Rhodamine-modified upconversion nanoprobe for distinguishing Cu2+ from Hg2+ and live cell imaging

Article abstract of DOI:10.1039/c5nj03010b

It is greatly important to seek a fast and sensitive method for the detection of Cu²⁺ ions because of their vital role in the human body. Fluorescent probes, especially the rhodamine derivatives, have been considered as promising candidates for detection of Cu²⁺ ions due to their attractive features. However, one problem frequently encountered in rhodamine-based fluorescent probes is that some of them could not distinguish Cu²⁺ from Hg²⁺ and these drawbacks limit their application in biological samples. In this paper, a rhodamine B derivative (RBH) was grafted to mesoporous silica coated upconversion nanoparticles (CS-UCNP) to fabricate a new organic-inorganic hybrid nanoprobe. On addition of Cu²⁺ ions, an emission band at about 580 nm appeared while the intensity of the green emission at about 545 nm of the nanoprobe decreased upon excitation of a 980 nm laser, implying that a luminescence resonance energy transfer (LRET) happened from the CS-UCNP to the RBH-Cu²⁺ complex. The obtained LRET nanoprobe could detect Cu²⁺ exclusively even in the presence of Hg²⁺ with a detection limit of 0.82 μM in absolute ethanol solution. Most importantly, this nanoprobe can be used for monitoring subcellular distribution of Cu²⁺ in living cells based on upconversion luminescence and downconversion fluorescence.

Full text of DOI:10.1039/c5nj03010b

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Rhodamine-Modified Upconversion Nanoprobe for Distinguish Cu2+ from Hg2+ and Living
Cells Imaging
Yanxia Xua, Huifang Lib, Xianfu Menga, Jinliang Liu*a, Lining Suna, Xiaolin Fanb and Liyi Shi*a
a Research Center of Nano Science and Technology, Shanghai University, 200444, China.
b
Key Laboratory of OrganoꢀPharmaceutical Chemistry, Gannan Normal University, Ganzhou
341000, P. R. China.
*To whom correspondence should be addressed:
Dr. Jinliang Liu
Research Center of Nano Science and Technology, Shanghai University, 200444, China.
Phone: +86ꢀ21ꢀ66137197
Fax: +86ꢀ21ꢀ66137197
Email: liujl@shu.edu.cn
Prof. Liyi Shi
Research Center of Nano Science and Technology, Shanghai University, 200444, China.
Phone: +86ꢀ21ꢀ66137208
Email: shiliyi@shu.edu.cn.

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Abstract
It is greatly important to seek a fast and sensitive method for the detection of Cu2 + ion because of
its vital role in human body. Fluorescent probes, especially the rhodamine derivatives, have been
considered as promising candidates for detection of Cu2+ ion due to their attractive features.
However, one problem frequently encountered in rhodamineꢀbased fluorescent probes is that some
of them could not distinguish Cu2+ from Hg2+ and these drawbacks limit their application in
biological samples. In this paper, a rhodamine B derivative (RBH) was grafted to the mesoporous
silica coated upconversion nanoparticles (CSꢀUCNP) to fabricate a new organicꢀinorganic hybrid
nanoprobe. On the addition of Cu2+ ion, an emission band at about 580 nm appeared while the
intensity of the green emission at about 545 nm of the nanoprobe decreased upon the excitation of a
980 nm laser, implying a luminescence resonance energy transfer (LRET) happened from the
CSꢀUCNP to the RBHꢀCu2+ complex. The obtained LRET nanoprobe could detect Cu2+ exclusively
even at the present of Hg2+ with a detection limit of 0.82 ꢁM in absolute ethanol solution. Most
importantly, this nanoprobe can be used for monitoring subcellular distribution of Cu2+ in living
cells based on upconversion luminescence and downconversion fluorescence.
Keywords: Upconversion nanoparticles; Detection; Cell imaging; Rhodamine B derivative

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1. Introduction
Copper is one of the most important trace element and plays a vital role in the body of human
beings, the dishomeostasis of coper ion could result in serious diseases, including Menkes and
Wilson diseases,1,2 Alzheimer’s disease,3 amyotrophic lateral sclerosis (ALS),4 and even death.
Accordingly, the development of probes for monitoring the trace level of Cu2+ ion in biological
samples has drawn much attention during these years. The traditional method of detecting copper
ion include atomic absorption spectrometry, inductively coupled plasma atomic emission
spectrometry (ICPꢀAES), inductively coupled plasma mass spectroscopy (ICPꢀMS). These methods
are always suffer from some drawbacks, such as low sensitivity, complicated pretreatment
procedures, expensive instrumentation dependence, making them not suitable for onꢀline
monitoring. Recently, fluorescent probes have been considered as promising candidates for
detection of Cu2+ ion because of their sample, rapid and nonꢀdestructive characteristics. Some
fluorescence sensors have been developed for detection of Cu2+ ion with high sensitivity and
selectivity.5ꢀ7 Among these report, rhodamine compounds have shown significant advantages over
some traditional fluorophores due to their attractive feature such as high photostability, wide
wavelength range and high fluorescence quantum yield, and thus become an extensive research
topic in the field of chemical and biological.8ꢀ14 However, one problem frequently encountered in
rhodamineꢀbased fluorescent probes is that, upon existence of Cu2+ and Hg2+ simultaneously, these
probes could not distinguish them selectively. These drawbacks limit their application in biological
samples.15ꢀ17
Compared to UV light, nearꢀinfrared (NIR) light has received increasing popularity as an
excitation source due to its deeper tissue penetration and less damage for biological specimens.18ꢀ21
Lanthanideꢀdoped upconversion nanoparticles (UCNPs), which are capable of converting NIR

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excitation light into shorter wavelength light have been proven to exhibit attractive optical and
chemical properties, such as large stokes shifts, low toxicity, weak autoꢀfluorescence backgrounds,
resistance to photoꢀbleaching22ꢀ27. In fact, these merits make UCNPs an ideal candidate as the
luminescence donor in luminescence resonance energy transfer (LRET) nanoprobe. For example,
Chu et al. assembled phospholipidꢀmodified upconversion nanoparticle with rhodamine B via the
hydrophobic van der Waals interactions for the detection of phospholipase D.28 Wang et al.
presented a novel dual LRET system for the detection of Hg2+ and Pb2+ simultaneously by using
gold nanoparticles (AuNPs) as efficient energy acceptors and two kinds of upconversion
nanoparticles as efficient energy donors.29 Li et al. constructed a biosensing platform by combining
the ssDNAꢀmodified UCNPs and graphene oxide (GO) materials acceptors via a strong πꢀπ stacking
effect between nucleobases of ssDNA and sp2 atoms of GO.30
Recently, Li and coꢀauthors’ reported a rhodamine B derivative 4 containing a highly
electronꢀrich S atom to serve as a fluorescence turnꢀon chemodosimeter for Cu2+. However, this
chemodosimeter could not distinguish Cu2+ from Hg2+ because both of them can arouse the similar
“turnꢀon” fluorescence and the color change from colorless to purple7. Considering that the S atom
in rhodamine B derivative 4 plays a key role in the response towards Hg2+, in this work, by
substituting S atom with O atom, a rhodamine B derivative was presented and then grafted to the
mesoporous silica (mSiO2) coated upconversion nanoparticles to fabricate an organicꢀinorganic
hybrid nanoprobe, in which the coreꢀshell structured upconversion nanoparticles
NaYF4:Yb,Er@NaYF4 (CSꢀUCNP) and rhodamine B derivative served as energy donor and energy
acceptor, respectively. Benefited from the oneꢀatom exchange (from S atom to O atom), the
obtained nanoprobe could detect Cu2+ exclusively even at the present of Hg2+, which was often
thought of as the most interferential ion to Cu2+. Most importantly, this nanoprobe can be used for

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monitoring subcellular distribution of Cu2+ in living cells based on upconversion luminescence
(UCL) and downconversion fluorescence.
2. Experimental
2.1 Materials
.
.
All the chemicals were used without further purification: YCl3 6H2O (99.99%), YbCl3 6H2O
.
(99.99%), ErCl3 6H2O (99.99%), tetraethyl orthosilicate (TEOS, >99%) were purchased from
Sigma Aldrich. Oleic acid (90%, technical grade) was purchased from Alfa Aesar. Octadecene (90%,
technical grade), methanol (99.5%), 3ꢀ(triethoxysilyl)propyl isothiocyanate (95%, Guaranteed
reagent), ammonium fluoride (NH4F, ACS, 98%), sodium hydroxide (NaOH, reagent grade, ≥98%),
and cetyltrimethylammonium bromide (CTAB, >99%) were purchased from Aladin Company.
Rhodamine B (RhB, 95%), hydrazine hydrate 80 wt.% solution in H2O were purchased from J & K
Technology Co., Ltd. Acetone (99.5%, analytial reagent), cyclohexane (99.5%, analytial reagent),
anhydrous ethanol (99.7%, analytial reagent), toluene (99.5%, analytial reagent), ammonium nitrate
(NH4NO3, ≥99.0%), and hydrochloric acid (HCl, 36%ꢀ38%, analytial reagent) were obtained from
Sinopharm Chemical Reagent Co., Ltd. Deionized water was used to prepare all aqueous solutions.
Solutions of Cu2+, Hg2+, Mn2+, Na+, K+, Fe3+, Cr3+, Mg2+, Ni2+, K+ and Ca2+ were prepared from
their chloride salts, solutions of Zn2+, Co2+, Fe2+, and Li+ were prepared from zinc acetate dihydrate,
cobaltous nitrate, ferrous sulfate, and lithium fluoride, respectively.
2.2 Synthesis of NaYF4:Yb,Er (UCNP) and NaYF4:Yb,Er@NaYF4 (CS-UCNP)
The βꢀphase NaYF4:Yb,Er nanocrystals was synthesized via a solvothermal method.31 1.56 mL
of YCl3 (1.0 M), 0.40 mL of YbCl3 (1.0 M) and 0.40 mL of ErCl3 (0.10 M) were added to a 100 mL
twoꢀnecked flask and the mixture was heated to 110 oC to evaporate the water, then, 12 mL of oleic
acid and 30 mL of 1ꢀoctadecene were added and the mixture were heated to 150 oC. After cooling to

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room temperature, 20 mL of methanol solution containing NH4F (0.3 g, 8 mmol) and NaOH (0.2 g,
o
5 mmol) was added and the solution was stirred at 110 C to remove methanol and residual water.
o
The resultant solution was then heated to 300 C under N2 atmosphere for 1h. The obtained
suspension was separated by centrifugation and washed with cyclohexane and acetone for three
times and then dispersed in 20 mL of cyclohexane. The synthesis procedure of
NaYF4:Yb,Er@NaYF4 nanoparticles was similar to that of NaYF4:Yb,Er except that 0.80 mL of
YbCl3 (1.0 M) was replaced by 5.0 mL of the prepared NaYF4:Yb,Er.
2.3 Synthesis of CS-UCNP@mSiO2 nanoparticles
0.1 g of CTAB was dissolved in 20 mL of deionized water, and then 2 mL of cyclohexane
solution containing the coreꢀshell nanoparticles (4 mg mLꢀ1) was added. The mixture was stirred
vigorously to evaporate the cyclohexane solvent. For silica coating, 10 mL of the aqueous CTAB
stabilized nanoparticles solution was added to a mixture of 20 mL of deionized water, 3.0 mL of
o
ethanol and 150 ꢁL of 2.0 M NaOH. The mixture was heated to 70 C under stirring. 200 ꢁL of
TEOS was then added dropwise and the heating was maintained for another 2 h with vigorous
stirring. After cooling to room temperature, the nanoparticles were centrifuged and washed with
anhydrous ethanol 4 times. The surfactants were removed via a fast and efficient ion exchange
method,32 and then dispersed in 10 mL of ethanol for further use.
2.4 Synthesis of Rhodamine B hydrazide (RBH)
RBH were prepared as previously reported.5 5 g (10 mmol) of rhodamine B was dissolved in
anhydrous ethanol and 12 mL (200 mmol) of hydrazine hydrate was added dropwise. The mixture
o
was heated to 100 C and maintained for 8 h with vigorous stirring under N2 atmosphere. After
cooling to room temperature, the solvent was removed under reduced pressure. The precipitate was
washed by deionized water and dried under reduced pressure. 1H NMR (CDCl3): δ (ppm) : 7.92 (d,

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1H, ArH), 7.44 (t, 2H, ArH)，7.09 (d, 1H, ArH), 6.46 (s, 2H, xantheneꢀH), 6.41 (d, 2H, xantheneꢀH),
6.29 (d, 2H, xantheneꢀH), 3.6 (s, 2H, NH2), 3.33 (q, 8H, NCH2CH3), 1.16 (t, 12H, NCH2CH3).
2.5 Synthesis of CS-UCNP@mSiO2-RBH nanoparticles
40 mg of CSꢀUCNP@mSiO2 nanoparticles were dissolved in 20 mL of toluene and 20 ꢁL of
3ꢀ(triethoxysilyl) propyl isothiocyanate was added. The mixture was stirred under argon for 24 h.
After cooling to room temperature, RBH (80 mg) was added and the mixture was refluxed for
another 24 h. The nanoparticles were separated by centrifugation at 8000 rpm for10 min, washed
several times with anhydrous ethanol. The obtained nanoparticles were dispersed in 10 mL of
anhydrous ethanol for further use.
2.6 Detection of Cu2+ ion
Stock solutions of Cu2+ (0.02 M), other metal ions (0.1 M) were prepared in deionized water.
The solution of CSꢀUCNP@mSiO2ꢀRBH nanoparticles (3 mg/mL) was prepared in absolute ethanol
solution. For titration experiments, the Cu2+ stock solution was added to 2.0 mL of
CSꢀUCNP@mSiO2ꢀRBH solution by using a micropipette, and then UVꢀVis absorption and
fluorescence spectra were recorded. For selectivity experiments, the test samples were prepared by
adding appropriate amounts of metal ions solution to 2.0 mL of the hybrid nanoꢀprobe
CSꢀUCNP@mSiO2ꢀRBH. For competition experiments, Cu2+ was added to the solutions containing
CSꢀUCNP@mSiO2ꢀRBH and other metal ions of interest. All solutions were stirred for 1 min
before the spectral test. For downconversion fluorescence measurements, excitation was fixed at
520 nm, and emission was collected from 535 to 700 nm, and for upconversion luminescence
measurements, excitation was fixed at 980 nm, and emission was collected from 450 to 680 nm.
2.7 Cell culture
Human cervical carcinoma HeLa cells were provided by the Institute of Biochemistry and Cell

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Biology, SIBS, CAS (China). The cells were grown in RPMI 1640 supplemented with 10% FBS at
37 oC and 5% CO2. Cells (1 ×105 cells mLꢀ1) were plated on 14 mm glass coverslips and allowed to
adhere for 24 h.
2.8 Cell imaging
Before the experiments, HeLa cells were washed with PBS buffer, and then incubated with 0.2
mg mLꢀ1 CSꢀUCNP@mSiO2ꢀRBH in PBS for 2 h at 37 oC, after that, the cells were incubated with
o
10 ꢁM of Cu2+ for 0.5 h at 37 C. Cell imaging was then carried out after washing with PBS.
Confocal fluorescence imaging was performed with an OLYMPUS IX81 laser scanning microscope
and a 60P oilꢀimmersion objective lens. Cells loaded with CSꢀUCNP@mSiO2ꢀRBH were excited at
543 nm using a HeNe laser. Emission was collected from 560 nm to 600 nm. Upconversion
luminescence imaging of Hela cells were executed on laser scanning upconversion luminescence
microscope (LSUCLM) under the excitation of CW 980 nm laser. Emission was collected from 520
nm to 560 nm.
2.9 Cytotoxicity assay
The methyl thiazolyl tetrazolium (MTT) assays were used to probe cellular viability. Hela cells
were cultivated in a 96ꢀwell cellꢀculture plate at a density of 104 per well under 100 % humidity for
12 h at 37 °C and 5% CO2. After that, the medium was substituted for fresh medium at different
concentrations of CSꢀUCNP@mSiO2ꢀRBH (0, 100, 200, 300, 400, 500 ꢁg mL−1). After 24 h, the
medium containing nanoparticles were removed and 10 mL, 3 mg mL−1 of MTT solutions
(dissolved in RPMI 1640) were added and the plate was incubated for an additional 4 h at the same
condition. The cellular viability was calculated by the following formula: Viability (%) = total
viable cells/total cells (viable and nonꢀviable) ×100.33
2.10 Characterization

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Sizes and morphologies of nanoparticles were determined at a JEMꢀ200CX transmission
electron microscope operated at 120 kV and 200 kV using a JEMꢀ2010F highꢀresolution
transmission electron microscope (HRꢀTEM). Samples of the asꢀprepared nanoparticles were
prepared by placing a drop of dilute aqueous dispersions on the surface of a copper grid. Xꢀray
diffraction (XRD) measurements were carried out on a D\maxꢀ2200 Xꢀray diffractometer using Cu
Kα (λ = 1.54 Å) radiation. Fourier transform infrared spectroscopy (FTꢀIR) spectra were acquired in
the spectral range from 4000 to 400 cmꢀ1 with an Avatar 370 by using pressed KBr pellet technique.
UVꢀVis absorption spectra were recorded on a 760CRT dual beam spectrophotometer. Fluorescence
spectra were determined on a LSꢀ55 fluorescence spectrometer. The upconversion luminescent
excitation and emission spectra were obtained on an Edinburgh FLS 920 luminescence spectrometer
1
equipped with an external 0ꢀ800 mW 980 nm adjustable CW laser. H NMR spectra were
performed on an AVANCE 500 MHz spectrometer. The measurements mentioned above were
conducted at room temperature (about 298K). Nitrogen (N2) adsorption/desorption isotherms were
measured by using a Nova l000 analyzer with nitrogen. The samples were outgassed for 4 h at
120 °C before the measurements. Surface areas were calculated by the BrunauerꢀEmmettꢀTeller
(BET) method and pore sizes by the BarrettꢀJoynerꢀHalenda (BJH) methods. Thermogravimetric
analysis (TGA) was performed on a PerkinꢀElmer Pyris Diamond thermal analyzer up to 800 oC at a
heating rate of 10 oC min ꢀ1 under N2.
2.11 Computational Details
Copper complexes were optimized with density functional theory (DFT)34 using the Becke’s
threeꢀparameter hybrid exchange functional combined with the LeeꢀYangꢀParr correlation
functional (B3LYP)35,36. The “doubleꢀζ” quality basis set LANL2DZ and corresponding effective
core potentials37ꢀ39 were used for Cu(II), while the 6ꢀ31G(p,d)40 basis set was used for all other

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atoms. All optimized configurations were confirmed to be minima on the potential energy surfaces
by performing vibrational frequency calculations at the same level. In addition, a conductorꢀlike
polarizable continuum model (CPCM)41 using ethanol (ε = 24.852) as the solvent was considered
for optimization calculations of the involved geometries. Calculations were performed with
Gaussian 09 (Revision D.01)42.
3. Results and discussion
3.1 Characterization of the nanoprobe CS-UCNP@mSiO2-RBH
The synthetic procedure for organicꢀinorganic hybrid nanoprobe (CSꢀUCNP@mSiO2ꢀRBH) is
presented in Scheme 1. The UCNP nanocrystals were hydrophobic and can be wellꢀdispersed in
cyclohexane. As shown in Fig. 1a, the nanocrystals were uniform with an average diameter of 28ꢀ30
nm. After coated with a NaYF4 layer, the obtained CSꢀUCNP were still fairly uniform and highly
monoꢀdisperse, and the particles size reached to 33ꢀ35 nm in diameter (Fig. 1b). Compared with the
corresponding single type nanoparticles, the luminescence of coreꢀshell nanoparticles was
significantly enhanced. As shown in Fig. S1 (See Supporting Information), both the UCNP and
CSꢀUCNP exhibited the similar emission peaks at 521, 542, and 654 nm when excited by 980 nm
laser, which were attributed to the 2H11/2→4I15/2, 4S3/2→4I15/2, and 4F9/2→4I15/2 transitions of Er3+ ion,
respectively.43 The luminescence intensity of CSꢀUCNP is about 8 times larger than that of UCNP,
demonstrated that the coreꢀshell structure nanoparticle can be serve as an ideal energy donor to
fabricate LRETꢀbased nanoprobe.

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Scheme 1. Schematic illustration of the synthetic procedure of the CSꢀUCNP@mSiO2ꢀRBH and the
proposed sensing mechanism of CSꢀUCNP@mSiO2ꢀRBH with Cu2+ ion.
Then, a mesoporous SiO2 layer was coated on CSꢀUCNP and furtherly grafted with RBH to
construct the nanoprobe. As shown in Fig. 1c, the mesoporous silica coated nanoparticles
CSꢀUCNP@mSiO2 were monoꢀdispersed and uniform in ethanol with an average diameter of
85ꢀ100 nm. Following immobilization of the RBH, the diameter of the CSꢀUCNP@mSiO2ꢀRBH
was not significantly changed, and no aggregates were observed (Fig. 1d).

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Fig. 1.TEM images of UCNP (a), CSꢀUCNP (b) dispersed in cyclohexane, CSꢀUCNP@mSiO2 (c),
CSꢀUCNP@mSiO2ꢀRBH (d) dispersed in ethanol. Inset: HRꢀTEM images of CSꢀUCNP.
The XRD patterns of CSꢀUCNP@mSiO2 and CSꢀUCNP@mSiO2ꢀRBH were shown in Fig.
S2A (See Supporting Information). Both of them show the similar diffraction peaks at the range of
10oꢀ90o. The broad peak centered around 23° in the XRD patterns may be ascribed to the coherent
diffusion of the siliceous layer of these nanoparticles, while the main diffraction peaks are ascribed
to the pure hexagonal NaYF4 structure (JCPDS No.16ꢀ0334). Smallꢀangle Xꢀray diffraction
(SAXRD) patterns of CSꢀUCNP@mSiO2 and CSꢀUCNP@mSiO2ꢀRBH samples were shown in Fig.
S2B (See Supporting Information). For CSꢀUCNP@mSiO2, two characteristic diffraction peaks (2θ
= 2.0o, 4.2o) were observed, which can be indexed to (100) and (110) diffractions and assigned to
the twoꢀdimensional hexagonal (P6mm) symmetry. For CSꢀUCNP@mSiO2ꢀRBH, the intensity of
the XRD pattern is decreased slightly, indicating a less ordered mesostructured achieved. This is
attributable to the attachment of the RBH to the mesoporous pores of the silica shell.
The mesoporous silica layer modification of CSꢀUCNP and the grafting with RBH were also

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confirmed by FTꢀIR spectroscopy. As shown in Fig. S3 (See Supporting Information), the peaks at
1080 cmꢀ1 and 550 cmꢀ1 in the FTꢀIR spectrum of CSꢀUCNP@mSiO2 and CSꢀUCNP@mSiO2ꢀRBH
can be ascribed to the stretching vibrations of the SiꢀOꢀSi and SiꢀO group of silica, demonstrating
the successful silica coating modification of CSꢀUCNP. For CSꢀUCNP@mSiO2ꢀRBH, the new
transmission bands at 2982 cmꢀ1 and 2920 cmꢀ1 were assigned to the asymmetric and symmetric
stretching vibrations of methylene (CH2), respectively, and the bands at 1630, 1590 and 1392 cmꢀ1
were attributed to the stretching vibration of the =CꢀH bond in the phenyl group of the attached
rhodamine, suggesting the successful grafting of RBH in the CSꢀUCNP@mSiO2 samples. The
nitrogen adsorptionꢀdesorption isotherms and poreꢀsize distribution data of the nanoparticles were
shown in Fig. 2A. The BET analysis results show that BET surface area and pore volume of
CSꢀUCNP@mSiO2 were 1039 m2gꢀ1 and 0.95 cm2gꢀ1, respectively. A narrow pore size distribution
of 2.78 nm was obtained using the BJH method. When RBH molecules were grafted into the
mesoporous pores of the silica shell, the surface area and pore size have decreased to 973 m2gꢀ1 and
2.53 nm, respectively. The decrease in surface area and pore diameter in CSꢀUCNP@mSiO2ꢀRBH
is attributed to the attachment of the RBH to the inner channels of the silica. Furthermore, The RBH
concentration of CSꢀUCNP@mSiO2ꢀRBH was calculated to be 5.3 wt% as determined by TGA of
CSꢀUCNP@mSiO2 and CSꢀUCNP@mSiO2ꢀRBH. (Fig. S4, See Supporting Information)
1000
(B)
(A)
1.0
0.8
0.6
0.4
0.2
0.0
adsorption of CS-UCNP@mSiO2
desorption of CS-UCNP@mSiO2
adsorption of CS-UCNP@mSiO2-RBH
desorption of CS-UCNP@mSiO2-RBH
2.73
2.53
800
600
400
200
0
CS-UCNP@mSiO2
CS-UCNP@mSiO2-RBH
0.0
0.2
0.4
0.6
0.8
)
1.0
2
3
4
5
6
7
8
9
Relative Pressure
(P/P0
Pore diameter/nm

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Fig. 2. The nitrogen adsorptionꢀdesorption isotherms (A) and poreꢀsize distribution data (B) of
CSꢀUCNP@mSiO2 and CSꢀUCNP@mSiO2ꢀRBH.
3.2 Detection of copper ion in vitro
The UVꢀVis absorption spectra of CSꢀUCNP@mSiO2ꢀRBH towards various metal ions were
illustrated in Fig. 3A. The spectra of CSꢀUCNP@mSiO2ꢀRBH in absolute ethanol exhibited only a
weak band above 500 nm, indicating the mainly existence of closed spirolactam ring of the
rhodamine fluorophore. Upon the addition of Cu2+, the absorbance was significantly enhanced and a
new peak at 555 nm was observed in the absorption spectrum, which was ascribed to the formation
of delocalized xanthane moiety of rhodamine and the ring opening of spirolactam. The proposed
sensing mechanism of CSꢀUCNP@mSiO2ꢀRBH with Cu2+ ion was shown in Scheme 1. Upon
addition of Cu2+, a 1:1 stoichiometry complex between CSꢀUCNP@mSiO2ꢀRBH and Cu2+ formed,
which gave rise to ringꢀopening reaction of the rhodamine group from the nanoprobe. The proposed
mechanism was in consistent with our computational results, in which copper complexes were
optimized with density functional theory (DFT) using the Becke’s threeꢀparameter hybrid exchange
functional combined with the LeeꢀYangꢀParr correlation functional (B3LYP). (Fig. S5, See
Supporting Information) Other metal ions, including Fe2+, Fe3+, Ca2+, Mg2+, Ni2+, Na+, Ba2+, Cr3+,
K+, Mn2+, Zn2+, Co2+, and Li+, had little interference, even Hg2+ which was often thought of as the
most interferential ion to Cu2+, indicating that this nanoprobe can detect Cu2+ exclusively. Fig. 3B
shows the UVꢀVis titration spectra of CSꢀUCNP@mSiO2ꢀRBH solution (3 mg/mL) upon the
addition of different concentrations of Cu2+ ion. Upon the addition of Cu2+ gradually, the intensity
of the absorption band centered at 555 nm increases evidently, indicating the undergoing of the
ringꢀopen process of the rhodamine B unit in CSꢀUCNP@mSiO2ꢀRBH. A linear increase of the
absorbance intensity can be observed with the increasing of Cu2+ in a wide range from 0 to 400 ꢁM

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(See inset of Fig. 3B) with a 15ꢀfold increase of absorbance. According to the formula, the detection
limit LOD = 3S0/S (where 3 is the factor at the 99% confidence level, S0 the standard deviation of
the blank measurements, and S is the slope of the calibration curve), the LOD of this nanoprobe
towards Cu2+ was calculated to be 2.56 µM (Fig. S6A, See Supporting Information). Furthermore,
the color change of CSꢀUCNP@mSiO2ꢀRBH solution upon the addition of various metal ions was
shown in Fig. 3C. Only an obvious purple color could be observed in the presence of Cu2+,
suggested that the nanoprobe CSꢀUCNP@mSiO2ꢀRBH could serve as “nakedꢀeye” chemosensor for
Cu2+.
1.0
CS-UCNP@mSiO2-RBH+Cu2+
0.8
(A)
(B)
0.7
1.998 mM
0.6
0.8
0.6
CS-UCNP@mSiO2-RBH only,
CS-UCNP@mSiO2-RBH+
0.4
0.2
0.0
Cu2+
0.5
0.4
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
Hg2+, Fe2+, Fe3+, Ca2+, Mg2+
,
Ni2+, Na+, Ba2+, Cr3+, K+,
Mn2+, Zn2+, Co2+, Li+
0
400 800 1200 1600 2000
Cu2+/µM
0
400
450
500
550
600
650
450
480
510
540
570
600
630
Warelength (nm)
Warelength (nm)
Fig. 3. (A) UVꢀvis absorption spectra of CSꢀUCNP@mSiO2ꢀRBH upon the addition of 20 ꢁL of
various metal ions (Hg2+, Ca2+, Mg2+, Ni2+, Na+, Ba2+, Cr3+, K+, Mn2+, Zn2+, Co2+, Li+ ,Cu2+, Fe2+,
Fe3+) in absolute ethanol. (B) Absorption changes of CSꢀUCNP@mSiO2ꢀRBH with increasing
amounts of Cu2+ ion. Inset: absorbance at 555 nm as a function of Cu2+ concentration. (C) The
colorimetric performance of the solution upon the addition of various metal ions.
The fluorescence spectra of CSꢀUCNP@mSiO2ꢀRBH towards various metal ions were

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illustrated in Fig. 4A. Only the addition of Cu2+ could induce a significant enhancement of
fluorescence intensity at 580 nm, which was consistent with the results from the UVꢀVis absorption
spectra. Correspondingly, significant enhancements in fluorescence intensity at 580 nm (above 10
fold) can be observed in the fluorescence titration spectra of CSꢀUCNP@mSiO2ꢀRBH solution (3
mg/mL) upon the addition of different concentrations Cu2+ ion (Fig. 4B), and an orange color was
obtained from the fluorescence image of CSꢀUCNP@mSiO2ꢀRBH solution in the presence of Cu2+
ion (Fig. 4C). The detection limit for Cu2+ was calculated to be 0.82 ꢁM, suggesting that the
nanoprobe could sensitively detect environmentally relevant levels of Cu2+ by using fluorescence
technic. (Fig. S6B, See Supporting Information) Moreover, the competition experiment revealed
that the Cu2+ꢀinduced absorbance intensity was not influenced by the subsequent addition of
competitive cations (Fig. S7, the red bars, See the Supporting Information). Obviously, these results
confirmed that the nanoprobe owned remarkably high sensitivity and selectivity toward Cu2+ ion
over other competitive cations.
1200
(A)
CS-UCNP@mSiO2-RBH+Cu2+
(B)
1000
800
600
400
200
0
1000
800
600
400
200
0
952 µM
Cu2+
1000
800
600
400
200
0
CS-UCNP@mSiO2-RBH only,
CS-UCNP@mSiO2-RBH
Hg2+, Fe2+, Fe3+, Ca2+, Mg2+
Ni2+, Na+, Ba2+, Cr3+, K+,
Mn2+, Zn2+, Co2+, Li+
+
,
0
0
500
1000
1500
2000
Cu2+/µM
540 560 580 600 620 640 660 680 700
Wavelength (nm)
540 560 580 600 620 640 660 680 700
Warelength (nm)
(C)

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Fig. 4. (A) Fluorescence spectra of CSꢀUCNP@mSiO2ꢀRBH upon the addition of 20 ꢁL of various
metal ions (Hg2+, Ca2+, Mg2+, Ni2+, Na+, Ba2+, Cr3+, K+, Mn2+, Zn2+, Co2+, Li+ ,Cu2+ Fe2+, Fe3+) in
,
absolute ethanol solution. (B) Fluorescence responses of CSꢀUCNP@mSiO2ꢀRBH with increasing
amounts of Cu2+ ion. Excitation was performed at 520 nm. Inset: fluorescence at 580 nm as a
function of Cu2+ ion concentration. (C) The fluorescence changes of the solution upon addition of
various metal ions.
Under the excitation of CW 980 nm laser, the synthesized CSꢀUCNP exhibited a strong green
UCL emission with the peaks at 534~561 nm, which well matched the main absorption of RBHꢀ
Cu2+ complex (Fig. 5A). Based on the high spectral overlap between them, in
CSꢀUCNP@mSiO2ꢀRBH nanoprobe, CSꢀUCNP was certainly arranged as energy donors that
transferred its green light energy to the attached RBHꢀCu2+ complex (Scheme 1 A). The UCL
emission spectra of CSꢀUCNP@mSiO2ꢀRBH under different concentrations of Cu2+ ion were
presented in Fig. 5B. It can be seen that the intensity of green emission at 526 nm and 545 nm of
CSꢀUCNP@mSiO2ꢀRBH decreases gradually with the increasing of Cu2+ ion concentration, a broad
emission band at about 580 nm appears, corresponding to the emission of the RBHꢀCu2+ complex.
Correspondingly, the photos of CSꢀUCNP@mSiO2ꢀRBH solution exhibited the green luminescence,
and after the addition of Cu2+, the color of the mixed solution changed to orange under 980 nm
irradiation. (Inset in Fig. 5B) The decreasing of the green emission and the emergence of the broad
band at about 580 nm demonstrated the energy transfer from the CSꢀUCNP to the RBHꢀCu2+
complex. As the ratio of the emission intensity at 580 nm (I580 nm) to the intensity of 545 nm (I545 nm
)
is dependent on the concentration of Cu2+, it is possible to use the CSꢀUCNP@mSiO2ꢀRBH as the
nano ratio probe for detection of Cu2+ ion.

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(A)
(B)
CS-UCNP
CS-UCNP@mSiO2-RBH
CS-UCNP@mSiO2-RBH+Cu2+
1200
900
600
300
0
570 580 590 600 610 620 630
Wavelength (nm)
400
450
500
550
600
400
450
500
550
600
Warelength (nm)
Wavelength (nm)
Fig. 5. (A) The upconversion emission spectrum of the CSꢀUCNP and the absorption spectra of
CSꢀUCNP@mSiO2ꢀRBH and CSꢀUCNP@mSiO2ꢀRBH upon addition of Cu2+. (B) Upconversion
emission spectra of CSꢀUCNP@mSiO2ꢀRBH upon addition of 0ꢀ690 ꢁM of Cu2+ ion in absolute
ethanol solution upon 980 nm excitation. Inset: the photos of CSꢀUCNP@mSiO2ꢀRBH solution
before (a) and after (b) the addition of Cu2+ ion under 980 nm irradiation.
The time dependence of the response of CSꢀUCNP@mSiO2ꢀRBH towrds Cu2+ was monitored
by means of fluorescence spectroscopy. As shown in Fig. 6A, the response of
CSꢀUCNP@mSiO2ꢀRBH (3 mg/mL) towards Cu2+ (950 µM) was rapid and a maximal signal was
obtained within 1 min. The results demonstrated that the nanoprobe could be used to monitor Cu2+
in real time. Furthermore, the pH dependence of the Cu2+ sensing by CSꢀUCNP@mSiO2ꢀRBH was
also studied. As shown in Fig. 6B, the fluorescence of the free nanoprobe CSꢀUCNP@mSiO2ꢀRBH
can be negligible under a pH range from 4.1 to 10. After the addition of Cu2+, the fluorescence of
nano probe at 580 nm rapidly increased to a maximum value. The results showed that the
nanoprobe can be worked within a wide pH range of 4.1ꢀ10.

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1000
(B)
900
1000 (A)
900
800
700
600
500
400
300
200
800
CS-UCNP@mSiO2-RBH
CS-UCNP@mSiO2-RBH +Cu2+
700
600
500
400
-20
4
5
6
7
8
9
10
0
20 40
100 120 140 160
60t/s80
PH
Fig. 6 (A) The fluorescence intensity at 580 nm of CSꢀUCNP@mSiO2ꢀRBH (3 mg/mL) with Cu2+
(950 µM) as a function of time. (B) The fluorescence intensity at 580 nm of
CSꢀUCNP@mSiO2ꢀRBH (3 mg/mL) in absolute ethanol solution of different pH in the absence
(bottom line, square) and presence (top line, circle) of 950 µM Cu2+. The pH of the solutions was
adjusted by addition of 0.1 mol Lꢀ1 HCl (or 0.1 mol L−1 NaOH).
3.3 Luminescence imaging of intracellular Cu2+
MTT assay was performed to assess the toxic nature of probe CSꢀUCNP@mSiO2ꢀRBH at the
experimental concentration (Fig. S8). Assuming that the viability of untreated HeLa cells was 100%,
o
the viabilities of HeLa cells still retained 90.0% after the incubation with probe for 24 h at 37 C,
even at a high concentration of 500 ꢁg /mL, indicating that this Cu2+ fluorescent probe can be
considered to be low cytotoxicity. These results demonstrate that CSꢀUCNP@mSiO2ꢀRBH can be
used for monitoring Cu2+ within biological samples. Therefore, laser scanning confocal microscopy
experiments were carried out to demonstrate the applicability of the CSꢀUCNP@mSiO2ꢀRBH in the
bioimaging of intracellular Cu2+. After incubating with 0.2 mg/mL of CSꢀUCNP@mSiO2ꢀRBH for
4 h at 37 oC and then supplemented with 10 ꢁM CuCl2 in the growth medium for 4 h at 37 oC, HeLa
cells showed the green UCL emission at 520ꢀ560 nm under the excitation of 980 nm (Fig. 7b), and
the emission at 560ꢀ600 nm was also observed obviously under the excitation of 543 nm with a
HeNe laser (Fig. 7c). The overlay of brightfield (Fig. 7a) and fluorescence pictures indicated that

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the nanoprobe could be used for monitoring subcellular distribution of Cu2+ in biological samples
based on upconversion luminescence and downconversion fluorescence (Fig. 7d).
Fig. 7. Laser scanning confocal microscopy and bright field images of HeLa cells. (a) Bright field
image, (b) upconversion luminescence image (λex= 980 nm), (c) downconversion fluorescence
image (λex= 543 nm), and (d) overlay image of HeLa cells stained with 0.2 mg/mL
o
CSꢀUCNP@mSiO2ꢀRBH for 4 h at 37 C and then incubated with 10 ꢁM CuCl2 in the growth
medium for 4 h at 37 oC.
4. Conclusion
In conclusion, we have reported a LRET nanoprobe in which coreꢀshell structured lanthanide
upconversion nanocrystals act as energy donor and a rhodamine B derivative (RBH) act as energy
acceptor for the detection of Cu2+. The nanoprobe caused an obvious color change for colorless to
pink that can be seen by nakedꢀeye and an enhancement of the fluorescence emission when upon
the addition of Cu2+. Upon 980 nm excitation, the intensity of green upconverting emission
decreases gradually while a new emission appears at 580 nm enhanced with the increasing
concentration of Cu2+, corresponding to the efficient LRET from CSꢀUCNP to the RBHꢀCu2+
complex. The obtained nanoprobe could detect Cu2+ exclusively even at the present of Hg2+
benefited by the one atom substituting in the rhodamine B derivative. Furthermore, laser scanning
confocal microscopy experiments have established the utility of CSꢀUCNP@mSiO2ꢀRBH probe in
monitoring Cu2+ within living cells, suggesting that this probe will be of great benefit to biomedical

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researchers for studying the effects of Cu2+ in biological systems.
Acknowledgement
We are grateful for the financial support from the National Natural Science Foundation of
China (Grant Nos. 21201117, 21231004), State Key Laboratory of Pollution Control and Resource
Reuse Foundation (NO. PCRRF13001).
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TOC Graphic
A new organic-inorganic hybrid nanoprobe based on luminescence resonance energy
transfer (LRET) from mesoporous silica coated upconversion nanoparticles to
rhodamine B derivative was prepared for distinguish Cu2+ from Hg2+ and living cells
imaging application.