folate-modified MoS2 nano-
■
*
ASSOCIATED CONTENT
S Supporting Information

sheets provided excellent biocompatibility, probe gene The Supporting Information is available free of charge on the
protection, and cancer cell targeting function. The results of ACS Publications website at DOI: 10.1021/acsami.7b18102.
miRNA-21 expression detection in living MCF-7 and Hela Calibration curve of Mo4+ for ICP-MS; XRD patterns of
cells demonstrated the feasibility of this MoS2-based nanop- pristine MoS2 and MoS2 nanosheets; high-resolution
TEM image of pristine MoS2 nanosheets; ζ potential
robes for in situ single-step miRNA detection at the single-cell
measurement; hemolysis activity testing; gray intensity
level, which could be a promising single-cell analysis platform analysis of Western blotting results; quenching stability
to monitor miRNA expression for fundamental research and testing; fluorescence stability testing against various ions,
clinical applications. pH, and amino acids; and qRT-PCR results (PDF)
358 DOI: 10.1021/acsami.7b18102
ACS Appl. Mater. Interfaces 2018, 10, 350−360
ACS Applied Materials & Interfaces Research Article

■ AUTHOR INFORMATION
Corresponding Authors
Silencing by RNA Interference: Principles, Challenges, and New
Strategies. Gene 2014, 538, 217−227.
(15) Ma, F.; Liu, M.; Tang, B.; Zhang, C.-Y. Rapid and Sensitive
*E-mail: [email protected]. Phone: +852-34008898. Quantification of microRNAs by Isothermal Helicase-Dependent
Fax: +852-23342429 (C.W.). Amplification. Anal. Chem. 2017, 89, 6182−6187.
*E-mail: [email protected]. Phone: +852-27664946. (16) Dong, M.; Tepp, W. H.; Johnson, E. A.; Chapman, E. R. Using
Fax: +852-23342429 (M.Y.). Fluorescent Sensors to Detect Botulinum Neurotoxin Activity in Vitro
and in Living Cells. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14701−
ORCID
14706.
Mo Yang: 0000-0002-3863-8187 (17) Ma, F.; Li, Y.; Tang, B.; Zhang, C.-Y. Fluorescent Biosensors
Notes Based on Single-Molecule Counting. Acc. Chem. Res. 2016, 49, 1722−
The authors declare no competing financial interest. 1730.

■
(18) Zhu, G.; Liang, L.; Zhang, C.-Y. Quencher-Free Fluorescent
Method for Homogeneously Sensitive Detection of microRNAs in
ACKNOWLEDGMENTS Human Lung Tissues. Anal. Chem. 2014, 86, 11410−11416.
This work was supported by the National Natural Science (19) Zhou, J.; Yang, Y.; Zhang, C.-Y. Toward Biocompatible
Foundation of China (NSFC) (Grant Nos. 81471747 and Semiconductor Quantum Dots: From Biosynthesis and Bioconjuga-
31771077), the Hong Kong Research Council General tion to Biomedical Application. Chem. Rev. 2015, 115, 11669−11717.
Research Grant (PolyU 152213/15E), and the Hong Kong (20) Wang, Q.; Wang, W.; Lei, J.; Xu, N.; Gao, F.; Ju, H.
Fluorescence Quenching of Carbon Nitride Nanosheet through Its
Polytechnic University Internal Fund (1-ZVJ7). This work was Interaction with DNA for Versatile Fluorescence Sensing. Anal. Chem.
also supported by the University Research Facility in Life 2013, 85, 12182−12188.
Sciences of the Hong Kong Polytechnic University.

■
(21) Shi, J.; Lyu, J.; Tian, F.; Yang, M. A Fluorescence Turn-On
Biosensor Based on Graphene Quantum Dots (GQDs) and
ABBREVIATIONS Molybdenum Disulfide (MoS2) Nanosheets for Epithelial Cell
Adhesion Molecule (EpCAM) Detection. Biosens. Bioelectron. 2017,
FA, folic acid; FRET, fluorescence resonance energy transfer;
93, 182−188.
MoS2, molybdenum disulfide; ssDNA, single-stranded DNA; (22) Shi, J.; Guo, J.; Bai, G.; Chan, C.; Liu, X.; Ye, W.; Hao, J.; Chen,
PEG, poly(ethylene glycol)

■
S.; Yang, M. A Graphene Oxide Based Fluorescence Resonance
Energy Transfer (FRET) Biosensor for Ultrasensitive Detection of
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