A Copper Nanocluster-Based Fluorescent Probe for Real-Time Imaging and Ratiometric Biosensing of Calcium Ions in Neurons

Article abstract of DOI:10.1021/acs.analchem.8b05360

Fluorescent calcium ion (Ca²⁺) sensing and imaging have become an essential technique for investigation of signaling pathways of Ca²⁺ and understanding the role of Ca²⁺ in neurodegenerative disease. Herein a copper nanocluster (CuNC)-based ratiometric fluorescent probe was developed for real-time sensing and imaging of Ca²⁺ in neurons, in which a specific Ca²⁺ ligand with two formaldehyde groups was synthesized and further conjugated with polyethylenimine (PEI) to form a new ligand molecule for the synthesis of CuNCs. Meanwhile, water-soluble Alex Fluor 660 NHS ester was immobilized onto CuNCs as a reference element. The developed ratiometric fluorescence nanoprobe demonstrated a good linearity with Ca²⁺ concentration in the range of 2-350 μM, and a detection limit down to 220 ± 11 nM was achieved. In addition, the response time of the present probe for Ca²⁺ was found to be less than 2 s with good stability and high selectivity. Taking advantage of the low cytotoxicity and good biocompatibility of the developed nanoprobe, it was discovered that the histamine-induced cytoplasmic Ca²⁺ increase in various parts of neurons was different. Moreover, it was found O₂^--induced cytoplasmic Ca²⁺ burst and O₂^--induced neuronal death possibly resulted from Ca²⁺ overload in the neurons.

Full text of DOI:10.1021/acs.analchem.8b05360

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Article
A Copper Nanocluster-Based Fluorescent Probe for Real-Time
Imaging and Ratiometric Biosensing of Calcium Ion in Neurons
Zhichao Liu, Xia Jin, Sanjun Zhang, and Yang Tian
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05360 • Publication Date (Web): 08 Jan 2019
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Analytical Chemistry
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A Copper Nanocluster-Based Fluorescent Probe for Real-Time Imag-
ing and Ratiometric Biosensing of Calcium Ion in Neurons
Zhichao Liu,† Xia Jin,† Sanjun Zhang,‡ and Yang Tian*, †, ‡
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†Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering,
East China Normal University, Dongchuan Road 500, Shanghai 200241, P. R. China
‡State Key Laboratory of Precision Spectroscopy, East China Normal University, North Zhongshan Road 3663, Shanghai
200062, P. R. China
ABSTRACT: Fluorescent calcium ions (Ca2+) sensing and imaging have become an essential technique for investigation of signaling
pathways of Ca2+and understanding the role of Ca2+ in neurodegenerative disease. Herein, a copper nanoclusters (CuNCs)-based
ratiometric fluorescent probe was developed for real-time sensing and imaging of Ca2+ in neurons, in which a specific Ca2+ ligand
with two formaldehyde groups was synthesized and further conjugated with polyethyleneimine (PEI) to form a new ligand molecular
for synthesis of CuNCs. Meanwhile, water-soluble Alex Fluor 660 NHS ester was immobilized onto CuNCs as a reference element.
The developed ratiometric fluorescence nanoprobe demonstrated a good linearity with Ca2+ concentration in the range of 2-350 μM,
and the detection limit was as achieved down to 22011 nM. In addition, the response time of the present probe for Ca2+ was found
to less than 2 s with good stability and high selectivity. Taking advantages of the developed nanoprobe including low cytotoxicity
and good biocompatibility, it was discovered that histamine-induced cytoplasmic Ca2+ increase in various parts of neurons was dif-
ferent. Moreover, it was found O2- induced cytoplasmic Ca2+ burst and O2 -induced neuronal death was possibly resulted from Ca2+
-
overload in neurons.
Calcium ion (Ca2+) as a significant intracellular signaling
molecule plays an indispensable role in signal transduction,1
memory formation,2 synaptic activity,3 gene transcription,1 and
oxidative stress process4. More importantly, dysregulation of
intracellular Ca2+ signaling has been implicated in the patho-
genesis of Alzheimer’s disease.5 Since the changes in cytosolic
Ca2+ concentration are transitory, it is important to track the dy-
namic changes of intracellular Ca2+ concentration and under-
stand the Ca2+ signal pathways in cells.
Fluorescence-based biosensors have attracted great interests
because they are simple and effective for cellular native species
sensing and imaging.6-10 Several elegant fluorescent Ca2+ probes
have been developed, such as genetically encoded fluorescent
proteins (GFP)11-13 , small organic molecule probes14-17, and up-
conversion nanoparticle-based probes18,19. Our group is very in-
terested in development of analytical methods for biosensing
and imaging of metal ions and reactive oxygen species (ROS)
in live brains, tissues, and cells. We have developed several in-
organic-organic ratiometric fluorescent probes for determina-
tion of Cu2+, Fe2+, pH and other oxidative stress-related ROS in
living cells and tissues.20-25 However, it is still a challenging
work to develop a fluorescent Ca2+ probe with high accuracy
and selectivity, as well as good biocompatibility and long-term
stability for fulfilling the requirements for selective imaging
and accurate biosensing of Ca2+ in live cells. In fact, inorganic-
organic composited probe may solve these problems well, be-
cause the designed organic molecules provide specific recogni-
tion for Ca2+ while fluorescent inorganic nanomaterials, like
metal nanoclusters and carbon dots, demonstrate low cytotoxi-
city and long-term fluorescence stability.26,27 Moreover, inner-
reference molecules can also be conjugated onto the surface of
nanomaterials to construct ratiometric probes with built-in cor-
rection, which shows high accuracy, independence of probe
concentrations, fluctuation of excitation light intensity or envi-
ronmental effects in complex samples.6,28,29 Therefore, it is
greatly desirable to develop an inorganic-organic ratiometric
fluorescent probe for Ca2+ with high selectivity, good compati-
bility and long-term stability.
In this work, a specific Ca2+ ligand (CaL) with two formalde-
hyde groups was synthesized (Scheme 1A). Then, CaL was fur-
ther attached onto polyethyleneimine (PEI) to develop a new
template molecule (PEI-CaL) by Schiff base reaction between -
CHO group of CaL and -NH2 group of PEI. PEI-CaL plays as
not only a specific recognition element for Ca2+, but also a lig-
and for synthesis of copper nanoclusters (CuNCs). Then,
CuNCs were synthesized using a template of PEI-CaL by re-
ducing Cu2+ in the presence of ascorbic acid (AA). Next, the
reference fluorescent molecule, Alexa Fluor 660 (AF660), was
designed and conjugated onto the surface of as-prepared CuNCs
to develop ratiometric fluorescent probe (CuNC@AF660) for
accurate determination of Ca2+ (Scheme 1B). The developed
CuNC@AF660 probe demonstrated two independent emission
peaks emerged at ~590 nm and ~690 nm upon excitation at 488
nm, respectively. Compared with previous reported fluorescent
Ca2+ probe, our developed ratiometric fluorescent
CuNC@AF660 probe shows excellent water solubility due to
the good water solubility of PEI-CaL. In addition, the devel-
oped CuNC@AF660 probe shows high selectivity owing to the
presence of CaL. Moreover, our developed ratiometric fluores-
cent nanoprobe shows high accuracy by using AF660 as refer-
ence signal. The emission peak at 590 nm increased with in-
creasing concentration of Ca2+ while the fluorescence intensity
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Scheme 1. (A) Synthesis routes of CaL and CaL ester. (B) Schematic illustration of working principle of CuNC@AF660 ratiometric
fluorescent probe for Ca2+ sensing.
collected with a JEM-2100F transmission electron microscope
(JEOL, Japan). The apoptosis assay was conducted at a FACS
at 690 nm kept unchanged, resulting in ratiometric determina-
tion of Ca2+ with high accuracy. The present ratiometric fluo-
rescent probe showed high selectivity against other metal ions,
amino acids, calcium-containing proteins and common ROS, as
well as long-term stability. In addition, taking advantages of
CuNC@AF660 probe including low cytotoxicity, good bio-
compatibility and rapid response dynamics, the developed flu-
orescent probe for Ca2+ was successfully applied in biosensing
and imaging of Ca2+ in neurons. Using this useful probe, it was
found that histamine-induced cytoplasmic Ca2+ increase in var-
ious parts of neurons was different. Furthermore, we can see
Calibur flow cytometry (Becton, Dickinson and Company,
U.S.A). The absorbance of cytotoxicity was recorded by a Vari-
oskan LUX multimode microplate reader (Thermo Fisher Sci-
entific, U.S.A). Fluorescence confocal imaging was performed
with a Leica TCS-SP8 confocal scanning microscope using a
63× oil objective and a numerical aperture of 1.40 (Leica, Ger-
many).
Synthesize of Ca2+ ligand (CaL). The synthetic route for CaL
was described in Scheme 1A, according to literatures with some
modification.30
that O2- induced cytoplasmic Ca2+ burst and O2 -induced neu-
-
ronal death was possibly resulted from Ca2+ overload in neurons.
Compound 1: A mixture of NaOH (4.8 g, 12 mmol), H2O (4.5
mL) and 2-nitrophenol (15.30 g, 11 mmol) in 30 mL DMF was
heated to 60 oC for 30 min with stirring. Then 1, 2-dibromoeth-
EXPERIMENTAL SECTION
o
ane (5.1 mL) was added and heated to 130 C for 3 h. After
Reagents and Chemicals. 2-nitrophenol, 1,2-dibromoethane,
carbon powder (C), ethylbromoacetate (C4H7BrO2), dimethyl
formamide (DMF) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe-
nyl-2-H-tetrazolium bromide (MTT) were purchased from
Aladdin Chemistry Co. Ltd. (Shanghai, China). Ferric trichlo-
ride (FeCl3), n-hexane, acetonitrile, methanol (CH3OH), ethyl
alcohol (EtOH), hydrazine hydrate (N2H4•H2O), sodium iodide
(NaI), methylbenzene, anhydrous sodium sulfate (Na2SO4),
phosphorus oxychloride (POCl3), sodium hydroxide (NaOH)
and sodium bicarbonate (NaHCO3) were provided by Si-
nopharm Chemical Reagent Co. Ltd. (Shanghai, China). Alexa
Fluor 660 NHS ester and Hank’s balanced salt solution (HBSS,
no calcium, no magnesium, no phenol red) was purchased from
Thermo Fisher Scientific (U.S.A). All chemicals were of ana-
lytical grade were used without further purification and modifi-
cation. All samples were prepared by deionized water purified
by Milli-Q water purification system.
cooling down to room temperature, the mixture was diluted to
100 mL with water and filtered. The precipitate was washed
with 10% NaHCO3 and water (w/w) for at least three times. Re-
crystallization from ethanol yielded 1.17 g compound 1. Yield:
70%. 1H NMR (DMSO, 500 MHz), δ: 7.85 (d, 2H, J=8.1), 7.65
(t, 2H, J=8.0), 7.43 (d, 2H, J=8.5), 7.14 (t, 2H, J=7.8), 4.54 (s,
4H).
Compound 2: A mixture of compound 1 (4.56 g, 15 mmol),
carbon powder (0.24 g, 20 mmol) and FeCl3 (0.032 g, 0.2 mmol)
was heated to 80 oC in 90% methanol (w/w, 45 g) under N2 at-
mosphere. Then, 85% N2H4•H2O (3 mL) was added dropwise
within 20 min. The mixture was refluxed for 5 h. It was filtered
while hot and washed with hot methanol. The combined filtrate
was evaporated and recrystallized from ethanol to give 2.20 g
faint yellow crystals. Yield: 60%. 1H NMR (DMSO, 500 MHz),
δ: 6.87 (dd, 2H, J=8.0), 6.71 (td, 2H, J=7.4), 6.66 (dd, 2H,
J=7.8), 6.53 (td, 2H, J=7.6), 4.66 (s, 4H), 4.28 (s, 4H).
Instruments and Methods. Nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker 500 MHz spectrom-
eter (Bruker, Germany). Mass spectra (MS) were recorded by
Agilent 6890 (Agilent, U.S.A). The fluorescence spectra and
UV-vis absorption spectra were obtained with a Hitachi F-4500
fluorescence spectrophotometer and Hitachi UH5300 spectro-
photometer (Hitachi, Japan). Fourier transform infrared spec-
troscopy (FTIR) spectra were obtained using a Thermo Scien-
tific Fourier Transform Infrared spectrometer at resolution of 4
cm-1 in the range of 500 - 4000 cm-1 (Thermo Fisher Scientific,
U.S.A). Atomic force microscope (AFM) images were recorded
in the ScanAsyst mode under ambient conditions (Bruker, Ger-
many). Transmission electron microscope (TEM) images were
Compound 3: A mixture of compound 2 (1.54 g, 6.32 mmol),
NaI (0.38 g, 2.53 mmol), K2HPO4 (7.212 g, 31.6 mmol) and
ethyl bromoacetate (4 mL, 36.4 mmol) in 25 mL anhydrous ac-
etonitrile was refluxed under nitrogen for 18 h. Toluene was
added after distilling the solvent. The solution was washed with
water and saturated brine. Organic layer was dried over Na2SO4
and evaporated. Recrystallization from ethanol gave 2.23 g
1
product as white powder. Yield: 60%. H NMR (DMSO, 500
MHz), δ: 6.99-6.91 (m, 2H), 6.86 (tt, 4H, J=7.4), 6.77-6.65 (m,
2H), 4.21 (s, 4H), 4.10 (s, 8H), 3.97 (m, 8H, J=7.1), 1.08 (t, 12H,
J=7.2).
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Analytical Chemistry
CaL ester: A mixture compound 3 (0.882 g, 1.5 mmol) and
and placed in ice-cold Ca2+ and Mg2+ free phosphate-buffered
saline (PBS). Tissues were dissected and incubated with Papain
for 15 min at 37 C, followed by trituration with fire-polished
glass pipettes, and plated in poly-D-lysine-coated 35-mm Petri
dishes with 20 mm bottom wells at a density of 1×106 cells per
dish. Neurons were cultured with Neurobasal medium supple-
mented with B27 and L-Glutamin, then maintained at 37 oC in
a humidified 5% CO2 atmosphere incubator. Cultures were fed
twice a week and used for all the assays 8-14 days after plating.
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150 μL pyridine was dissolved in 3 mL DMF. Then, 1.2 mL
o
o
POCl3 was dropped slowly at 0 C. The reaction mixture was
o
stirred at room temperature for 30 min and heated to 60 C for
1 h and then back to room temperature for 20 h. The mixture
was poured into aqueous NaOH mixed with ice. The aqueous
layer was extracted with CH2Cl2 for three times. The combined
organic layer was washed with water and saturated brine for
three times. After drying with Na2SO4 and evaporation, the res-
idue was chromatographed on silica in petroleum ether: ethyl
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acetate = 4:1 (v/v) to yield 193 mg CaL ester. Yield: 20%. H
Cytotoxicity and apoptosis assay. For cytotoxicity assay, dif-
ferent concentrations of CuNC@AF660 probe were added into
pre-incubated neurons in 96-well plates and cultured for 24 and
48 h, respectively. Subsequently, 20 μL MTT were added to
each well in dark. After reaction for 4 h, the mixed solution was
removed and 80 μL DMSO was added. After shaking for 5 min,
the absorbance was measured at 490 nm. Cell viability values
were determined according to the following formulae: cell via-
bility (%) = absorbance of the experimental group/ absorbance
of the blank control group × 100%. For apoptosis assay, differ-
ent concentrations of CuNC@AF660 probe were cultured with
pre-incubated neurons for 24 h. After removing the culture me-
dia, the cells were collected with the help of EDTA-free trypsin.
After washing with HBSS, the cells were re-suspended in 300
μL binding buffer and incubated with 5 μL FITC-Annexin V
and 5 μL propidium iodide solution for 30 min in dark. Apop-
tosis assay was detected at an excitation wavelength of 480 nm.
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NMR (DMSO, 500 MHz), δ: 9.78 (s, 2H), 7.45 (dd, 2H, J=8.3),
7.38 (d, 2H, J=1.8), 6.73 (d, 2H, J=8.3), 4.23 (d, 12H, J=10.4),
3.97 (q, 8H, J=7.1), 1.05 (t, 12H, J=7.1). 13C NMR (DMSO, 500
MHz): δ = 191.10, 170.70, 149.00, 144.98, 129.19, 126.12,
116.37, 111.62, 67.44, 60.89, 53.92, 14.22 ppm.
CaL: CaL ester (130 mg, 0.2 mmol) was added into 3 mL
KOH solution (1 M, dissolved in EtOH), then 1 mL CH2Cl2 was
added. The mixture was stirred at 37oC for 24 h. After that, the
mixture was adjusted to a pH range of 4-5 by HCl. The solid
was filtered and product was extracted with abundant CH2Cl2.
After drying with Na2SO4 and evaporation, 65 mg CaL ester
1
was obtained. Yield: 60%. H NMR (DMSO, 500 MHz), δ:
12.56 (s, 4H), 9.75 (s, 2H), 7.44 (dd, 2H, J=8.4), 7.38 (d, 2H,
J=1.8), 6.71 (d, 2H, J=8.4), 4.30 (s, 4H), 4.16 (s, 8H). 13C NMR
(DMSO, 500 MHz): δ = 191.00, 172.37, 148.75, 145.41, 128.78,
126.13, 116.07, 112.88, 67.49, 54.39 ppm.
Preparation of CuNCs and CuNC@AF660. In order to syn-
thesize CuNCs, PEI-CaL was first prepared by the reaction be-
tween PEI and CaL. In brief, 1 mL PEI (5 mM, WM: 1800) was
dissolved in ethanol, then 1 mL CaL solution (30 mM, dissolved
in CH2Cl2) was added, the mixture was reacted at room temper-
ature for 12 h. PEI-CaL was obtained by removing CH2Cl2
through reduction vaporization and unreacted CaL was re-
moved by dialysis. Next, PEI-CaL solution was adjusted to pH
5 by acetic acid, then 3 mL Cu(NO3)2 solution (1 mM) was
added under stirring. After 10 min, 10 mg ascorbic acid was
added and reacted for another 8 h. The obtained CuNCs capped
with PEI-CaL were purified by ultrafiltration with a Millipore
(30 K, molecular weight cutoffs) with 16000 rpm for 10 min
Fluorescence confocal imaging. For Ca2+ imaging,
CuNC@AF660 probe (90 μg/mL) was cultured with neurons
for 1 h in Hanks’ balanced salt solution with no Ca2+ and Mg2+
(HBSS), then washed by HBSS for twice and different concen-
trations of Ca2+ with 5 μM calcimycin (a divalent cation iono-
phore which allows Ca2+ to cross the cell membrane) and cul-
tured for another 30 min in HBSS, cell imaging was carried out
after cells were washed with HBSS for twice. For histamine or
-
O2 -stimulating imaging, CuNC@AF660 probe was first cul-
tured with neurons for 1 h in HBSS, then the neurons were
washed by HBSS for twice. After that, the neurons were cul-
tured in neurobasal medium, histamine (50 μM) or O2- (80 μM)
was added and the images were obtained with time interval of
20 s.
o
and stored at 4 C for further using. For preparing the rati-
ometric fluorescence probe (CuNC@AF660), the obtained
CuNCs (1 mL) were mixed with appropriate volume of AF660
NHS ester solution in PBS buffer (pH 7.4), and reacted at room
temperature under stirring overnight. Finally, CuNC@AF660
was purified by ultrafiltration.
RESULTS AND DISCUSSION
Preparation and characterization of CuNCs. In order to re-
alize Ca2+ detection with high selectivity, a Ca2+ ligand (CaL)
with two formaldehyde groups was first designed and synthe-
sized. The structures of CaL and intermediates were confirmed
1
by H NMR (Figures S1-S5) and 13C NMR (Figures S6, S7),
Calculation of the mass concentrations of CuNCs and
CuNC@AF660. In order to determine the concentration of
CuNCs, three pre-weighed tubes (m0) were added with 0.5 mL
of CuNCs, respectively. Then, the tubes were dried to evaporate
water. Finally, the tubes containing dry CuNCs were weighed
again (m1). The mass concentration of CuNCs was calculated
by dividing the difference in masses (m1-m0) by 0.5 mL and
taking the average. The mass concentration of CuNC@AF660
was obtained by the same method with that of CuNCs.
and the molecular weights of CaL and CaL ester were charac-
terized by mass spectroscopy (Figures S8, S9). Fourier trans-
form infrared spectroscopy (FTIR) spectrum of CaL shows two
distinct peaks at 2760 and 1740 cm-1, assigned to the stretching
vibration of C-H and C=O, respectively, indicating the presence
of -CHO group in CaL (Figure 1A). Other characteristic groups
of CaL can also be found from FTIR spectra (Figure S10). Then,
CaL was further conjugated onto PEI to develop a new template
molecule (PEI-CaL) by Schiff base reaction between -CHO
group of CaL and -NH2 group of PEI for synthesizing CuNCs.
From FTIR spectrum, PEI shows two distinct peaks emerged at
1650 and 1311 cm-1, which belongs to N-H stretching vibration
and bending vibration of amino groups. After CaL was conju-
gated onto PEI to form PEI-CaL, a new peak at 1572 cm-1 was
observed, which pertained to the stretching vibration of C=N,
Primary culture of mouse cortical neurons. The experimental
protocols were approved by Animal Care and Use Committee
of East China Normal University, Shanghai, China. Primary
cultures of mouse cortical neurons were prepared as described
previously.23 Briefly, postnatal day 1 C57BL/6 wild-type mice
were anesthetized with halothane. Brains were removed rapidly
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The results demonstrated that the synthesized CuNCs through
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PEI-CaL template were stable against oxidation in air.
In addition, considering the high salt concentrations in cells,
the fluorescence of CuNCs exposed to different concentrations
of NaCl were also checked. The fluorescence intensity of the
present CuNCs showed no obvious change even in 1 M of NaCl
(< 4.1%) (Figure S14A). Moreover, different thiol compounds
and phosphate had no apparent influence on the fluorescence of
CuNCs (< 3.5%) (Figure S14B). All these results suggest good
stability of our developed CuNCs.
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Synthesis and characterization of CuNC@AF660 probe for
Ca2+ biosensing. For improving the accuracy of Ca2+ biosens-
ing in live cells, Alexa Fluor 660 (AF660) was then attached
onto CuNCs to develop CuNC@AF660 ratiometric fluorescent
probe. FTIR spectra were used to characterize the assembly of
CuNC@AF660. Individual FTIR spectrum of CuNCs shows
one board peak emerged at 3374 cm-1 and one narrow peak
around 1643 cm-1 (Figure 2A), which belong to the stretching
vibrations of N-H. In addition, a peak located at 1400 cm-1 was
also observed, which attributes to the bending vibration of N-H,
promising the presence of amino group on CuNCs. On the other
hand, AF660 NHS ester shows a distinct peak at 1663 cm-1,
which pertains to the stretching vibration of C=O from amide I
band, demonstrating the presence of carboxylic NHS ester in
AF660 NHS ester. However, after AF660 NHS ester was con-
jugated onto CuNCs to form CuNC@AF660, the stretching vi-
bration of C=O from amide I band blue-shifted to 1653 cm-1.
Furthermore, a new peak located at 1566 cm-1 was observed,
which belongs to the bending vibration of N-H from amide II
band, confirming the successful attachment of AF660 NHS es-
ter onto CuNCs by forming amide bond. In addition, dynamic
light scattering (DLS) results showed that the average diameter
of individual CuNCs was estimated to 98±12 nm (Figure S15).
However, after the AF660 was conjugated onto CuNCs to form
CuNC@AF660 probe, the average diameter of CuNC@AF660
probes increased to 110±10 nm. The results further prove that
AF660 was successfully conjugated onto CuNCs. Moreover,
CuNC@AF660 display two emissions at ~590 nm and ~690 nm
upon excitation at 488 nm, respectively, ascribed to that of
CuNCs and AF660 NHS ester (Figure 2B).
Next, the stability of developed CuNC@AF660 probe was
investigated. The fluorescence ratio of CuNC@AF660 probe
(F510-650/F660-780) shows no obvious change even after the probe
was incubated with different solutions for 5 h (< 1%) (Figure
S16), indicating the high stability of developed CuNC@AF660
probe. It should be pointed out that cell imaging experiments
were conducted within 2 h, thus the detection time is long
enough for cell imaging. Meanwhile, the influence of concen-
tration ratio of CuNCs to AF660 on the Ca2+ sensing was also
investigated. The fluorescence enhancement efficiency (R) of
CuNC@AF660 was obtained after 300 μM Ca2+ was added into
the solution of CuNC@AF660 probe at different concentration
ratio of CuNCs to AF660. The value of R increased with the
increasing concentration ratio of CuNCs to AF660, and the R
value reached stable when the concentration ratio of CuNCs to
AF660 was 15:1 (Figure S17A). Moreover, no obvious differ-
ence (< 1%) was observed for the fluorescence intensity ratio
(F510-650/F660-780) of various batches of CuNC@AF660 probes
(Figure S17B). Furthermore, the effect of pH on the ratiometric
fluorescent response of CuNC@AF660 to Ca2+ was also inves-
tigated. R value of CuNC@AF660 probe increased 5.04% when
pH increased from 5.0 to 7.0 after addition of 250 μM Ca2+ into
CuNC@AF660 solution (Figure S18). Meanwhile, R value of
Figure 1. (A) FTIR spectra of CaL, PEI, and PEI-CaL. (B) TEM
image of CuNCs. Inset shows diameter distributions of CuNCs. (C)
AFM image of CuNCs deposited on freshly cleaved mica. Inset
shows height distributions of CuNCs along the line shown in C. (D)
UV-vis absorption spectrum and fluorescence spectra of CuNCs
(90 μg/mL) at excitation wavelengths from 500 nm to 570 nm with
interval of 10 nm. Insets show photographs of CuNCs solution (90
μg/mL) under ambient light (left) and illuminated by an UV lamp
of 365 nm (right).
indicating PEI-CaL was successfully conjugated by forming
Schiff base complexes.31 Next, CuNCs were synthesized using
a template of PEI-CaL. As shown in Figure 1B, transmission
electron microscopy (TEM) image shows that CuNCs are mon-
odispersed with averaging size about 1.1±0.4 nm (Insert in Fig-
ure 1B). The typical atomic force microscope (AFM) image of
CuNCs demonstrates many dot-like compositions with cross-
sectional heights of ~1 nm (Figure 1C). On the other hand,
CuNCs display three well-resolved absorption peaks at 284, 350
and 476 nm as well as the bright jacinth fluorescent CuNCs (un-
der a 365 nm UV lamb) exhibit excitation-dependent fluores-
cence emissions (Figure 1D and insert). In addition, the fluores-
cence intensity of CuNCs increased with the increasing concen-
tration ratio of PEI-CaL to Cu2+, and the fluorescence intensity
reached stable when the concentration ratio of PEI-CaL to Cu2+
was 2:1 (Figure S11A). Furthermore, the fluorescence intensity
of various batches of CuNCs was comparable (< 1%) (Figure
S11B). Compared with previously reported CuNCs,32 the opti-
mal emission of our developed CuNCs red-shifted by ~70 nm.
The fluorescence quantum yield (QY) of the developed CuNCs
was 20% by using fluorescein as a reference chromophore
(Figure S12). In order to confirm the valence state of Cu in
CuNCs, X-ray photoelectron spectroscopy (XPS) was em-
ployed to characterize the prepared CuNCs. XPS spectrum
shows typical elements of C, N, O and Cu in CuNCs (Figure
S13A). XPS spectrum of the developed CuNCs in Cu 2p region
demonstrates two intense peaks at 932.2 and 952.0 eV (Figure
S13B), assigned to the binding energies of 2p3/2 and 2p1/2 elec-
trons of Cu (0). Meanwhile, no peak was observed at 942.0 eV,
which belongs to binding energy of Cu (II) electrons.33 It is of
great importance to mention that 2p3/2 binding energy of Cu (0)
is only ~0.1 eV away from that of Cu (I). Therefore, the valence
state of the obtained CuNCs most likely lies between 0 and +1.
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CuNC@AF660 probe decreased 4.04% when pH changes from
7.0 to 9.0 after addition of 250 μM Ca2+ into CuNC@AF660
solution. Thus, pH between 5.0-9.0 had no obvious influence
on ratiometric fluorescent response of CuNC@AF660 to Ca2+.
The results proved that the developed CuNC@AF660 probe can
be used in Ca2+ sensing and imaging in cytoplasm.
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5
In order to confirm the working principle, ratiometric fluo-
rescent probe was further used to detect Ca2+ in cell lysis buffer.
As shown in Figure 2C, with increasing concentration of Ca2+,
the fluorescent intensity of CuNCs (F510-650: 510-650 nm) in-
creased apparently while the fluorescence intensity of AF660
(F660-780: 660-780 nm) kept no obvious change. Thus, the fluo-
rescence from AF660 provided an inner reference for built-in
correction, which may eliminate the environmental interference
for determination of Ca2+. In consequence, the fluorescent sig-
nal ratio (F510-650/F660-780) gradually enhanced with increasing
concentration of Ca2+, and displayed a good linearity with Ca2+
concentration in the range of 2 - 350 μM (Figure 2D). The de-
tection limit was calculated as 22011 nM (S/N = 3), which can
meet the detection requirement in live cells.34 More importantly,
the response time of our developed probe towards Ca2+ was less
than 2 s (Figure S19), revealing fast response dynamics of the
developed nanoprobe towards Ca2+. In addition, both of the flu-
orescence intensities of CuNCs and AF660 from
CuNC@AF660 showed no obvious change (< 5.1%) upon illu-
mination of a Xe lamp (90 W) for 2.5 h, indicating long-term
photostability of CuNC@AF660 probe. (Figure S20).
It has been reported that both of metal core and surface ligand
shell contribute to the fluorescence of metal nanoclusters.35
Specifically, the surface ligand shell can influence the fluores-
cence of metal nanoclusters by charge transfer or electron do-
nation from the ligands to clusters core.36 In the absence of Ca2+
ion, UV-vis absorption spectrum of CuNCs shows three well-
resolved absorption peaks at 284, 350 and 476 nm (Figure 1D),
which may be due to interband electronic transitions of the Cu
clusters from discrete energy levels.33 The fluorescence of
CuNCs show typical emission peak around 590 nm. However,
after addition of Ca2+, UV-vis absorption spectra of CuNCs in-
creased while the fluorescence of CuNCs enhanced (Figure
S21A, B). Meanwhile, the fluorescence lifetime of CuNCs pro-
longed from 0.83 ns [7.615 ns (4%); 0.543 ns (96%)] to 6.47 ns
(100%) after addition of 400 μM Ca2+ (Figure S21C). In order
to evaluate the mechanism of fluorescence enhancement of
CuNCs, dimethyl sulfoxide (DMSO) was first added in the so-
lution. Since DMSO is hydrogen-bond-breaking polar sol-
vents,37 the entanglements between the polymer chains can be
broken due to the weakening or absence of the hydrogen-bond
effect in DMSO. With the increase of the volume fraction of
DMSO in the mixed blank solvent (f = VolDMSO / VolDMSO + water),
the fluorescence intensity of CuNCs gradually increased (Fig-
ure S22A, B). The fluorescence quantum yield of CuNCs in
DMSO (f = 1) increased to 30.7% (Figure S22C), much higher
than that (20%) in water solution. Meanwhile, dynamic light
scattering (DLS) results demonstrated that the hydrodynamic
diameter of CuNCs in DMSO (f = 1) obviously decreased
from98±12 nm (f = 0) to 20±11 nm (Figure S22D). Thus, the
fluorescent enhancement of CuNCs was considered to be as-
cribed to aggregation induced emission because Ca2+-induced
aggregation of CuNCs through carboxyl groups in the polymer
chain on CuNCs core, leading to the extension of conjugation
and increase in the rigidity of the molecular conformation
through space electronic interactions, namely, overlap of π and
lone pair (n) electrons among carboxyl groups on the polymer.37
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Figure 2. (A) FTIR spectra of CuNCs (a), AF660 NHS ester (b)
and CuNC@AF660 (c). (B) Fluorescence spectra of CuNCs (a),
AF660 NHS ester (b) and CuNC@AF660 probe (c). (C) Fluores-
cence responses of CuNC@AF660 (90 μg/mL) with the addition of
various concentrations of Ca2+ (0, 2, 25, 80, 110, 160, 205, 215,
280, 300, 350, 380, 400 μM) in cell lysis buffer. (D) Calibration
curve between F510-650/F660-780 and various concentrations of Ca2+.
Data were obtained from Figure 2C.
Actually, during imaging and biosensing of Ca2+ in live cells,
not only the sensitivity and detection linearity range but selec-
tivity is also extremely important during analytical processes.
Thus, the effect of intracellular potential interferences on the
developed sensing probe was studied. The common potential
interferences, such as metal ions, physiological relevant amino
acids, calcium-containing proteins as well as typical ROS were
investigated. In the selectivity test (Figure S23), negligible in-
terferences (< 5.1%) were observed from other metal ions like
Mg2+ (10 mM), K+ (100 mM), Na+ (50 mM), Cu2+, Fe2+, Fe3+,
Pb2+, Zn2+, Ag+, Cd2+, Cr3+, Ni2+, amino acids, calcium-contain-
ing proteins like troponin, calpain, and ROS including H2O2,
-
-
O2 , NO, ROO•, ClO4 and •OH. Furthermore, negligible influ-
ence (< 4.2%) was observed determination of Ca2+ followed by
addition of other metal ions, amino acids, calcium-containing
proteins and ROS, indicating the high selectively for Ca2+ de-
termination against other metal ions, amino acids, calcium-con-
taining proteins and ROS.
Imaging and biosensing of Ca2+ in neurons. Before imaging
and biosensing of Ca2+ in neurons, cytotoxicity and biocompat-
ibility of the developed nanoprobe were in advance evaluated.
Cell viability was higher than 90% even at nanoprobe concen-
tration up to 270 μg/mL (Figure S24), which is 3 times as that
used in cell imaging. In addition, flow cytometry experimental
results showed that no significant increase of apoptotic cells
was observed after the neurons were incubated with different
concentrations of CuNC@AF660 probe (Figure S25). All these
results indicate low cytotoxicity and good biocompatibility of
our developed nanoprobe. Moreover, co-localization experi-
mental results demonstrated that the fluorescence of the nano-
probe merged well with that of commercial cytoplasmic probe
(CellTracker Green CMFDA), and the Pearson’s coefficient
was calculated as 0.91 (Figure S26A). It should be pointed out
that a small neuronal nucleus was observed in this imaging
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Figure 4. (A) Time-tracking of confocal fluorescence microscopic
images of neurons were stimulated by 50 μM histamine for differ-
ent times. (B) The enlarged image of neurons stimulated by hista-
mine for 160 s. The different zones (1-4) represent the soma, prox-
imal axon, distal axon and dendrite of neuron, respectively. (C)
Fgreen/Fred value of different zones in neurons after the neurons were
stimulated by 50 μM histamine for different times. Fgreen and Fred
represent the average fluorescence intensity collected from green
channel (510-650 nm) and red channel (660-780 nm), respectively.
The excitation wavelength was 488 nm. Fgreen = F510-650, Fred = F660-
780. Scale bars = 25 μm.
Figure 3. (A) Confocal fluorescence microscopy images of neu-
rons collected from different channels after the neurons were co-
incubated with CuNC@AF660 probe (90 μg/mL) in the presence
of different concentration of Ca2+ (0, 100, 200 and 300 μM), re-
spectively. (B) Average fluorescence intensities of green channel
(Fgreen) and red channel (Fred) at different concentrations of Ca2+.
Fgreen and Fred represent the average fluorescence intensity collected
from green channel (510-650 nm) and red channel (660-780 nm),
respectively. (C) Fgreen/Fred values verse different concentration of
Ca2+. Fgreen = F510-650, Fred = F660-780. Scale = 25 μm.
changed from blue to red, suggesting our probe can be used for
intracellular Ca2+ detection. The average fluorescence intensity
ratio of green channel to red channel (Fgreen/Fred) increased from
0.85 to 1.44 (Figure 3C). It should be noted that there were
many bright domains (inside the blue circles) in cytoplasm,
which belongs to the Ca2+-rich domains in neurons, suggests the
distribution of cytoplasmic Ca2+ was inhomogeneous.
plane (Z1 position) (Figure S26A), which is due to specific cell
morphology of neurons.38 Generally, the neuronal nucleus is lo-
cated in soma of cortical neurons with diameter of 3-18 μm.
Meanwhile, the diameter of soma is 30-150 μm. Because the
diameters of dendrite and distal axon(0.5∼2 μm) are much
smaller than those of the soma and neuronal nucleus, the soma
and neuronal nucleus are hard to be observed in the same imag-
ing planes as those of dendrite and distal axon. To obtain the
distribution of cytoplasm in soma, dendrite, and distal axon
simultaneously, the suitable imaging plane (Z1 position) was
chosen during imaging. In this context, neuronal nucleus can
hardly be observed in this imaging plane (Z1 position). However,
when the Z2 position was chosen as imaging plane (Figure
S26B), the neuronal nucleus was clearly observed, but the den-
drite and distal axon of neuron can hardly be observed. The flu-
orescence of the present nanoprobe also merged well with that
of CellTracker CMFDA in this imaging plane (Z2 position) and
the Person’s correlation coefficient was calculated as 0.92.
However, the fluorescence of nanoprobe seldom merged with
that of 4’,6-diamidino-2-phenylindole (DAPI), and the Person’s
correlation coefficient was calculated as 0.15. All the results
demonstrated that the developed CuNC@AF660 probe entered
into cells and primary located in cytoplasm.
More interestingly, it has been reported that histamine can
mobilize intracellular Ca2+ stores and arise Ca2+ levels in live
cells. Then, the concentration change of Ca2+ was investigated
using our developed CuNC@AF660 probe in neurons stimu-
lated by histamine. As shown in Figure 4A, after the neurons
were stimulated by 50 μM histamine for 40 s, green channel
turned into brighter while red channel kept unchanged. Subse-
quently, the fluorescence intensity of green channel was contin-
uously enhanced and remained stability after the neurons were
stimulated by histamine for 120 s. Interestingly, it was found
that histamine-induced Ca2+ increase of fluorescent ratio in dif-
ferent regions of neuron was diverse (Figure 4B). As shown in
Figure 4C, before the neurons were stimulated by histamine,
Fgreen/Fred values were basically the same in different regions of
the neuron (~0.79±0.04). After the neurons were stimulated by
histamine for 120 s, Fgreen/Fred values in soma and proximal axon
were 1.16±0.05 and 1.12±0.03, respectively. Meanwhile,
Fgreen/Fred value in distal axon was 0.98±0.04. These results sug-
gested that histamine-induced Ca2+ concentration increase was
similar in soma and proximal axon, and the increased Ca2+ con-
centration in distal axon of neurons was much lower than that
in soma. Unexpectedly, Fgreen/Fred value in dendrite of neurons
was 0.80±0.05 after the neurons were stimulated by histamine
for 120 s, implying that histamine stimulation had no obvious
effect on Ca2+ concentration in dendrite.
Then, this nanoprobe was applied for Ca2+ sensing and imag-
ing in cytoplasm of neurons. As shown in Figure 3A, with in-
creasing concentration of Ca2+ from 0 to 300 μM, green channel
became brighter while red channel kept unchanged, demonstrat-
ing the fluorescence intensity of Fgreen (Fgreen = F510-650) increased
while Fred (Fred = F660-780) kept stable (Figure 3B). From the
merged channel we can see that the pseudo color of neurons
Real-time monitoring of Ca2+ level induced by superoxide
-
anion (O2 ). Increasing evidence has indicated that homeo-
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static and physiological ROS levels are indispensable in regu-
lating Ca2+ burst production, leading to increase in oxidative
stress or even cell death,39 which may be strongly related to
many diseases, like cardiovascular diseases and neurodegener-
ative diseases.40 O2- as the primary species of ROS and precur-
sor of other ROS, has highly oxidative activity.21,24,41 Excessive
accumulation of O2- in cells can cause oxidative damage to pro-
teins, DNA, liposomes, and even cell death.42,43 Based on the
established fluorescent CuNC@AF660 probe as a selective and
accurate probe for imaging and biosensing of intracellular Ca2+,
the effect of O2- on cellular Ca2+ during stimulation was inves-
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tigated. The influence of O2 on neuronal viability was first
evaluated. Neuronal viability obviously decreased with increas-
ing concentration of O2 as well as extension of stimulation
-
time (Figure 5A). Higher concentrations of O2- resulted in re-
duced cell viability. It is well-known that, under normal physi-
ological conditions, O2- undergoes disproportionation by non-
catalytic and enzymatic reactions, resulting in a rather low
physiological concentration (10-8-10-7 M).44 Increases in the ac-
tivity of O2- occur in response to traumatic brain injury ische-
mia-reperfusion, hypoxia, and environmental stresses, the con-
centration of O2- may increase to ∼10-4 M.39,41,45,46 As shown in
Figure 5A, neuron viability was only ~15±5% after the neurons
Figure 5. (A) Summarized data of neuron viability after the neu-
rons were stimulated by various concentrations of O2 (0, 25, 50
-
and 80 μM) for different times (0, 1, 5 and 15 h). (B) Time-tracking
of Fgreen/Fred values obtained every 20 s after the neurons were stim-
ulated by 80 μM O2-. (C) The typical confocal fluorescence micro-
scopic images of neurons collected from different channels after
-
the neurons were stimulated by 80 μM O2 for different times.
Fgreen and Fred represent the average fluorescence intensity col-
lected from green channel (510-650 nm) and red channel (660-780
-
stimulated by 80 μM O2 for 15 h. In order to understand the
-
nm), respectively. The excitation wavelength was 488 nm. Fgreen
=
mechanism of O2 -induced neuronal death, we then real-time
monitored the cytoplasmic Ca2+ changes when the neurons were
F510-650, Fred = F660-780. Scale = 25 μm.
-
stimulated by 80 μM O2 . As shown in Figure 5B, Fgreen/Fred
found to be similar with that in soma. Meanwhile, Ca2+ change
in neuronal distal axon was less than that in soma, and Ca2+ con-
centration in dendrite had no obvious change after the neurons
were stimulated by histamine. More importantly, we have found
value clearly increased after the neurons were stimulated by
O2- for 240 s, and Fgreen/Fred value increased from 0.81±0.02 to
-
1.36±0.04 after the neurons were stimulated by O2 for 360 s.
According to the established calibration curve in cell lysis (Fig-
ure 2C), cytoplasmic Ca2+ increased to ~250 μM after the neu-
rons were stimulated by O2- for 360 s. It should be
-
that O2 -induced neuronal death might be resulted from the
Ca2+ overload in neurons, providing a new point of view to un-
derstand the mechanism of oxidative stress-related diseases.
This study has provided a methodology to develop metal clus-
ter-based inorganic-organic fluorescent probes for metal ions,
ROS, and other biological species in neurons.
pointed out that normal concentration of Ca2+ in cytoplasm was
about 0.2 μM.47 These results strongly indicated that O2 -in-
-
duced Ca2+ burst in neurons. Consistently, the green channel be-
came significantly brighter after the neurons were stimulated by
80 μM O2- while the red channel kept no obvious change (Fig-
ASSOCIATED CONTENT
-
ure 5C). This O2 -induced cytoplasmic Ca2+ burst suggested
-
that O2 -induced neuronal death was possibly resulted from
Supporting Information
Ca2+ overload in neurons, which may cause Ca2+ disorders in
neurons and unfavorable for neuron viability.
The Supporting Information is available free of charge on the ACS
Publications website. Characterization of compound 1, compound
2, compound 3, CaL ester and CaL; Fluorescence quantum yield,
XPS spectra and stability investigation of CuNCs. Fluorescence en-
hancement mechanism research. Selectivity, cytotoxicity and bio-
compatibility study of CuNC@AF660 probe. Co-localization im-
aging results.
CONCLUSIONS
In summary, a CuNCs-based ratiometric fluorescent nanoprobe
has been developed for real-time biosensing and bioimaging of
cytoplasmic Ca2+ in neurons, in which a specific CaL was syn-
thesized and conjugated to PEI to prepare PEI-CaL. PEI-CaL
plays not only as a recognition element for Ca2+, but also as a
new ligand molecular for synthesis of fluorescent CuNCs. Fur-
thermore, AF660 was attached onto the surface of CuNCs to act
as a reference element, resulting in a built-in correction to im-
prove the accuracy. This inorganic-organic ratiometric fluores-
cent nanoprobe has demonstrated long-term stability, as well as
high selectivity against other metal ions, amino acids, calcium-
containing proteins and ROS. Accordingly, the developed fluo-
rescent nanoprobe with low cytotoxicity and good biocompati-
bility has been successfully applied in multicolor imaging and
biosensing of cytoplasmic Ca2+ in neurons. Taking advantages
of rapid response dynamics of our developed probe, it has been
found that Ca2+ in cytoplasm was inhomogeneous. Besides, his-
tamine-induced Ca2+ increase in proximal axon of neurons was
AUTHOR INFORMATION
Corresponding Author
*Phone + 86 21 54341041; fax +86 21 54341041; E-mail:
ytian@chem.ecnu.edu.cn.
ORCID
Yang Tian: 0000-0001-8850-0349
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors greatly appreciate the financial support from NSFC
(21635003 and 21827814) and The Program of Shanghai Subject
Chief Scientist (15XD1501600). This work also was supported by
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Innovation Program of Shanghai Municipal Education Commis-
sion (201701070005E00020).
(26) Shi, W.; Li, X.; Ma, H. Angew. Chem. Int. Ed. 2012,
51, 6432-6435.
1
2
3
4
5
6
7
8
9
(27) Liu, J.-M.; Chen, J.-T.; Yan, X.-P. Anal. Chem. 2013,
85, 3238-3245.
(28) Pan, W.; Wang, H.; Yang, L.; Yu, Z.; Li, N.; Tang,
B. Anal. Chem. 2016, 88, 6743-6748.
(29) Li, P.; Fang, L.; Zhou, H.; Zhang, W.; Wang, X.; Li,
N.; Zhong, H.; Tang, B. Chem. Eur. J. 2011, 17, 10520-
10523.
(30) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol.
Chem. 1985, 260, 3440-3450.
(31) Rama Rao, A. V.; Laxma Reddy, K.; Machender
Reddy, M. Tetrahedron Lett. 1994, 35, 5039-5042.
(32) Liu, Z.-C.; Qi, J.-W.; Hu, C.; Zhang, L.; Song, W.;
Liang, R.-P.; Qiu, J.-D. Anal. Chim. Acta 2015, 895, 95-
103.
REFERENCES
(1) Clapham, D. E. Cell 2007, 131, 1047-1058.
(2) Berridge, M. J. Nature 1993, 361, 315-325.
(3) Plattner, H.; Verkhratsky, A. Cell Calcium 2015, 57,
123-132.
(4) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. Rev.
Drug Discovery 2004, 3, 205-214.
(5) LaFerla, F. M. Nat. Rev. Neurosci. 2002, 3, 862-872.
(6) Wan, Q.; Chen, S.; Shi, W.; Li, L.; Ma, H. Angew.
Chem. Int. Ed. 2014, 53, 10916-10920.
(7) Kim, S.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J.
Am. Chem. Soc. 2014, 136, 11707-11715.
(8) Setsukinai, K.-i.; Urano, Y.; Kakinuma, K.; Majima,
H. J.; Nagano, T. J. Biol. Chem. 2003, 278, 3170-3175.
(9) Peng, L.; Xu, S.; Zheng, X.; Cheng, X.; Zhang, R.;
Liu, J.; Liu, B.; Tong, A. Anal. Chem. 2017, 89, 3162-
3168.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(33) Wei, W.; Lu, Y.; Chen, W.; Chen, S. J. Am. Chem.
Soc. 2011, 133, 2060-2063.
(34) Montero, M.; Alonso, M. T.; Carnicero, E.; Cuchillo-
Ibanez, I.; Albillos, A.; Garcia, A. G.; Garcia-Sancho, J.;
Alvarez, J. Nat. Cell Biol. 2000, 2, 57-61.
(35) Chen, Y.; Yang, T.; Pan, H.; Yuan, Y.; Chen, L.; Liu,
M.; Zhang, K.; Zhang, S.; Wu, P.; Xu, J. J. Am. Chem.
Soc. 2014, 136, 1686-1689.
(36) Wu, Z.; Jin, R. Nano Lett. 2010, 10, 2568-2573.
(37) Yang, T.; Dai, S.; Yang, S.; Chen, L.; Liu, P.; Dong,
K.; Zhou, J.; Chen, Y.; Pan, H.; Zhang, S. J. Phys. Chem.
Lett. 2017, 8, 3980-3985.
(10) Zhang, W.; Li, P.; Yang, F.; Hu, X.; Sun, C.; Zhang,
W.; Chen, D.; Tang, B. J. Am. Chem. Soc. 2013, 135,
14956-14959.
(11) Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J. M.
Nature 1997, 388, 882.
(12) Heim, N.; Griesbeck, O. J. Biol. Chem. 2004, 279,
14280-14286.
(13) Ohkura, M.; Matsuzaki, M.; Kasai, H.; Imoto, K.;
Nakai, J. Anal. Chem. 2005, 77, 5861-5869.
(14) Tour, O.; Adams, S. R.; Kerr, R. A.; Meijer, R. M.;
Sejnowski, T. J.; Tsien, R. W.; Tsien, R. Y. Nat. Chem.
Biol. 2007, 3, 423-431.
(15) Agarwal, H. K.; Janicek, R.; Chi, S.-H.; Perry, J. W.;
Niggli, E.; Ellis-Davies, G. C. R. J. Am. Chem. Soc. 2016,
138, 3687-3693.
(16) Egawa, T.; Hirabayashi, K.; Koide, Y.; Kobayashi,
C.; Takahashi, N.; Mineno, T.; Terai, T.; Ueno, T.;
Komatsu, T.; Ikegaya, Y.; Matsuki, N.; Nagano, T.;
Hanaoka, K. Angew. Chem. Int. Ed. 2013, 52, 3874-3877.
(17) Mishra, A.; Jiang, Y.; Roberts, S.; Ntziachristos, V.;
Westmeyer, G. G. Anal. Chem. 2016, 88, 10785-10789.
(18) Li, Z.; Lv, S.; Wang, Y.; Chen, S.; Liu, Z. J. Am.
Chem. Soc. 2015, 137, 3421-3427.
(19) Song, X.; Yue, Z.; Zhang, J.; Jiang, Y.; Wang, Z.;
Zhang, S. Chem. Eur. J. 2018, 24, 6458-6463.
(20) Zhu, A.; Qu, Q.; Shao, X.; Kong, B.; Tian, Y. Angew.
Chem. Int. Ed. 2012, 51, 7185-7189.
(38) Liu, Z.; Pei, H.; Zhang, L.; Tian, Y. ACS Nano 2018,
12, 12357-12368.
(39) Wang, W.; Fang, H.; Groom, L.; Cheng, A.; Zhang,
W.; Liu, J.; Wang, X.; Li, K.; Han, P.; Zheng, M.; Yin, J.;
Wang, W.; Mattson, M. P.; Kao, J. P. Y.; Lakatta, E. G.;
Sheu, S.-S.; Ouyang, K.; Chen, J.; Dirksen, R. T.; Cheng,
H. Cell 2008, 134, 279-290.
(40) Li, P.; Zhang, W.; Li, K.; Liu, X.; Xiao, H.; Zhang,
W.; Tang, B. Anal. Chem. 2013, 85, 9877-9881.
(41) Turrens, J. F. J. Physiol. 2003, 552, 335-344.
(42) Liou, G.-Y.; Storz, P. Free. Radical. Res. 2010, 44,
479-496.
(43) Fridovich, I. Annu. Rev. Biochem. 1995, 64, 97-112.
(44) Cadenas, E.; Davies, K. J. Free. Radical. Bio. Med
2000, 29, 222-230.
(45) Deng, Z.; Rui, Q.; Yin, X.; Liu, H.; Tian, Y. Anal.
Chem. 2008, 80, 5839-5846.
(46) Gorrini, C.; Harris, I. S.; Mak, T. W. Nat. Rev. Drug
Discovery 2013, 12, 931-947.
(47) Coll, K. E.; Joseph, S.; Corkey, B.; Williamson, J. J.
Biol. Chem. 1982, 257, 8696-8704.
(21) Gao, X.; Ding, C.; Zhu, A.; Tian, Y. Anal. Chem.
2014, 86, 7071-7078.
(22) Han, Y.; Ding, C.; Zhou, J.; Tian, Y. Anal. Chem.
2015, 87, 5333-5339.
(23) Liu, Z.; Wang, S.; Li, W.; Tian, Y. Anal. Chem. 2018,
90, 2816-2825.
(24) Huang, H.; Dong, F.; Tian, Y. Anal. Chem. 2016, 88,
12294-12302.
(25) Zhuang, M.; Ding, C.; Zhu, A.; Tian, Y. Anal. Chem.
2014, 86, 1829-1836.
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