Dual-Emitting Fluorescent Metal-Organic Framework Nanocomposites as a Broad-Range pH Sensor for Fluorescence Imaging

Article abstract of DOI:10.1021/acs.analchem.8b01455

pH plays an important role in understanding physiological/pathologic processes, and abnormal pH is a symbol of many common diseases such as cancer, stroke, and Alzheimer's disease. In this work, an effective dual-emission fluorescent metal-organic framework nanocomposite probe (denoted as RB-PCN) has been constructed for sensitive and broad-range detection of pH. RB-PCN was prepared by encapsulating the DBI-PEG-NH₂-functionalized Fe₃O₄ into Zr-MOFs and then further reacting it with rhodamine B isothiocyanates (RBITC). In RB-PCN, RBITC is capable of sensing changes in pH in acidic solutions. Zr-MOFs not only enrich the target analyte but also exhibit a fluorescence response to pH changes in alkaline solutions. Based on the above structural and compositional features, RB-PCN could detect a wide range of pH changes. Importantly, such a nanoprobe could "see" the intracellular pH changes by fluorescence confocal imaging as well as "measure" the wider range of pH in actual samples by fluorescence spectroscopy. To the best of our knowledge, this is the first time a MOF-based dual-emitting fluorescent nanoprobe has been used for a wide range of pH detection.

Full text of DOI:10.1021/acs.analchem.8b01455

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Article
Dual-Emitting Fluorescent Metal-Organic Framework Nanocomposites
as a Broad-Range pH sensor for Fluorescence Imaging
Haiyong Chen, Jing Wang, Duoliang Shan, Jing Chen, Shouting Zhang, and Xiaoquan Lu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01455 • Publication Date (Web): 04 May 2018
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Dual-Emitting Fluorescent Metal-Organic
Framework Nanocomposites as a Broad-Range pH
sensor for Fluorescence Imaging
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Haiyong Chena, Jing Wanga, Duoliang Shana, Jing Chena, Shouting Zhanga,b, Xiaoquan Lua, b*
a Key Laboratory of Bioelectrochemistry and Environmental Analysis of Gansu Province, College
of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070, P. R.
China.
b
Tianjin Key Laboratory of Molecular Optoelectronic, Department of Chemistry, Tianjin
University, Tianjin, 300072, P. R. China.
*Corresponding author: Xiaoquan Lu
E-mail: luxq@tju.edu.cn; luxq@nwnu.edu.cn
Tel: +86 931 7971276/Fax: +86 931 797 1276
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ABSTRACT
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pH plays an important role in understanding physiological/pathologic
processes and abnormal pH is a symbol of many common diseases such
as cancer, stroke, and Alzheimer’s disease. In this work, an effective
dual-emission fluorescent metal-organic framework nanocomposites
probe (denoted as RB-PCN) has been constructed for sensitive and broad
range detection of pH. RB-PCN was prepared by encapsulating the
DBI-PEG-NH2-functionalized Fe3O4 into the Zr-MOFs and then further
reacted with rhodamine B isothiocyanates (RBITC). In the RB-PCN,
RBITC is capable of sensing changes of pH in the acidic solutions.
Zr-MOFs not only enrich the target analyte, but also exhibit a
fluorescence response to pH changes in alkaline solutions. Based on
above structural and compositional features, RB-PCN could be detected a
wide range of pH changes. Importantly, such nanoprobe could “see” the
intracellular pH changes by fluorescence confocal imaging as well as
“measure” the wider range of pH in actual samples by fluorescence
spectra. To the best of our knowledge, this is the first time using MOF
based dual-emitting fluorescent nanoprobe for wide range of pH
detection.
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INTRODUCTION
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pH plays an irreplaceable regulatory role in maintaining the stability,
the normal morphology and function of cells and organisms.1-4 Abnormal
intracellular pH can affect the cell differentiation and apoptosis,5,6 muscle
contraction,7 ion transport and internal environment stability,8-10 causing a
series of adverse reactions; even some of the disease also derive from the
pH anomaly.11,12 Therefore, the study of pH changes in living cells is
important for revealing the mechanism of cell metabolism and different
physiological and pathological processes and intracellular drug delivery
process.13,14 Fluorescence spectroscopy15-17 is under a unique advantage in
detecting pH changes in spatiotemporal distribution, as compared to pH
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measurement methods such as proton permeable microelectrode,18 31
spectroscopy19 spectroscopy.20
Also,
P
NMR
and
absorption
fluorescence-based techniques21 have the advantages of simple operation,
fast response, high signal-to-noise ratio, high sensitivity and in most cases
without damage to cells. Generally speaking, two kinds of fluorescent pH
probes have been exploited, one that is cytoplasmic probes which work in
the pH range of 6.8 - 7.4,22,23 and the other is probes for acidic organelles
such as lysosome which work in the pH range of 4.5 - 6.0.24,25 However,
new probes for use in broad-range pH sensors that are highly sensitive
and biocompatible are still highly required.
Metal-organic frameworks (MOFs) are kind of porous materials
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consisting of metal ions or clusters and organic ligands.26,27 They are
considered to be one of the foremost candidates for sensing materials
because of their high specific surface area, multifunctional framework
composition and exposed active sites.28 It is useful to noting that
luminescent metal-organic frameworks (LMOFs) have been successfully
applied to analytical chemistry.29-31 As far as we know, there are little pH
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sensors based on MOFs have been reported: Rocha et al. depicted an
Eu-based MOFs which shows linear pH-dependence across the range
33
from 5 - 7.5. Zhou et al.
reported that the PCN-224 based on the
organic ligand porphyrin was used as pH-dependent fluorescence probe
in the range from 1 to 11. However, the PCN-224 makes a significant
fluorescence change only under alkaline conditions. RBITC34,35 is a
commonly used pH probe. However, it has only a significant fluorescence
response to the acidic medium. We assume that integrating MOFs and
RBITC in one system will broaden the scope of the pH.
In this work, for the first time, we developed a dual-emission
fluorescent MOFs-based probe for real-time monitoring pH changes over
a
wide range (Scheme
1
and Figure S1). First, the
DBI-PEG-NH2-functionalized Fe3O4 NPs were encapsulated into the
ocket-channel frameworks (PCN-224), and then RBITC was modified
onto the surface of encapsulated Fe3O4 NPs to obtain target probe
(denoted as RB-PCN). The resulting core-shell-like RB-PCN exhibits a
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single-excitation, dual-emission fluorescence properties, one from
organic ligand porphyrin of PCN and the other from the RBITC.
Therefore, RB-PCN probe for pH sensing has the following advantages:
(1) The RB-PCN probe can be present in the most wide pH solution and
exhibit excellent chemical stability in solutions with the pH range from 1
to 11.36 (2) The porous structure and open channels of PCN allow H+ to
rapidly and freely diffuse to provide a fast response.37 (3) The covalent
conjugation of RBITC on the surface of Fe3O4 nanoparticles encapsulated
in PMOF prevents dye leaching, thereby eliminating background
disturbances caused by dye release during pH testing. As expected,
RB-PCN has excellent photostability and is capable of imaging in cells.
RB-PCN can be susceptible to pH changes both in the range 1.7 - 7.0 and
7.0 - 11.3, accompanying by different fluorescence changes. Moreover,
the resultant RB-PCN can be used for seeing pH changes in living cells as
well as measuring pH in real samples.
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EXPERIMENTAL SECTION
Materials and Reagents. Pyrrole, methyl p-formylbenzoate, Iron (III)
2,4-pentanedionate,
1-Amino-9-octadecene,
Dibenzyl
ether,
3,4-dihydroxybenzaldehyde, polyethene glycol (PEG) (MW=4000) were
purchased from Sigma-Aldrich and implemented without further
purification. Zirconium Oxychloride Octahydrate (ZrOCl2·8H2O) and
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Benzoic
Acid
were
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Aladdin.
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Meso-tetra-(4-carbomethoxyphenyl) porphyrin (TCPP-OMe), meso-Tetra
(4-carboxyphenyl) porphyrin (TCPP), DBI-PEG-NH2 and Fe3O4 NPs
were prepared according to the literature with minor modifications. The
solution of metal ions (Mg(NO3)2, Zn(NO3)2, KCl, CuCl2, NaCl,
Al(NO3)3, BaCl2, Ni(NO3)2, Ca(NO3)2, AgNO3; Na2SO4, NaNO3,
Na2HPO4, NaH2PO4, Na2SO3, Na2CO3, CH3COONa, NaHCO3, NaI, NaBr,
Glucose, L-cysteine, L-glutamic acid, Ascorbic Acid) were dissolved in
ultrapure water. TCPP was synthesized according to published methods
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with
minor
modifications.31-33
Fe3O4-DBI-PEG-NH2
and
Fe3O4-DBI-PEG-NH2. were synthesized according to the previous
method.38
Apparatus. Field-emission scanning electron microscope (FE-SEM,
FEI, Sirion 200) and Transmission electron microscopy (TEM, Philips EM
420 (120 kV)) were utilized to confirm the morphology of RB-PCN.
Bruker AXS D8-Advanced diffractometer with Cu Kα radiation
(λ=1.5418Å) was used to record X-ray powder diffraction (XRD) patterns
of the nanomaterials. FT-IR characterization was recorded on a Bruker
Vertex 70 FT-IR spectrometer. UV-vis absorbance measurements were
performed on Shimadzu UV-1750. Fluorescence spectroscopy was
performed on a Shimadzu RF-5301 spectrofluoro Photo-meter.
Fluorescence images of cell imaging were made using a Zeiss Leica
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inverted epifluorescence/reflectance laser scanning confocal microscope.
Synthesis of Meso-tetra-(4-carbomethoxyphenyl) porphyrin
(TCPP-OMe). Methyl p-formylbenzoate (16.5 g, 0.1 mol) was dissolved
in propionic acid (250 mL) in a 500 mL three-necked flask and heated to
140 ℃ with stirring. Pyrrole (6.7 mL, 0.1 mol) dissolved in 30 mL of
propionic acid was then added drop wise over 30 min and the solution
was refluxed for 2 h. After the reaction, 150 mL of ethanol was added to
the mixture which cooled to room temperature. Finally, the precipitates
were collected by suction-filtration and washed with methanol, ethyl
acetate and THF, respectively. After drying in an oven for 12 h, purple
solid was obtained.
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Synthesis of Meso-Tetra (4-carboxyphenyl) porphyrin (TCPP). In a
250-mL single-necked flask, the obtained TCPP-OMe (0.164 g, 0.2 mmol)
was stirred in 150 ml mixture of THF and MeOH (v: v = 2: 1), to which a
solution of 12 ml NaOH (40%) was introduced. The mixture as refluxed
for 2 h at 40 ℃. After the reaction, the solution was adjusted to pH 4 - 5
with hydrochloric acid. The solution was then extracted 2 - 3 times with a
mixed solvent of THF and CH2Cl2 (v: v = 1 : 1), and the organic phase
was distilled off to remove the solvent. Then the product was placed in an
oven at 40 ℃ for dry, and violet purple red target products was obtained.
Synthesis of Fe3O4 nanoparticles (NPs). The synthesis of Fe3O4 NPs
is based on the literature method.38 Firstly, acetylacetonate (2 mmol) was
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dissolved in a mixed solution of oleylamine (10 mL) and dibenzyl ether
(10 mL), and the reaction was carried out with nitrogen 120 ℃ for 1 h.
After heating to 300 ℃ and keeping at this temperature for 2 h, the
reaction mixture was cooled to room temperature. The Fe3O4 NPs were
precipitated by adding ethanol (40 mL) and collected by centrifugation.
The prepared Fe3O4 NPs were redispersed in n-hexane and preserved at
low temperature.
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Synthesis of Fe3O4 NPs@PCN-224.39-42 In a typical synthesis, 30 mg
ZrOCl2·8H2O, 10 mg TCPP and 300 mg benzoic acid were dissolved in 6
mL DMF and sonicated for 10 min. Then, 0.8 mg Fe3O4-DBI-PEG-NH2
was added to the above solution. The mixture was sonicated for 10 min
before heating in oven at 120 ℃ for 24 h. After cooling to room
temperature, the obtained precipitate was centrifuged (5000 rpm, 3 min)
and washed three times with DMF. Finally, by soaked with acetone, the
solid was centrifuged and dried in a vacuum oven for further use.
Post-synthetic loading of rhodamineisothiocyanate (RB-PCN). 1
mL anhydrous ethanol solution of RBITC (1 mg/mL) was added to 2 mL
anhydrous ethanol solution with 4 mg Fe3O4 NPs@PCN-224. After the
mixture stirred at room temperature in the dark for 24 h to afford
RB-PCN, the product was collected by centrifugation at 5000 rpm and
washed with ethanol.
Spectral Measurements. The stock solution of RB-PCN was prepared
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with ultrapure water. In the pH titration experiment, the stock solution
was diluted to 100 mg/L with ultrapure water, and the pH of the solution
required was adjusted by adding a certain amount of NaOH or HC1. Then,
the resulting solution was transferred to a quartz cell to acquire
fluorescence spectrum with λex = 520 nm.
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In the study of the reversibility of RB-PCN, the pH of the RB-PCN
(100 mg/L) solution was converted between 4.0 and 11.0 with 1 M NaOH
or HCl. The interference of other ions /bio-molecules (Mg2+, Zn2+ , K+,
2-
-
-
2-
Cu2+, Na+, Al3+, Ba2+, Ni2+, Ca2+, Ag+; SO4 , H2PO4 , HCO3 , SO3 ,
2-
-
2-
HPO4 , NO3 , CO3 , Cl-, AcO-, Br-, I-, S2-; Glucose, L-cysteine,
L-glutamic acid, Ascorbic acid) to the fluorescence intensity of RB-PCN
was detected at pH 6.9 and 11.0, respectively.
Cytotoxicity Assay. The evaluation about in vitro cytotoxicity of probe
RB-PCN performed by MTT assay in the HeLa cells incubated with the
probe RB-PCN. Cells were seeded into a 96-well plate (5 × 104 cells/well)
containing 10% FBS (fetal bovine serum) in DMEM (Dulbecco's
modified eagle's medium) under 5% CO2 at 37 °C for 24 h. The cells
were then incubated with RB-PCN for 24 h and 48 h at 37 ℃ under 5%
CO2, respectively, where the concentrations of RB-PCN were 20, 40, 60,
80, 100, 120 mg/L in DMEN. Cells in a culture medium without RB-PCN
were used as a control. Next, MTT (20 ꢀL/well, 5 mg/mL) was added to
each well plate and the plate was incubated at 37 ℃ and 5% CO2 for 4 h.
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After adding 100 ꢀL/well DMSO (dimethylsulfoxide), the plate of cells
was permitted to maintain 15 min at 37 °C. Finally, the optical density at
492 nm (A492) of the extraction liquid was detected using a microplate
reader.
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Flow cytometry assay. Cells were treated as described above. Before
measurement by flow cytometry, cells were scraped off gently and
collected into a clean centrifuge tube. Then, cells were centrifuged (800
rpm, room temperature, 3 min). After removing the supernatant, 1 mL
PBS was slowly added to resuspend the cell pellet. Finally, cells were
analyzed on a Beckman flow cytometer equipped with a 488 nm Ar laser,
and fluorescence was collected by PE channel.
Fluorescence confocal imaging. The HeLa cells were seeded into a
6-well plate (5 × 105 cells/well) containing 10% FBS in DMEM
(Dulbecco's modified eagle's medium) under 5% CO2 at 37 °C for 24 h.
The cells were then incubated with RB-PCN (final concentration of 40
mg/L) and the lysosome dye LysoBlue (final concentration of 1 ꢀM,
KeyGEM,
China)
or
the
cytoplasm
tracker
CMAC
(7-amino-4-chloro-O-methyl coumarin, final concentration of 1 ꢀM,
Invitrogen, United States) for 1 h under standard culture conditions. Then
the cells were washed with PBS three times and incubated with PBS
buffer solutions at pH 5.0, 7.0 or 8.0 for 20 min containing 100 ꢀM
Nigericin, 41 and observed by confocal microscopy (λex=360 nm and λem=
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460 nm for LysoBlue or CMAC; λex=520 nm and λem= 575 nm or 641 nm
for RB-PCN, Olympus, Japan).
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Analysis of real Sample. We use the Yellow River (Lanzhou, China)
and tap water as actual water samples for testing. The samples were
centrifuged at 13 000 rpm for 3 min and filtered through a membrane to
eliminate impurities, and then used to prepare solutions at different pH
values.
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RESULTS AND DISCUSSION
Characterization of RB-PCN. Figures 1a shows SEM images of
RB-PCN. It can be seen that the resultant RB-PCN still maintained
uniform cube sphere crystals with an average size of 330 nm (Figure S2),
which is maintained the morphology of PCN-224 (Figure S3).
Transmission electron microscopy (TEM) image of RB-PCN revealed
their well-defined micro-structure (Figure 1b, Figure S4). As shown in
Figure S5, it can be seen that RB-PCN contains Fe, Zr, Cl, O elements,
indicating the successful preparation of RB-PCN. According to the
literature,43 the similar powder X-ray diffraction (PXRD) curves show
that the crystalline and structural integrity of PCN-224 is not destroyed
after encapsulation of Fe3O4 nanoparticles (Figure 1c). The peaks
observed at 30.1°, 35.3°, 42.9°, 53.5°, 57.0°, and 62.5° were assigned to
(220), (311), (400), (422), (511) and (440) planes of cubic structure of
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Fe3O4 crystal (JCPDS No.75-1609). The simultaneous existence of the
characteristic peaks of Fe3O4 NPs38 and PCN-22443,44 in its PXRD pattern
indicates the successful formation of RB-PCN nanocomposites. FTIR
demonstrated that the RB-PCN was comprised of Fe3O4-DBA-PEG-NH2,
PCN-224 and RBITC (Figure S6).
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Identification performance of RB-PCN in different pH. Firstly, we
tested the UV-vis absorption spectra of RB-PCN in different pH solution.
Figure 2 shows the absorption spectra at pH 3.0, 7.0 and 10.0,
respectively. In the acidic condition, the characteristic absorption peak of
RBITC appeared at 450 nm, and this peak position appeared blue shift
and finally disappeared with the increase of pH to alkaline. When the pH
arrived to 10, the characteristic absorption peak of organic ligand
porphyrin at 420 nm was appeared. Also, we observed that RB-PCN
undergoes a slight change from pink to yellow-green with pH increasing
in aqueous solution.
Moreover, we carried out standard fluorescence pH titration analysis of
the aqueous solution of RB-PCN at a concentration of 100 mg/L. Figure
3a shows the fluorescence emission spectra under acidic conditions.
RB-PCN has weak fluorescence intensity at 575 nm at neutral solution.
With the decrease of pH value, the fluorescence intensity of RB-PCN at
575 nm gradually increased, which was result of the ring-opening effect
of RBITC34 (Scheme 2a). In the pH range of 1.7 - 7.0, the relationship
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between pH (X axis) and fluorescence intensity (Y axis) can be described
by a perfect linear regression relationship with R2 = 0.9952 (Figure 3b).
The linear regression relationship equation is as follows: pH=17.099 -
0.079y (y=Fluorescence intensity). Any sample of pH range under acidic
conditions can be calculated by this formula. We still choose 520 nm as
the excitation wavelength under alkaline conditions. Under alkaline
conditions, the fluorescence emission intensity of RB-PCN at 641nm
gradually increases with the increase of pH (Figure 3c). The enhancement
of the fluorescence intensity is due to the deprotonation of the imino
groups of the porphyrin ligand in the PCN-224 framework under alkaline
conditions31 (Scheme 2b). This is of great significance for studying the
pH change in the chemical system.
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Figure 4 indicates the reversibility of the probe in the acidic range and
the alkaline range, respectively. After four cycles, the probe still works
well, indicating that RB-PCN is a highly reversible probe for sensing pH.
At the same time, it was proved that RB-PCN had excellent stability by
detecting the change of fluorescence intensity at pH 4.0, 7.0 and 11.0
(Figure 4, Figure S7).
To further explore the performance of RB-PCN, we selected some
metal ions, anions and bio-molecules as interference objects to explore
the anti-interference ability of RB-PCN in the complex environment. The
measurement was carried out by adding separate interfering ions at a
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concentration of 100 ꢀM to the RB-PCN solution at pH 7.0 and 11.0. As
shown in Figure 5 and Figure S8, the addition of interference reagents did
not significantly affect the fluorescence intensity at 575 nm and 641 nm.
The results indicated the high selectivity and selectivity of RB-PCN to
pH.
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Cell Toxicity and Fluorescence confocal imaging. HeLa cells were
used to evaluate the toxicity of RB-PCN probe. As shown in Figure S9,
RB-PCN presented relatively low toxicity to the cells at different times.
When the incubation time of RB-PCN reached to 48 h, the cell survival
rate was higher than 70%, which indicated that RB-PCN had low toxicity
and could be employed in fluorescence imaging in vitro. To verify the
probe RB-PCN located into cells, flow cytometry analysis was performed
to observe the change of fluorescence inside cells. As shown in Figure
S10, significant enhancement of fluorescence emission was observed
after incubating with RB-PCN, suggesting the probe RB-PCN can be
used for cell imaging.
Next, we explored to use RB-PCN to sense the pH fluctuation in cells
by confocal microscopy. The HeLa cells were incubated with RB-PCN
(40 mg/L) for 1 h, and then treated by PBS at pH 5.0 (Figure 6a-e), 7.0
(Figure 6f-j), and 8.0 (Figure 6k-o) for 20 min in the presence of 100 ꢀM
Nigericin. As shown in Figure 6b, at pH 5.0 the cells mainly emitted the
strong bright yellow fluorescence. When the pH increased to 7.0, yellow
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fluorescence was dimmed out and red fluorescence enhanced. When the
pH arrived at 8.0, the cells mainly emitted the strong red fluorescence.
Moreover, both the red and the bright yellow fluorescence of RB-PCN
could overlap the fluorescence of the lysosome tracker dye LysoBlue,
indicating that RB-PCN was localized in the lysosome (Figure 6). These
results showed that RB-PCN is suitable for fluorescence imaging to
reflect lysosome pH changes in living cells.
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Further, we stained HeLa cells with RB-PCN and the cytoplasm tracker
dye CMAC, and found that RB-PCN can image in the cytoplasm (Figure
7). The changes in fluorescence at different pH values are consistent with
changes in lysosomes. In addition, the red and bright yellow fluorescence
of RB-PCN can overlap with that of the cytoplasmic tracking dye CMAC.
Simultaneously, by semi-quantifaction by Image J software revealed that
the red and yellow fluorescence intensity had turn-on and turn-off
behaviors, respectively (Figure S11). The statistical significance results
showed that RBITC channel at pH 5.0 (R=20.9 2.52) exhibited an
obvious difference from that at pH 7.0(5.21 2.01, p < 0.01). And the
PCN channel at pH 7.0 (6.67 3.15) also exhibited an obvious difference
from that at pH 8.0 (9.46 0.62, p < 0.05). In short, RB-PCN has the ideal
sensitivity for detecting cell pH.
The application of the RB-PCN in the actual water sample
detection. In order to further assess the anti-interference ability of
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fluorescent probes, we selected the Yellow River water, tap water and
deionized water to prepare the solutions with different pH values, and
evaluated the differences in the detection of different water samples. As
shown in the Figure 8, the fluorescence intensity of the solution gradually
increased with the decrease of pH value in tap water, Yellow River water
and deionized water. Moreover, the fluorescence intensity values of
RB-PCN in the three water samples were the same as those of the pH
value, and no significant difference was observed. And fluorescence
intensity was linear with pH. It is shown that the detection system is the
same as the result of deionized water detection in laboratory conditions,
and can effectively eliminate the interference of coexisting ions, organic
pollutants and so on, and prove the excellent selectivity of RB-PCN.
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Conclusion
In summary, we successfully prepared a dual-emission fluorescent
MOFs-based nanoprobe (RB-PCN) for rapid and ultrasensitive detection
of pH in abroad range from 1 - 11. This nanocomposites was synthesized
by encapsulating the DBI-PEG-NH2-functionalized Fe3O4 NPs into the
PCN-224 and then further reacted with RBITC, in which the RBITC was
modified on the surface of Fe3O4 NPs. The resulting core-shell-like
RB-PCN exhibited two emissions at 575 nm for RBITC in response to
the acidic range and 641 nm for PCN-224 in response to the alkaline
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range under the same excitation wavelength. Importantly, PCN-224 shell
as a protective layer can not only prevent the aggregation of nanoparticles,
but also enrich the target analyte, thereby amplifying the fluorescence
signal. Moreover, the resultant RB-PCN probe could “see” the pH change
in in living cells and “measure” the pH values in actual water sample.
This work opens up a new strategy to develop a dual emission
fluorescence MOFs based- probes for wide range of pH detection.
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ASSOCIATED CONTENT
Supporting Information
Synthetic route of RB-PCN and Figures S1-S11. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgments
Thanks a lot to Prof. Wujian Miao worked in the University of Southern
Mississippi, Prof. Chunyang Zhang worked in Shenzhen advanced institute of science
and technology, Prof. Baodui Wang worked in the University of Lanzhou, associate
Prof. Qilin Yu worked in Nankai University and associate Prof. Yao Meng worked in
Northwestern Polytechnical University for their kind discussion.
This work was supported by Natural Science Foundation of China (Grant Nos.
21575115, 21327005, 21705117); the Program for Chang Jiang Scholars and
Innovative Research Team, Ministry of Education, China (Grant No. IRT-16R61) and
the Program of Gansu Provincial Higher Education Research Project (Grant No.
2017-D-01).
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Scheme 1. Design Strategy and Fluorescence Changes of RB-PCN upon pH Variation.
Figure 1. (a) SEM images of RB-PCN, (b) TEM images of RB-PCN, (c) PXRD patterns of
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synthesized RB-PCN(blue), PCN-224(red), Fe3O4(black).
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Figure 2. UV-vis absorption spectra of RB-PCN in aqueous solutions with different pH values.
Figure 3. (a) Fluorescence spectra (λex = 520 nm) of the RB-PCN with 100 mg/L in acidic
aqueous solutions. (b) Linear relationship of the fluorescence intensity (I575 nm) versus pH values.
(c) Fluorescence spectra (λex = 520 nm) of the RB-PCN with 100mg/L in basic aqueous solutions.
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(d) Fluorescence intensity (I641 nm) versus pH values.
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Scheme 2. Proposed sensing mechanism of RB-PCN. (a) Open ring of rhodamine B under acidic
conditions. (b) Protonation processes of porphyrin involved in RB-PCN framework.
Figure 4. (a) The fluorescence reversibility of RB-PCN (100 mg/L) in aqueous solutions between
pH 7.0 and 4.0 at 575 nm. (b) When the pH value is 4.0 and 7.0, the fluorescence intensity of
RB-PCN changes with time. The fluorescence reversibility study (c) and the time courses of
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fluorescence intensity at 641 nm (d).
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Figure 5. (a) Relative fluorescence intensity changes at 575 nm of 100mg/L RB-PCN in the
presence of different metal ions at pH 7.0. (b) Relative fluorescence intensity changes at 641 nm
of 100 mg/L RB-PCN in the presence of different metal ions at pH 11.0. 1, blank ; 2, Mg2+; 3,
Zn2+; 4, K+; 5, Cu2+; 6, Na+; 7, Al3+; 8, Ba2+ 9, Ni2+; 10, Ca2+; 11, Ag+. λex = 520 nm. Error bars
are s.d.
Figure 6. Fluorescence images of HeLa cells after incubation with RB-PCN and LysoBlue
(lysosome dye) at different pH (5.0, 7.0 and 8.0). The cells were then observed by confocal
microscopy (λex=360 nm and λem= 460 nm for LysoBlue; λex=520 nm and λem= 575 nm or 641 nm
for RB-PCN). Merge (P+L), the merging image of PCN and LysoBlue; Merge (R+L), the merging
image of RBITC and LysoBlue.
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Figure 7. Confocal fluorescence images of HeLa cells after incubation with RB-PCN at different
pH (5.0 (a-d), 7.0 (c-h) and 8.0 (i-l)). HeLa cells were stained with the Cell Tracker Blue CMAC
(7-amino-4-chlor-omethylcoumarin). The images in the first row and second row were collected in
the ranges of 620-660 nm and 540-580 nm, respectively. The images in the fourth row were the
merged ones of PCN, RBITC and CMAC channels.
Figure 8. Fluorescence intensity of RB-PCN solution in different water samples with different
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