Silica Nanoparticles with Up-conversion Fluorescence Based on Triplet-Triplet Annihilation Mechanism for Specific Recognition of Apoptosis Cells

Article abstract of DOI:10.1021/acs.analchem.8b04784

Discrimination of live and apoptotic cells is a crucial task in the research of pharmacology, biology, pathology, and medicine science. Recently, up-conversion (UC) luminescent materials have appealed much attention due to their unique ability to convert low energy excitation photons to high energy ones. However, UC fluorescence has not been employed in the field of discrimination of live and apoptotic cells. We present a facile and costless St?ber method to fabricate robust silica nanoparticles (SiO₂ UCNPs) exhibiting several merits, such as narrow size distribution and UC luminescence. SiO₂ UCNPs could discriminate live and apoptosis cells by taking advantage of the unique surface property of SiO₂ UCNPs for the first time. This work is also the first demonstration of the use of single photon excited UC fluorescence derived from nanoparticles for biological recognition of a specific type of cells.

Full text of DOI:10.1021/acs.analchem.8b04784

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Article
Silica Nanoparticles with Up-conversion Fluorescence Based on Triplet-
Triplet Annihilation Mechanism for Specific Recognition of Apoptosis Cells
Yujing Zuo, Minggang Tian, Jie Sun, Tingxin Yang, Yu Zhang, and Weiying Lin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04784 • Publication Date (Web): 26 Nov 2018
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Page 1 of 10
Analytical Chemistry
Silica Nanoparticles with Up-conversion Fluorescence Based on
Triplet-Triplet Annihilation Mechanism for Specific Recognition of
Apoptosis Cells
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Yujing Zuo ‡, Minggang Tian ‡, Jie Sun, Tingxin Yang, Yu Zhang, and Weiying Lin *
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Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials
Science and Engineering, University of Jinan, Shandong 250022, P.R. China
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Email: weiyinglin2013@163.com
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ABSTRACT: Discriminating of live and apoptotic cells is a crucial task in the research of pharmacology, biology, pathology, and
medicine science. Recently, up conversion (UC) luminescent materials have appealed much attention due to their unique ability to
convert low energy excitation photons to high energy ones. However, UC fluorescence has not been employed in the field of
discriminating of live and apoptotic cells. We presented a facile, and cost-less Stöber method to fabricate robust silica nanoparticles
(SiO2 UCNPs) exhibiting several merits, such as narrow size distribution, and UC luminescence. SiO2 UCNPs could discriminate
live and apoptosis cells by taking advantage of the unique surface property of SiO2 UCNPs for the first time. This work is also the
first demonstration of the use of single photon excited UC fluorescence derived from nanoparticles for biological recognition of a
specific type of cells.
1. Introduction
Among the bioimaging categories, discriminating
of live and apoptotic cells is a crucial task in the
research of pharmacology, biology, pathology, and
medicine science.20-22Apoptosis is the programmed cell
death which occurs under physiological conditions and
immune reactions to maintain the homeostasis of cell
population in tissues. Abnormal levels of apoptosis can
lead to neurodegenerative diseases, cancer, and other
pathological conditions.23 As a feedback to external
environment or drug treatment, monitoring the cell
vitality is of vital significance.24 Despite several
examples of in vitro imaging with TTA-emission, to
date, none of them have been used for the imaging of
apoptotic cells. Taking advantage of DIBP-Py UC
system, our first goal was to establish a theoretically
feasible TTA-UC system that can be operated as a UC
imaging agent for imaging applications. However, the
complicated physiological conditions are likely to
impact on the TTA-emission. The presence of reactive
oxygen species (ROS) within the cells may quench the
TTA emitting, thus; for imaging applications under
complicated biological conditions, more stable matrix
was required. Besides, for the enhanced cellular
permeability of the apoptotic cells, it is tough for small
molecular dyes to selectively penetrate to certain types
of cells. Silica nanoparticles have easily tunable size
with good cyto-compatibility.25, 26 More importantly,
the relative stable Si-O-Si bridged structure may render
additional environment protection for UC dyes. To
overwhelm those difficulties, we have developed a
novel loading method based on silica nanoparticles,
which may lead to the formation of stable dyes
contained nanoparticles retaining excellent fluorescent
properties compared with the previously reported
TTA-UC systems.
Luminescent materials are applied in many devices
of importance such as tunable lasers, displays and
amplifiers for optical communication.1-5 In recent
years, up conversion (UC) luminescent materials have
appealed much attention due to their unique ability to
convert low energy excitation photons to high energy
ones.6-8 Much previous research has been focused on
lanthanide (Ln)-based upconversion systems with
lanthanide ions as emission centers and the two-photon
absorption upconversion systems.8-10 Lu and coworkers
reported a series of upconversion nanocrystals with
Tm3+ doped showed separate lifetime identities.
Furthermore, these nanocrystals were used to prove the
viability of lifetime encoding.11 In our previous work, a
number of two photon absorptive molecules or
materials have been designed.12-14 The most
characteristic property of the UC luminescent materials
is the wavelength conversion ability that can convert
short wavelength excitations into long wavelength
emissions. Therefore, UC materials produce negligible
auto-fluorescence and meanwhile provide low noise,
which make them suitable for bioimaging.15
However, those methods suffer from several
fundamental disadvantages. For example, high-power
mode locked laser is needed to excite two-photon
fluorescence which can bring large damage to
biosample and limit its use in biosystems. Recently,
upconversion based on triplet-triplet annihilation
(TTA) mechanism is appealing for its facile tunable
excitation or emission wavelength, high quantum yield
and low excitation power density.16-18 Compared with
the above mentioned upconversion systems, TTA-UC
ones exhibited several outstanding advantages. The
excitation wavelength of the TTA-UC process can be
gradually tuned from visible to NIR regions by using
appropriate sensitizers.19 Besides, UC process could
achieve under relative low spectral power density;
thus, TTA-based luminescent materials also attracted
attention in bioimaging.20
In this report, we have developed a strategy of
loading both sensitizer and annihilator into one silica
nanoparticle (Scheme 1) to build TTA UC SiO2
nanoparticle (denoted as SiO2 UCNPs). SiO2 UCNPs
presented blue upconversion luminescence (λem = 460
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nm). It was worth noting that SiO2 UCNPs were Under the protection of nitrogen atmosphere, 533
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successfully used for discriminating live and apoptotic
cells with upconversion luminescence under low
power density excitation at 514 nm for the first time.
The ability of SiO2 UCNPs to selectively target
apoptotic HeLa cells, while not penetrate into lived
ones, can be exploited for the specific labeling of
apoptotic cells. Compared with the traditional method
for the detection of apoptosis cells, SiO2 UCNPs
exhibited several outstanding advantages. First is the
low toxicity towards lived cells. Secondly, the
synthesis method is very convenient and cost-less.
Moreover, UC process could achieve under relative
low spectral power density; thus, the novel method we
designed is quite attractive.
mg (4.0 mmol) of benzaldehyde was added into a
round-bottom flask filled with 30 mL dichloromethane.
0.84 mL (8.4 mmol) of 2,4-dimethylpyrrole was then
added to the above system. The mixture was stirred at
room temperature for 24
h
to consume the
benzaldehyde. After that, 0.91 g (4.0 mmol) 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone was solved in
30 mL dichloromethane and added to the above system
dropwise. The mixture was stirred for another 6 h
before the addition of 18 mL triethylamine and 18 mL
BF3•OEt2. After the system was stirred for additional
12 h, the reaction was accomplished. The mixture was
poured into ice water and extracted with
dichloromethane three times. The organic layer was
combined together, dried with anhydrous MgSO4, and
concentrated under reduced pressure. The final product
was purified and obtained by column chromatography
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1
as yellow-red powder (yield, 40 %). H NMR (400
MHz, Chloroform-d) δ = 7.51 (qd, J = 4.9, 1.8 Hz,
3H), 7.37 – 7.26 (m, 2H), 6.00 (s, 2H), 2.58 (s, 6H),
1.39 (s, 6H).
2.4 Synthesis of 2,6-diiodo-1,3,5,7-tetramethyl-8-
phenyl-4,4-difluoroboradiazaindacene (DIBP)
The synthesis route for DIBP was illustrated in
Scheme S1. To a round-bottom flask with 30 mL
dichloromethane, 100 mg (0.31 mmol) of compound 1
was added. The mixture was stirred for 5 min, and 140
mg (0.62 mmol) N-Iodosuccinimide (NIS) in 10 mL
dichloromethane was added dropwise into the above
system. The reaction can be finished at room
temperature for 6 h, and after that the reaction solution
was concentrated under reduced pressure. The purified
product was obtained via column chromatography as
red powder (yield, 88%). 1H NMR (400 MHz,
Chloroform-d) δ 7.57 – 7.52 (m, 3H), 7.28 – 7.25 (m,
2H), 2.67 (d, 6H), 1.40 (s, 6H). HR MS, calcd
([C19H17BF2I2N2]-) m/z = 575.9542, found m/z =
575.9547.
Scheme 1. Illustration for the fabrication of silica
nanoparticles (SiO2 UCNPs).
2. Experimental Section
2.1 Materials
Tetraethyl orthosilicate (TEOS) was obtained as a
commercial product and used directly. Benzaldehyde
(98 %), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(98 %), and perylene (98 %) were purchased from
Energy Chemical Co., Ltd. and used as received.
Dichloromethane and ethanol were purchased from
Tianjin Fuyu Chemical Co., Ltd. and distilled over
sodium before use.
2.5 Preparation of NP0 solution
2.2 Characterization and measurements
Py (1.009 mg) was dissolved in 4 mL THF to get
Py solution (1 mM), and DIBP (2.303 mg) was
dissolved in 4 mL THF to obtain DIBP solution (1
mM). Then 0.8 mL Py solution and 0.2 mL DIBP
solution were mixed to obtain 1 mL NP0 solution.
Nuclear magnetic resonance (1H NMR, 13C NMR)
spectra were recorded on a Bruker AVANCE 400
spectrometer using CDCl3 as the solvent and with
tetramethylsilane as an interior label. Scanning
electronic microscopy (SEM) images were obtained
using Hitachi S-4800. NPs were coated with a thin
layer of gold before the investigation. The
luminescence (excitation and emission) spectra of the
samples were determined with a Hitachi F-4500
2.6 Fabricate of silica nanoparticles (SiO2 UCNPs)
To take SiO2 UCNP3 as an example, 5 mL of
TEOS and 1 mL of NP0 were dissolved in 5 mL
anhydrous ethanol at room temperature to get solution
1. Then, 5 mL of ammonia and 5 mL deionized water
were added into 75 mL anhydrous ethanol. The
mixture was stirred at room temperature for 0.5 h.
Then, Solution 1 was then added dropwise into the
mixture, and stirred at room temperature for 12 h.
Finally, the mixed solution was centrifuged (8000
r/min, 10 min) with a centrifuge. The light pink
fluorescence
spectrophotometer
using
a
monochromated Xe lamp as an excitation source. The
excitation and emission slits measured 5 nm and 5 nm,
respectively. The fluorescence imaging of cells was
performed using a Nikon A1MP confocal microscope.
2.3 Synthesis of 1,3,5,7-tetramethyl-8-phenyl-4,4-
difluoroboradiazaindacene (1)
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powder (SiO2 UCNP3) was obtained after dried at For imaging, at first, 20 mg of SiO2 UCNPs were
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room temperature.
suspended in 5 mL of ethanol. HeLa cells were
cultured in 1mL of culture medium with 20 μL of NPs
turbid liquid added for 30 min. Then, the culture
medium was removed, and the HeLa cells were
washed with PBS for three times. HeLa cells were
imaged immediately after washing.
2.7 Measurement method of the loading quantity of Py
and DIBP
The loading quantity of the dyes were mesured
according to the Lambert beer's law and were listed in
Table 1. To take SiO2 UCNP3 as an example, 5 mL of
TEOS and 1 mL of NP0 were dissolved in 5 mL
anhydrous ethanol at room temperature to get solution
1. Then, 5 mL of ammonia and 5 mL deionized water
were added into 75 mL anhydrous ethanol. The
mixture was stirred at room temperature for 0.5 h.
Then, solution 1 was then added dropwise into the
mixture, and stirred at room temperature for 1 min to
obtain a solution before sol-gel formation. The
absorption of the solution was then tested by using of a
Ultraviolet Spectrometer (UVS). The result was shown
in Figure S9.The absorption of Py was defined as A0py,
while the absorption of DIBP was defined as A0dibp.
Then the solution was stirred for another 12 h. Finally,
the mixed solution was centrifuged (8000 r/min, 10
min) with a centrifuge. The absorption of the
supernatant after sol gel process was then tested. The
result was illustrated in Figure S10. The absorption of
Py was defined as A1py, while the absorption of DIBP
was defined as A1dibp. The loading quantity of Py was
defined as Qpy and was calculated by the followed
equation:
2.9 H2O2 Pretreatment
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Lived HeLa cells were treated with H2O2 to
obtained apoptosis cells. Stock solutions of 5 M H2O2
was first prepared in PBS buffer solutions. 1μL of the
stock solutions was added into 1 mL of culture
medium to obtain the culture medium with 5mM of
H2O2. The culture medium with H2O2 was used to
incubate live HeLa cells for 4 h. Then, the culture
medium was removed, and the cells were washed with
PBS buffer solutions for three times. 20 μL of SiO2
UCNP1 in ethanol solution prepared in section 2.8 was
then used to stain these cells for another 30 min. After
rinsing with PBS three times, cells were imaged
immediately.
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2.10 Fluorescent Imaging Methods
Confocal fluorescence images were obtained with a
Nikon A1R confocal laser scanning microscope. In up
conversion experiments, excitation wavelengths were
514 nm from a Ar+ ion laser laser source, and the
incident power on samples was ∼1 mW. A barrier filter
of 510-520 nm was used to block the incident light. The
down conversion (DC) images were taken with 405 nm
Ar+ ion laser.
푄푝푦 = 퐷푝푦 ∗ (퐴0
)
푝푦 ― 퐴1푝푦
(1)
퐴0푝푦
The loading quantity of DIBP was defined as Qdibp and
was calculated by the similar equation:
3. Results and discussion
푑푖푏푝 ― 퐴1푑푖푏푝
푄푑푖푏푝 = 퐷푑푖푏푝 ∗ (퐴0
)
For design of an UC luminescent materials, to find
a proper UC luminescent molecular couple is of the
primary concern. In this work, an organic triplet
sensitizer, 2,6-diiodo-1,3,5,7-tetramethyl-8-phenyl-4,4-
difluoroboradiazaindacene (DIBP) was chosen due to
the unique advantages such as apparent absorption of
visible light, weak non-radiative decay of the excited
state, and excellent light stability. Meanwhile, Py was
used as the emitter to achieve TTA UC process.
However, the TTA mechanism between DIBP and Py
were easily quenched by ROS. Loading into the silica
nanoparticles may render protection for dyes. Locked
dyes into the Si-O-Si networks reduced the distance
between DIBP and Py, which facilitated their energy
transfer; therefore, silica nanoparticles were used to
load DIBP and Py to increase the UC efficiency, and
to realize the selectivity towards apoptosis cells. The
size of SiO2 UCNPs is facilely tuned, and the
hydrophilic property of their surface makes the SiO2
UCNPs (SiO2-based up conversion nanoparticles)
impermeable to intact plasma membrane of live cells.
In comparison, plasma membranes of apoptosis cells
were destructed, which allowed the entry of SiO2
UCNPs with proper size.
(2)
퐴0푑푖푏푝
Table 1. Various ratios of NPs complexes.
Py
(10-
4mmol
)
DIBP
(10-
Yield
(%)
Molar
ratios
Py:
NP0
TEOS
(g)
4mmol)
(mL)
DIBP
Calculated
4:1
4:1
4:1
5.00
5.00
5.00
0.3
0.5
1.0
2.06
3.67
4.44
0.63
0.94
1.20
84
87
79
NP1
NP2
NP3
2.8 Cell culture and staining methods
HeLa cells were cultured with DMEM medium
and incubated at 37 °C in air atmosphere (5% CO2).
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Stöber process was taken as the most efficient particle size to other UCNPs. As can be found from
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and simplest way to prepare monodisperse silica
nanoparticles for the normal reactants and the
regulable reaction condition, which was easy to be
carried out. The Stöber approach also allows the
preparation of siloxane-based materials with small
organic molecules doped by the polymerization of
silica
Figure 1, SiO2 UCNPs were assembled into regular
nanoparticles whose dimensions were about 150 nm.
The XRD diffractogram of SiO2 UCNP1 was shown in
Figure 1(e). SiO2 UCNP1 were amorphous at the
range of 10° to 70°. Broad peaks were observed which
were correspond to the existence of a short-range order
in the nanoparticles. The broad peaks centered on 23°
for SiO2 UCNPs. The characteristic peaks in the XRD
patterns were attributed to the coherent diffraction of
the Si-O skeleton. The lack of the crystalline regions in
NPs ascribed to the presence of organic small
molecules in the host inorganic framework. The results
illustrated that neither Py, nor DIBP crystallized
throughout the range of SiO2 UCNPs. Moreover,
altering the dosage of Py and DIBP did not affect the
morphology of the obtained nanoparticles. Py and
DIBP hardly impacted on the microstructure in these
hybrids. The formation of silica SiO2 UCNPs involved
alkoxides or hydrolysis at ambient condition.27
We designed blue emitting upconversion silica
nanoparticles fabricated by Stöber process (Scheme 1)
with an annihilator of Py (λem = 460 nm) and a
sensitizer of DIBP designedly imported, to further
consider if blue-emissive upconversion nanoparticles
can be used for specifically recognition of apoptosis
cells.
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（ b（
（ a（
a
controlled aggregation mechanism. At first,
subparticles were obtained through TEOS hydrolysis
catalyzed by ammonia. Then, Py and DIBP absorbed
onto the subparticles, which were further aggregated to
nanoparticles. For conjugation, that Py and DIBP
directly adsorbed onto the subparticle surfaces was
mediated mainly by physical absorption. Py and DIBP
were further fixed to the Si-O-Si networks through
aggregation process.
100 nm
1000 nm
（ c（
（ d（
1000 nm
100 nm
To evaluate the effect of the Si-O-Si network on
the energy transfer efficiency of the dye couples, a
controlled trial was performed. The emission of the
dye solution in ethanol with a mixture of Py and DIBP
(molar ratio = 4:1, concentration: Py = 400 uM,
DIBP= 100 uM) was tested by an excitation
wavelength of 405 nm. As shown in Figure S6, peaks
at 450 to 500 nm was found, which should be
attributed to the emission of Py. However, the red
emission ascribe to DIBP was not observed. We could
draw a conclusion that neglectable FRET occurred in
the dilute solutions.
（ e（
SiO2 UCNP1
Fluorescence studies of the SiO2 UCNPs were
undertaken. SiO2 UCNPs were photoexcited by 405
nm wavelength at room temperature in their solid
states. Two major peaks located at 450 to 500 nm and
550 to 600 nm were observed from the spectral. These
two emission peaks of the SiO2 UCNPs were
corresponding to the emission peak of Py and DIBP,
respectively. With the increase of the dosage of the
mixed dye solution (Py and DIBP), the fluorescent
intensity enhanced. As shown in Figure 2(b),
according to the CIE 1931 RGB system, blue
fluorescence of NPs could be easily observed by the
naked eye upon excitation with a wavelength of 405
nm. The CIE result was consistent with the inserted
photo in Figure 2(a), which demonstrated the
photoluminescence of SiO2 UCNPs under a 365 nm
UV light.
Figure 1. SEM images of SiO2 UCNPs, (a) and (b) for SiO2
UCNP1, (c) and (d) for SiO2 UCNP3, (e) XRD spectra for
SiO2 UCNP1 from 10 ° to 70 °.
As shown in Scheme 1, SiO2 nanoparticles were
prepared from Stöber process. The measured
tetraethoxysilane (TEOS), Py and DIBP were added
into the growth solution simultaneously, and then
formed uniform nanoparticles with dyes loaded. The
scanning electron microscopy (SEM) analysis of SiO2
UCNPs could render some characteristics of the
morphology. To take SiO2 UCNP1 and UCNP 3 as
example, Figure 1 demonstrated that mono-dispersed
nanoparticles were obtained. Figure 1(a) and (b)
corresponded to NP1 (adding 0.3 mL NP0), while
Figure 1(c) and (d) were obtained from SiO2 UCNP3.
The morphologies of SiO2 UCNP2 were illustrated in
Figure S1, which showed similar morphology and
The FRET mechanism was illustrated in Figure
3(a). Py absorbed the energy when excited by a
wavelength of 405 nm and then transferred to DIBP.
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The FRET efficiency of NPs was illustrated in Figure
3(c), and the efficiencies ranged from 91 % to 94 %,
which indicated that efficient intermolecular FRET
existed in the SiO2 UCNPs. The distance between
DIBP and the Py moiety was reduced due to the
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Figure 2. (a) solid-state photoluminescence spectra of SiO2 UCNPs excited by 405 nm wavelength, the inserted
picture: the photoluminescence images of SiO2 UCNP1 excited by 365 nm UV light, (b) the photograph of the CIE
chromaticity diagrams for SiO2 UCNP1 excited by 405 nm wavelength.
6
푅6
0 퐸 ― 푅60
(3)
r =
Where
E
represented the energy transfer
efficiency, while R0 was the Förster distance. the
Förster distance in Å was calculated by28:
1
푅0 = 0.211 휅 푛 푄퐷퐽(휆 ) 6(푖푛 Å)
2
―4
(
)
(4)
Where κ2 was always assumed to be equal to 2/3,
n was the refractive index of SiO2 and equal to 1.458,
QD was the quantum yield of Py in the absence of the
acceptor DIBP. J(λ) was the overlap integral which
indicated the extent of fluorescence spectral overlap
between the acceptor absorption (DIBP) and the donor
emission (Py), which was obtained from28:
∫∞퐹퐷(휆)휀퐴(휆)휆4푑휆
0
J(λ) = ∫∞퐹퐷(휆)휀퐴(휆)휆4푑휆 =
Figure 3. (a). Simplified mechanism explaining the
FRET down conversion emission, (b). the calculated
distances between DIBP and the Py moiety of SiO2
UCNPs, (c). the FRET efficiency between DIBP and
the Py moiety of SiO2 UCNPs.
∫∞퐹퐷(휆)푑휆
0
0
(5)
formation of SiO2 UCNPs; therefore, the distance
requirement for FRET could be satisfied. FRET
mechanism was then used to measure the distance
between Py and DIBP. FRET was well understood by
considering a single donor and acceptor separated by a
distance (r). The distance r was given by28:
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410-700 nm; (f) λex = 514 nm, λem = 410-490 nm; (g)
λex = 514 nm, λem = 520-700 nm.
1
2
The fluorescence of SiO2 UCNPs in their solid powder
state were studied by using of a Laser Scanning
Confocal Microscopy (LSCM). Green excitation light
(λexc=514 nm) with relative low optical power (about 1
mW in LSCM) generated TTA-UC fluorescence. Real-
color images at full spectra were depicted in Figure
4(a). Bright blue and red emissions were captured at
450 and 560 nm, which corresponded to the
upconversion emission of Py and the enhanced down
conversion emission of DIBP, respectively. As shown
in Figure 4(d), the in situ emission spectra of SiO2
UCNP3 clearly revealed the upconverted blue
emission of perylene at 400 to 500 nm and the down
conversion emission of DIBP beyond 520 nm. The
nanoparticle base rendered a benign medium for
intermolecular energy transfer. The mechanism for the
emission of SiO2 UCNPs was illustrated in Figure
4(e). The upconversion process was known as the
triplet–triplet annihilation (TTA) upconversion, which
took advantages of the triplet state of dyes as the
intermediate excited state. Triplet sensitizer (DIBP)
and acceptor (Py) were the two components of TTA
upconversion. The excitation energy was derived from
DIBP, and the energy was transferred to triplet
acceptor via triplet-triplet energy transfer (TTET).
DIBP absorbed energy from the excitation light (514
nm) and transferred it to Py, then brought an excited
triplet state (3T1). When DIBP and Py at the excited
states contacted within the NP systems, the triplet–
triplet annihilation drove Py to the excited singlet
states (1S1), and resulting in efficient emission of Py.
Finally, the fluorescence of Py was observed. As a
result, SiO2 UCNPs were ideal materials for
investigating the effects of FRET and TTA
upconversion within the intermolecular forces driving
adjacent photosensitizer/triplet acceptors.
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Figure 4. Real-color emission images of SiO2 UCNP2
powder, (a) full spectra, (b) λem = 450 nm, (c) λem
560 nm, (d) the corresponding in situ emission spectra
(a), λex = 514 nm; bar = 10 μm. (e) simplified
mechanism explaining the TTA upconversion
emission.
=
Where FD(λ) is the corrected fluorescence
intensity of Py at the wavelength range from λ to λ +
Δλ with the total intensity. εA(λ) was the extinction
coefficient of DIBP at λ. The distances (r) were
calculated and listed in Figure 3(b). The distance of
SiO2 UCNP1 was about 23.5 Å, while for SiO2
UCNP2 was 22.0 Å, for SiO2 UCNP3 was about 24.1
Å. The distances meet the requirement of the
occurrence of FRET effect. Due to the fantastic FRET
effect occurred in SiO2 UCNPs, we anticipated that
TTA UC may take place.
As illustrated in Figure 5(a), the photosensitizer
DIBP and the acceptor Py were initially utilized to
incubate live HeLa cells, without the support of SiO2
UCNPs. Considering the highly hydrophobic
properties of DIBP and Py, we expected that the two
fluorogens could be enriched in lipid droplets.
Indicated by the real-color image in Figure 5(b) and
in-situ emission spectra in Figure 5(d), both DIBP and
Py can target lipid droplets in live cells, and with the
excitation of 405 nm, dual emissions were obtained
peaked at 450 nm and 560 nm corresponding to Py and
DIBP, respectively [Figure 5(c)]. Since DIBP cannot
be excited by 405 nm, the fluorescence peaked at 560
nm was generated via the FRET process, indicating the
short contact of Py and DIBP in lipid droplets. HeLa
cells were then excited with 514 nm and the
fluorescence signals were captured in the wavelength
range of 410-700 nm, to check the UC behavior.
Shown in Figure 5(e), (f), and (g), with the excitation
of 514 nm, HeLa cells displayed intense DC emission
peaked at 560 nm, while showed extremely weak UC
fluorescence in the wavelength range of 410-490 nm.
Figure 5. (a) Scheme of the photosensitizer DIBP and
the acceptor Py were initially utilized to incubate live
HeLa cells, without the support of SiO2 UCNPs, and
the real-color fluorescent images (b, d, f, g) and the in-
situ emission spectra (c, e) of HeLa cells incubated
with 2 μM DIBP and 10 μM Py for 30 min. (b,c) λex =
405 nm, λem = 410-700 nm; (d,e) λex = 514 nm, λem
=
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The complex constituents such as ROS in lipid
droplets may be responsible for the quenching of UC
process.
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Afterwards, the bioimaging applications of the
SiO2 UCNPs containing Py and DIBP were
investigated. Due to the highly hydrophilic nature of
the silica SiO2 UCNPs and their sizes in nanometer
scale, the SiO2 UCNPs display poor permeability to
intact plasma membrane and cannot stain live cells.
Since apoptosis cells have destructive cell membranes,
the SiO2 UCNPs may be able to discriminate apoptosis
cells. However, it is widely accepted that silica
nanoparticles were easily uptaken by cells. Some
previous reports showed that those SiO2 nanoparticles
always exhibited smaller diameter. Moreover, the
surfaces of those SiO2 nanoparticles were modified by
some specific functional groups. For example, the SiO2
nanoparticles prepared by Xie’s group29 exhibited
smaller diameter (about 50 to 70 nm), which facilitated
their uptaken by cells. Moreover, the SiO2
nanoparticles
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Figure 6. The DIC (a), fluorescent images (b, c), and in-situ emission spectra (d) of apoptotic HeLa cells incubated
with the SiO2 UCNPs and PI for 30 min; (e) fluorescent images of live HeLa cells incubated with the SiO2 UCNPs
for 30 min; the real-color fluorescent image (f) and in-situ emission spectra (g) of HeLa cells pretreated with 8 mM
hydrogen peroxide for 2 h and then incubated with the SiO2 UCNPs for 30 min. (b) λex = 514 nm, λem = 445-455
nm; (c) λex = 514 nm, λem = 615-625 nm; (e, f, g) λex = 514 nm, λem = 400-700 nm. Bar = 10 μm.
prepared by Xie’s group has been modified by PEG,
which could enhance the lipotropy of the SiO2
nanoparticles. Therefore, the SiO2 nanoparticles
synthesized by Xie et. al could penetrate the living cell
membranes more easily.
HeLa cells were thus co-stained with the SiO2
UCNPs and propidium iodide (PI), a commercialized
reagent selectively staining apoptosis cells. In Figure
6, only two cells showed intense fluorescence peaked
at 460 nm, 540 nm, and 620 nm, corresponding to Py,
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DIBP, and PI, respectively. The fluorescence from PI
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demonstrates the apoptosis state of the two cells. In
order to understand the imaging property of SiO2
UCNPs in live cells, cell images were obtained with
SiO2 UCNP1 incubation. As shown in Figure 6(e),
after the incubation with SiO2 UCNP1 for 20 min, no
emission of lived HeLa cells was observed with the
excitation of 514 nm. The result indicated that SiO2
UCNP1 could not penetrate intact cell membrane in
live cells. The UC fluorescence could only be detected
in the two cells, indicating that the SiO2 UCNPs can
exclusively label apoptosis cells.
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Synthesis route of DIBP, H-NMR, 13C-NMR of DIBP, and the
absorption or the emission spectra of the SiO2 UCNPs.
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AUTHOR INFORMATION
Corresponding Author
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* Weiying Lin
Weiyinglin2013@163.com
Hydrogen peroxide is a byproduct of the aerobic
respiration chain, and excess amount of hydrogen
peroxide can in-return inhibit the respiration reaction
and lead to cell apoptosis. To confirm the selectivity to
apoptosis cells and explore the potential application of
the SiO2 UCNPs, HeLa cells were pretreated with
hydrogen peroxide and then incubated with the SiO2
UCNPs. In Figure 6(f), the hydrogen peroxide-
induced apoptosis cells could be stained with the SiO2
UCNPs to display intense fluorescence. The in-situ
emission spectra illustrate that in apoptosis cells the
SiO2 UCNPs can emit both DC and UC fluorescence
peaked at 460 nm and 540 nm, respectively. It should
be noted that with the support of SiO2 UCNPs, strong
UC fluorescence could be detected in the cells.
Compared with the results in Figure 5, the SiO2
UCNPs should have a protection effects to the UC
process against the complex components in
physiological conditions. Moreover, Py and DIBP
were always trapped in the SiO2 UCNPs during the
staining process, and the trapped effect can largely
increase the short contacts between the two kinds of
molecules, which is also favorable to the UC process.
These results further confirmed that the SiO2 UCNPs
could selectively light up apoptosis cells with UC and
DC fluorescence, and also demonstrated the potential
of the SiO2 UCNPs in the investigation of cell
apoptosis.
Author Contributions
All authors have given approval to the final version of the
manuscript. / ‡Yujing Zuo and Minggang Tian contributed
equally.
ACKNOWLEDGMENT
This work was financially supported by NSFC (21472067,
21672083, 21877048), Natural Science Foundation of Shandong
Province (ZR2018BB022, ZR2018BB058), Taishan Scholar
Foundation (TS201511041), and the startup fund of University of
Jinan (309-10004, 1009428).
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