Metal ion induced fluorescence resonance energy transfer between crown ether functionalized quantum dots and rhodamine B: Selectivity of K+ ion

Article abstract of DOI:10.1039/c4ra10925b

We report a ratiometric fluorescent metal ion sensor based on the mechanism of fluorescence resonance energy transfer (FRET) between synthesized 15-crown-5-ether capped CdSe/ZnS quantum dots (QDCE) and 15-crown-5-ether attached rhodamine B (RBCE) in pH 8.3 buffer solution. Fluorescence titration with different metal ions in pH 8.3 buffer solution of the QDCE-RBCE conjugate showed a decrease and an increase in the fluorescence intensity for QDCE and RBCE moieties respectively due to FRET from QDCE to RBCE. This sensor system shows excellent selectivity towards K⁺ ions resulting in increasing efficiency of FRET. Energy transfer efficiency depends on the affinity between metal ions and crown ether functionalized with QDCE/RBCE. The detailed analysis of FRET was explored. This water soluble ratiometric sensor system can act as a good FRET probe for sensing applications especially in biological systems.

Full text of DOI:10.1039/c4ra10925b

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: K. Lin, H. Lee and
D. Namasivayam, RSC Adv., 2014, DOI: 10.1039/C4RA10925B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
3
38x190mm (96 x 96 DPI)

RSC Advances
Page 2 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Metal ion Induced Fluorescence Resonance Energy Transfer
between Crown Ether Functionalized Quantum dots and
+
Rhodamine B: Selectivity of K ion
a
a
HsinꢀLung Lee, Namasivayam Dhenadhayalan and KingꢀChuen Lin*
Department of Chemistry, National Taiwan University, Taipei 106, and
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.
Abstract
We report a ratiometric fluorescent metal ions sensor based on the mechanism
of fluorescence resonance energy transfer (FRET) between synthesized 15ꢀcrownꢀ5ꢀ
ether capped CdSe/ZnS quantum dot (QDCE) and 15ꢀcrownꢀ5ꢀether attached
rhodamine B (RBCE) in pH 8.3 buffer solution. Fluorescence titration with different
metal ions in pH 8.3 buffer solution of QDCEꢀRBCE conjugate showed decrease and
an increase in the fluorescence intensity for QDCE and RBCE moiety respectively
due to FRET from QDCE to RBCE. This sensor system shows excellent selectivity
+
towards K ion resulted in increasing efficiency of FRET. Energy transfer efficiency
depends on the affinity between metal ions and crown ether functionalized with
QDCE/RBCE. The detailed analysis of FRET was explored. This water soluble
ratiometric sensor system can act as a good FRET probe for sensing applications
especially in biological systems.
Keywords: FRET, Metal ion sensor, Quantum dots, Rhodamine B

Page 3 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Introduction
Alkali and alkaline earth metal ions are the most abundant metal ions in
physiological systems and necessary for the function of all living cells where they
activate many enzymes, participate in the oxidation of glucose to produce adenosine
1
triphosphate and also participate in the transmission of nerve signals. Among them,
+
+
2+
2+
the importance of K , Na , Mg and Ca metal ions in biological systems are even
2
more significant. Potassium ion plays many important roles in the chemical processes
3
of life, including maintenance of ion balance and nerve impulse transmission. A
+
deficiency of K ion can cause decreased levels of oxygen, which will reduce the
+
efficiency of cell function. K ion has an essential role in the activation of many
4
,5
+
growth related enzymes in plants. In Photosynthesis, activation of enzymes by K
ion is essential for production of adenosine triphosphate which is an important energy
source for many chemical processes taking place in plant issues. Due to their
biological and chemical importance so far, several variety of analytical methods have
+
6
been implemented for the detection of K ions such as fluorescence spectroscopy,
7,8
9
10,11
12
electrochemistry, surface plasmon resonance, chromatography,
colorimetry,
13
and ionꢀselective electrodes. Though considerable efforts are being made to develop
+
a simple, sensitive and selective method for K ions detection.
Fluorescence techniques are desirable approaches for the measurement of
chemosensors, because this technique is reliable, rapidly performed and highly
14,15
sensitive.
photoinduced electron or charge transfer, fluorescence resonance energy transfer
FRET), and excimer formation responsible for the photophysical changes (such as
There are different photophysical processes primarily including
(
fluorescence intensity, spectral changes and lifetimes) upon metal ions binding with
1
6
fluorophores. Among them, FRET has grown more extensive attentions and
17
promising selectivity and sensitivity.
Therefore we have focused on the
+
development of new ratiometric FRET based sensor for metal ions (K ) in the
aqueous medium. For studying the FRET phenomena, energy donor and energy

RSC Advances
Page 4 of 28
View Article Online
DOI: 10.1039/C4RA10925B
acceptor must be in close contact to each other. An important type of fluorescent
metal ions sensor is based on the molecular recognition of metal ions by crown ethers
which were covalently conjugated to fluorophores. Crown ether has been known to
form sandwich complex with alkali metal ions and it has tendency to bring the donor
18
and acceptor molecules into close proximity. Various fluorescence sensors such as
benzoꢀ15ꢀcrownꢀ5 and diazaꢀ18ꢀcrownꢀ6 fluoroionophores, pyrenylꢀlinked benzoꢀ15ꢀ
crownꢀ5, BODIPYꢀlinked azacrown ethers, benzofuran isophthalate and so on were
+
16d,19
widely used previously for K ion detection.
With a number of unique optical
properties, quantum dots were used as fluorophore for the detection of metal ions
20,21
based on FRET process.
their broad absorption band with large absorption cross sections.
et al. reported that FRET based potassium ion detection by using different size of
Generally quantum dots can act as energy donors due to
22,23
Previously Chen
2
1
CdSe/ZnS quantum dots. There are very few reports to sensing metal ions by using
24,25
rhodamineꢀcrown ether conjugate.
In the present work, we have synthesized the 15ꢀcrownꢀ5ꢀether attached
rhodamine B and 15ꢀcrownꢀ5ꢀether capped CdSe/ZnS quantum dots. In addition, the
synthesized QDCE was capped with two different ligands to enhance the solubility of
QDs in aqueous solution. The potential application of modified QDs and rhodamine
might be controlled as FRET probes to sensing the metal ions. This work aims to
probe the effect of alkali and alkaline earth metal ion on the FRET kinetics between
QDCE and RBCE. Herein, QDCE and RBCE used as an energy donor and energy
acceptor respectively. The study of FRET based metal ion selectivity was performed
for QDCEꢀRBCE conjugate system with various metal ions using fluorescence
titration method. The analysis of FRET effect on sensing metal ions was explored in
+
more detailed. Herein, the detection limit of 4.3 µM was obtained for K ion. The
+
determined value for K ion was found to be lower than those reported
1
7a,18e,26,27
17b,21,28,29
previously,
and close to some earlier literatures.
Note that the

Page 5 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
+
sensing of K ion by using colorimetric methods exhibited lower detection limit in the
3
0,31
nM range.
Results and Discussion
The synthesis of 15ꢀcrownꢀ5ꢀether attached rhodamine B was carried out as
shown in Scheme 1. To synthesize the Bocꢀ6ꢀAhxꢀ15ꢀcrownꢀ5 compound (iii), 6ꢀ
2
aminohexanoic acid was treated with diꢀtertꢀbutyl dicarbonate (Boc O) to protect the
amino group in the presence of sodium hydroxide to yield Bocꢀ6ꢀaminohexanoic acid
32
(ii).
Then 2ꢀhydroxymethylꢀ15ꢀcrownꢀ5ꢀether (1000 mg, 4 mmol) and 4ꢀ
dimethylaminopyridine (DMAP, 30 mg, 0.27 mmol) were added to the above
solution. The reaction mixture was cooled down to 0 °C. Thereafter, N,N'ꢀ
dicyclohexylcarbodiimide (DCC, 825 mg, 4 mmol) dissolved in dichloromethane
(
DCM, 15 mL) was slowly added to the mixture. After stirring at room temperature
under N atmosphere for 12 h, the solution was filtered and the filtrate was washed
with 0.05 N HCl and brine. The organic phase was dried with dry MgSO and
2
4
concentrated in vacuum. The residue was purified by silica gel column
chromatography using ethyl acetate/hexanes (1:1, v/v) which afforded the Bocꢀ6ꢀAhxꢀ
1
5ꢀcrownꢀ5 product. The Boc amino protecting group was then removed by
trifluoroacetic acid (TFA)/DCM (1:1, v/v) for 1 h, and concentrated in vacuum to give
ꢀAhxꢀ15ꢀcrownꢀ5 (iv).
The mixture of DCC (144 mg, 0.7 mmol), hydroxybenzotriazole (HOBt, 95
6
mg, 0.7 mmol) and N,Nꢀdiisopropylethylamine (DIPEA, 90 mg, 0.7 mmol) in cold
DCM were added to a solution of rhodamine B (304 mg, 0.63 mmol) dissolved in
cold DCM (2 mL). After 5 min, 6ꢀAhxꢀ15ꢀcrownꢀ5 (254 mg, 0.7 mmol) was added to
2
the above solution. The mixture was stirred at room temperature under N atmosphere
for 12 h, the resultant mixture were filtered through a glass frit and concentrated in
vacuum to remove the DCM. The residue was dissolved in ethyl acetate and washed
with 1 M K CO . The organic phase was dried with dry MgSO and concentrated
2
3(aq)
4

RSC Advances
Page 6 of 28
View Article Online
DOI: 10.1039/C4RA10925B
in vacuum. The residue was purified by silica gel column chromatography using
methanol/dichloromethane (1:20 to 1:9, v/v) to afford a pink liquid (214 mg, 41%
1
13
yield). The final product RBCE was characterized by H, C NMR and ESIꢀMS (Fig.
S2, ESI†).
The 15ꢀcrownꢀ5ꢀether capped CdSe/ZnS QDs was synthesized by three steps
2
1
33
using the procedure reported by Chen et al. and Mattoussi et al. In order to
enhance the solubility of QDs in buffer solution, the modification was applied. In the
present work, the CdSe/ZnS was capped with dihydrolipoic acid (DHLA) along with
1
5ꢀcrownꢀ5ꢀether as illustrated in Scheme 2. At first step, the 15ꢀcrownꢀ5ꢀlipoic acid
2
1
was prepared following literature method. In brief, lipoic acid (A, 908 mg, 4.4
mmol) was added to a solution of 2ꢀhydroxymethylꢀ15ꢀcrownꢀ5ꢀether (1 g, 4 mmol)
2
in DCM (15 mL), and stirring for 15 min at 0 °C under N . Then Nꢀ(3ꢀ
dimethylaminopropyl)ꢀN'ꢀethylcarbodiimide (EDC) (0.77 mL, 4.4 mmol) and DMAP
49 mg, 0.4 mmol) were added into above solution. The reaction mixture was stirred
(
2
for 18 h under N at room temperature, the resulting mixture was washed with
Millipore water and brine. The organic phase was dried with dry MgSO and
4
concentrated in vacuum. The residue was purified by silica gel column
chromatography using ethyl acetate/hexanes (1:1, v/v) to afford a yellow liquid (1.8 g,
9
3% yield). In the second step, 15ꢀcrownꢀ5ꢀDHLA (C) and DHLA (D) was prepared
by sodium borohydride reduction method. 15ꢀcrownꢀ5ꢀDHLA was characterized by
1
13
H, C NMR and MS (Fig. S3, ESI†). Finally, 5 ꢁL of each 15ꢀcrownꢀ5ꢀDHLA and
DHLA (1:1, v:v) was mixed with CdSe/ZnS QDs solution (5 mg dissolved in 1 mL of
CHCl ). After stirring under dark condition for 12 h at room temperature, the 1 mL of
3
TrisꢀHCl buffer (pH 8.3) was added to form two phases. The addition of
tetramethylammonium hydroxide pentahydrate (TMAH, 25 mg) rendered all QDs
transfer to water phase, and the QDs solution was stirred for 1 h at room temperature.
To remove excess of free surface ligands and base, the QDs solution was washed with
chloroform, and the product was purified by dialyzing twice with Amicon Ultraꢀ15

Page 7 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
centrifugal filter (NMWL of 50 kDa) and characterized by TEM and FTIR (Fig. S4
and S5, ESI†).
In the study of FRET, the synthesized QDCE and RBCE was used as an
energy donor and acceptor respectively. The absorption maximum of QDCE and
RBCE was found to be ~515 and 554 nm respectively. The fluorescence spectrum of
QDCEꢀRBCE conjugate exhibits dual emissions at a visible wavelength range of
~
530 and 575 nm corresponding to the QDCE and RBCE moieties respectively. Fig. 1
shows the fluorescence spectra of QDCE, RBCE and QDCEꢀRBCE conjugate in Trisꢀ
HCl pH 8.3 buffer solution. When the QDCE and RBCE were mixed together in pH
8
.3 buffer solution, the fluorescence intensity of QDCE was found to decrease
whereas the fluorescence intensity of RBCE was found to increase. There are two
essential features responsible for energy transfer process such as (a) the distance
between the energy donor and acceptor, and (b) the spectral overlap between the
emission spectrum of the energy donor and the absorption spectrum of the energy
34
acceptor. In the present study, it is observed that the strong spectral overlap between
the absorption spectrum of RBCE and the emission spectrum of QDCE (inset Fig. 1).
Hence, the observed fluorescence quenching and enhancement is attributed to the
energy transfer from QDCE to RBCE. The study of FRET based metal ion selectivity
was performed using fluorescence titration method. Fluorescence titration of QDCEꢀ
+
RBCE conjugate with different concentration of K ion in pH 8.3 buffer solution was
+
carried out (Fig. 2). Interestingly, addition of K ion facilitates the FRET process.
Both crown ether attached with quantum dot and rhodamine B moieties show reactive
+
with K ion.
It was found that a slight red shift (~4 nm) along with an enhancement of
fluorescence intensity at 575 nm corresponding to RBCE whereas decreases in the
fluorescence intensity at 530 nm corresponding to QDCE with increasing
+
concentration of K ion. On the contrary, the absorption bands of QDCEꢀRBCE
+
conjugate (~515 and 554 nm) remains unchanged on the addition of K ion. These

RSC Advances
Page 8 of 28
View Article Online
DOI: 10.1039/C4RA10925B
+
results confirm that the FRET process is more feasible by the presence of K ion in
QDCEꢀRBCE conjugate system. Moreover, the energy transfer may take place in an
excited state rather than in ground state. To further evidence for FRET, we have
carried out the absorption and fluorescence measurements in varying the
+
concentration of K ion with nonꢀconjugate systems (sole QDCE and RBCE). In both
the systems, the absorption and fluorescence spectra does not much affected even in
+
the presence of highest concentration of K ion (Fig. S6, ESI†). This result reveals
that the observed fluorescence intensity changes in the conjugate system is merely due
to FRET between QDCE and RBCE. The timeꢀresolved fluorescence measurements
were carried out by measuring the fluorescence decay of QDCEꢀRBCE conjugate in
+
the absence and presence of K ion to characterize the energy transfer. The
fluorescence decays were fitted well with triꢀexponential function. The fluorescence
+
lifetimes of QDCEꢀRBCE conjugate in an absence of K ion, were found to be 2.8
+
(τ
1
), 7.3 (τ
2
3
) and 19.2 ns (τ ) with an average lifetime of 12.1 ns. In the presence of K
ion (1 mM), fluorescence lifetimes were found to be 3.1 (τ
1
), 6.8 (τ
2
3
) and 17.8 ns (τ )
with an average lifetime of 10.6 ns. This result shows that the energy donor QDCE
lifetimes (τ
2
and τ
3
1
) were reduced and the energy acceptor RBCE lifetime (τ ) was
+
increased in the presence of K ion. Thus, the fluorescence lifetime studies indicate
35
the nonꢀradiative energy transfer from QDCE to RBCE.
Several positive charged metal ions form stable chelation complexes by
1
8d,36,37
strongly bind with crown ethers.
The affinity of the crown ether with metal ions
is mainly influenced by the denticity of the polyether. Due to the strong binding
+
affinity between the K ion and crown ether moieties in QDCE and RBCE, sandwich
+
18a,18d,21
complex of crown ethers with K is formed.
Consequently both the QD and
rhodamine dye moieties are located in close proximity to each other which leads to
FRET between QDCE and RBCE. The stoichiometry of interaction of QDCEꢀRBCE
+
38
conjugate with K ion was determined by Job’s plot analysis based on the changes in
the fluorescence intensity. The Job’s plot shows the maximum changes at the mole

Page 9 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
+
fraction of about 0.5, which indicates 1:1 interaction of K ion with QDCEꢀRBCE
+
conjugate (Fig. 3). The process of energy transfer and binding mode of K ion with
QDCEꢀRBCE conjugate is depicted in Scheme 3. Initially the conjugate of QDCEꢀ
RBCE was formed due to electrostatic interaction between QDCE and RBCE. The
conjugation induced a FRET from QDCE to RBCE as a result of the overlapping
between emission spectrum of QDCE and absorption spectrum of RBCE. Then
introducing metal ions into QDCEꢀRBCE conjugate system, metal ion forms a
sandwich complex with crown ethers (each one of QDCE and RBCE). Due to metal
ion trapped by crown ethers, the QDCE and RBCE moieties are located closely to
each other which leads to more effective of FRET process. The detection limit was
determined on the relation 3(σ/slope), where σ is the standard deviation of the
−6
response. The detection limit of 4.3×10 M was determined from a linear correlation
observed over the concentration range of 0 – 50 µM. This sensor apparently shows a
+
high selectivity and sensitivity for the detection of K ion. For comparison, the
+
detection limit of 10 µM for K ion was estimated based on an ionꢀselective crown
17a
ether and energy transfer from carbon dots to graphene,
and of 16 µM was
18e
recognized by benzoꢀ15ꢀcrownꢀ5 fluoroionophore/γꢀcyclodextrin complex sensors.
To gain more insight into the FRET based metal ion selectivity, we performed
similar fluorescence measurements for a series of metal ions (alkali metal ions such as
+
+
+
2+
2+
2+
2+
Li , Na , Rb and alkalineꢀearth metal ions like Mg , Ca , Sr , Ba ) with QDCEꢀ
RBCE conjugate under identical experimental conditions (Fig. S7, ESI†).
+
2+
2+
Interestingly, addition of Na , Mg and Ca metal ions showed considerable effects
in selectivity and energy transfer process, which is evident from the metal ion
selectivity plot. Binding affinity of crown ethers mainly depends on the ionic size and
coordination number of the metal ion. For instance calcium ions generally bind to
3
9
oxygen atoms and their preferred coordination numbers range from 6 to 8. So
calcium ion may not be strongly bind with 15ꢀcrownꢀ5ꢀether. The selectivity plot (ꢀF
n+
=
0
F – F versus [M ]) of QDECꢀRBCE conjugate towards different metal ions is

RSC Advances
Page 10 of 28
View Article Online
DOI: 10.1039/C4RA10925B
shown in Fig. 4. F and F are the fluorescence intensity ratio of (F /F ) in the
0
575 530
absence and presence of metal ion respectively. Addition of other metal ions such as
+
+
2+
2+
Li , Rb , Sr and Ba causes the FRET process to be weakened.
The efficiency of FRET strongly depends on the separation distance between
34
the QDCE donor and RBCE acceptor moieties. The energy transfer efficiency was
calculated using the following equation (1)
(1)
in which R
0
is the Forster distance and r is distance between donor and acceptor. The
Forster distance was estimated based on following equation (2),
(2)
where Q
D
is the quantum yield of the donor in the absence of the acceptor, n is the
2
refractive index of the medium, k is the orientation factor and generally assumed to
be equal to 2/3, and J(λ) is the overlap integral, which expresses the degree of spectral
overlap between the donor emission and acceptor absorption. Table 1 shows the
values of calculated distance between donor and acceptor and Forster distance for all
metal ion systems. The calculated distance between donor and acceptor (r) is 46.12 Å
for an above system, which is shorter distance than that of other metal ion sensing
+
systems. The Forster distance was found to be 44.34 Å for QDCEꢀRBCEꢀK ion
sensing system. This calculated Forster distance was found to be lower compared to
40ꢀ42
previously reported values.
0
Freeman et al. have reported the calculated R value
40
of 53.0 Å for the FRET between CdSe/ZnS QDs and rhodhamine B dye. Recently,
0
Hu et al. have reported the R value of 41.0 Å for the system of QDs and R6G
43
derivative mercury conjugate which is close to our R value. The calculated
0
efficiency of FRET is shown in Fig. 5. The FRET efficiency of QDCEꢀRBCE/metal
+
2+
+
ion systems was found to be 44.13, 34.30, 24.21, 26.32 and 1.80% for K , Mg , Na ,
2
+
2+
+
Ca and Ba respectively. K ion exhibits the higher efficiency of FRET compared
with that of all other metal ions. This results further reveal that the donor moiety of

Page 11 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
QDCE and acceptor moiety of RBCE brought in close proximity due to the formation
+
of sandwich complex of crown ethers with K ion which enhances the FRET
efficiency.
The rate constant of energy transfer [kET (r)] between QDCE and RBCE in the
34
presence of metal ions was calculated by using following equation and the values
are compiled in Table 1.
(3)
D
where τ is the fluorescence lifetime of the donor in the absence of the acceptor. The
7
ꢀ1
+
kET (r) was found to be 5.55×10 s for K ion and the rate constant of energy transfer
2+
decreases upon addition of other metal ions. For example, the rate constant for Ba
7
ꢀ1
ion system was found to be 0.13×10 s . The rate of energy transfer depends on the
distance between the donor and acceptor, and spectral overlap. In this present
+
systems, the calculated higher and lower rate constant values respectively for K and
2
+
Ba systems are well evidence for the observed results of distance between the donor
+
2+
and acceptor (r = 46.12 Å for K and 90.10 Å for Ba ). This result confirms that the
+
energy transfer is more feasible when an addition of K ion to QDCEꢀRBCE
conjugate.
Finally, we have estimated the binding constant (log K) by using the Benesi–
44
n+
0
Hildebrand method. The Benesi–Hildebrand plot of 1/(F − F ) versus 1/[M ]
exhibits a straight line, which is further confirms the 1:1 stoichiometry (Fig. S8,
ESI†). The binding constant values are determined by dividing the intercept by the
slope of the straight line. The log K was estimated to be 3.09, 2.77, 2.45, 1.90, 1.77,
+
2+
+
2+
+
+
and 1.62 for K , Mg , Na , Ca , Rb and Li metal ions respectively. The binding
2
+
2+
constant of other metal ions (Sr , Ba ) could not be estimated due to minor changes
in the corresponding fluorescence spectra. Based on all results, it may conclude that
the synthesized fluorescent QDCE and RBCE performs as a great selective FRETꢀ
+
based ratiometric fluorescence probe towards K ion.

RSC Advances
Page 12 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Conclusions
We have established a FRET based metal ion sensors. A varying metal ion
concentrations was introduced to the FRET sensor system, FRET from QDCE to
RBCE was induced by metal ions. Efficiency of energy transfer depends on the
affinity between metal ions and crown ether functionalized with QDCE/RBCE. The
+
sensor shows excellent selectivity towards K ion resulting in the enhanced FRET
between QDCE and RBCE conjugate, which evident from the observed higher
+
efficiency and rate constant of FRET for K ions compared with that of other metal
+
ions. The stoichiometry of interaction of QDCEꢀRBCE conjugate with K ion was
+
found to be 1:1 which reveals that the K ion formed sandwich like complex with
+
crown ethers of QDCEꢀRBCE conjugate. Due to high selectivity for K , the QDCEꢀ
RBCE conjugate sensor system have good potential for an application in the
determination of this metal ion in biological systems.
Experimental Section
Materials and Methods
All commercially available organic reagents were used without further
2 2
purification. Dichloromethane was distilled from CaH under N . Flash column
chromatography was carried out with silica gel 60 (Merck). Thin layer
chromatography was performed with silica gel 60 F254 plates purchased from Merck.
The hydrophobic CdSe/ZnS QDs capped with octadecylamine were obtained from
Ocean NanoTech, USA, and used as without further purification.
UV–Visible absorption spectra were recorded on a Thermo Scientific evolution
2
20 spectrophotometer. Fluorescence spectra were recorded on a PerkinꢀElmer LS45
spectrophotometer. Fluorescence decays were recorded by using the timeꢀcorrelated
45 1
13
singleꢀphoton counting technique as reported elsewhere. H NMR and C NMR
spectra were recorded on a Bruker DPX 400MHz NMR spectrometer in deuterated

Page 13 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
solvents. All NMR spectra were assigned, and the chemical shifts are reported in ppm
downfield relative to the chemical shift. Electrospray ionization mass spectrometry
(ESIꢀMS) experiments were carried out on a Bruker microTOFꢀQII mass
spectrometer. Infrared spectra were recorded on a Thermo Scientific Nicolet iS5 FTꢀIR
spectrometer. TEM observations were conducted on a Hitachi Hꢀ7100 transmission
electron microscope. All measurement were recorded at ambient temperature.
Characterization of RBCE and QDCE
Boc-6-Ahx-15-crown-5 [Scheme 1, (iii)]
1
3
H NMR (400 MHz, CDCl ): δ = 4.53 (s, NH), 4.22ꢀ4.04 (m, 2H), 3.83ꢀ3.52 (m,
1
3
1
9H), 3.08 (q, 2H), 2.31 (t, 2H), 1.65ꢀ1.29 (m, 15H). C NMR (100 MHz, CDCl ): δ
3
=
173.43, 79.10, 71.12, 70.93, 70.85, 70.78, 70.56, 70.44, 70.22, 64.06, 40.37, 34.06,
2
9.73, 28.42, 26.26, 24.55 ppm.
1
5-crown-5-ether attached Rhodamine B, RBCE [Scheme 1, (v)]
1
H NMR (400 MHz, CD OD): δ = 7.85ꢀ7.82 (m, 1H), 7.53ꢀ7.48 (m, 2H), 7.05ꢀ
3
7
3
.02 (m, 1H), 6.42ꢀ6.23 (m, 6H), 4.26ꢀ4.08 (m, 2H), 3.81ꢀ3.55 (m, 19H), 3.36 (q, 8H),
13
.04 (m, 2H), 2.14 (t, 2H), 1.37 (t, 2H), 1.14 (t, 12H), 1.06 (tt, 4H). C NMR (100
MHz, CD
3
OD): δ = 174.73, 169.81, 154.87, 154.77, 150.38, 133.96, 132.51, 129.68,
1
6
1
29.49, 125.09, 123.39, 109.50, 106.50, 99.03, 77.63, 70.50, 70.37, 70.23, 70.05,
9.93, 69.80, 69.77, 69.53, 66.98, 63.46, 45.37, 41.01, 34.57, 28.87, 27.46, 25.46,
+
+
2.88 ppm. ESIꢀMS: m/z calcd. for [M + Na ] 810.4300, found 810.4289.
1
5-crown-5- DHLA [Scheme 2, (C)]
1
3
H NMR (400 MHz, CDCl ): δ = 4.21ꢀ4.03 (m, 2H), 3.82ꢀ3.51 (m, 19H), 3.15ꢀ
3
1
.06 (m, 2H), 2.42 (sext, 1H), 2.31 (t, 2H), 1.87 (sext, 1H), 1.69ꢀ1.59 (m, 4H), 1.48ꢀ
1
3
3
.35 (m, 2H). C NMR (100 MHz, CDCl ): δ = 173.12, 70.91, 70.72, 70.62, 70.56,

RSC Advances
Page 14 of 28
View Article Online
DOI: 10.1039/C4RA10925B
7
0.36, 70.28, 70.21, 70.04, 63.93, 56.13, 40.03, 38.30, 34.40, 33.79, 28.52, 24.47
+
+
ppm. ESIꢀMS: m/z calcd. for [M + Na ] 461.1638, found 461.1633.
General procedure for sensing of metal ion
Metal chloride stock solutions were prepared in pH 8.3 buffer solutions, and the
fluorescence titration measurements were carried out by adding small volumes (upto 5
ꢁl) of the metal ion to the QDCEꢀRBCE conjugate solution in a quartz cuvette. After
an addition of metal ion to the cuvette, the solution was shaken well and kept 2 min
before measurement. QDCE and RBCE stock solutions were prepared in pH 8.3
buffer solutions.
Acknowledgements
This work was supported by Ministry of Science and Technology of Taiwan,
Republic of China under contract no. NSC 99ꢀ2113ꢀMꢀ002ꢀ010ꢀMY3.
1
13
Electronic Supplementary Information (ESI) available: H NMR, C NMR, FTIR
spectra and fluorescence spectral study figures. See DOI:
Notes and references
a
Equal contribution
*
To whom correspondence should be addressed.
Department of Chemistry, National Taiwan University, Taipei 106, and Institute of
Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.
Email: kclin@ntu.edu.tw; Fax: +886ꢀ2ꢀ2362ꢀ1483; Tel: +886ꢀ2ꢀ3366ꢀ1162.
1
(a) W. Maret and A. Wedd, Binding, Transport and Storage of Metal Ions in
Biological Cells, RSC, Cambridge, UK, 2014; (b) J. A. Cowan, Inorganic
biochemistry: An introduction, WileyꢀVCH, Inc., USA, 1997; (c) J. B. Ames, K. B.

Page 15 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Hendricks, T. Strahl, I. G. Huttner, N. Hamasaki and J. Thorner, Biochemistry,
000, 39, 12149−12161; (d) J. C. Henquin, T. Tamagawa, M. Nenquin and M.
Cogneau, Nature, 1983, 301, 73−74; (e) B. Valeur and I. Leray, Coord. Chem.
Rev., 2000, 205, 3–40; (f) Y. Xiang and Y. Lu, Inorg. Chem., 2014, 53
925−1942.
2
,
1
2
(a) J. Lee, H.ꢀJ. Kim and J. Kim, J. Am. Chem. Soc., 2008, 130, 5010–5011; (b) I.
Leray, B. Valeur, F. O’Reilly, J.ꢀL. Habib Jiwan, J.ꢀPh. Soumillion and B. Valeur,
Chem. Commun., 1999, 795−796; (c) H. Ueyama, M. Takagi and S. Takenaka, J.
Am. Chem. Soc., 2002, 124, 14286−14287; (d) H. Ma, E. A. Gibson, P. J. Dittmer,
R. Jimenez and A. E. Palmer, J. Am. Chem. Soc., 2012, 134, 2488–2491; (e) N. B.
Sankaran, S. Nishizawa, M. Watanabe, T. Uchidaa and N. Teramae, J. Mater.
Chem., 2005, 15, 2755−2761; (f) H. Wang, D. M. Wang, M. X. Gao, J. Wang and
C. Z. Huang, Anal. Methods, 2014, 6, 7415−7419; (g) E. Arunkumar, P. Chithra
and A. Ajayaghosh, J. Am. Chem. Soc., 2004, 126, 6590−6598.
rd
3
4
5
6
L. D. Stryer, Biochemistry, 3 ed; Freeman, New York, 1988, pp 949−974.
I. Cakmak, J. Plant Nutr. Soil Sci., 2005, 168, 521–530.
P. Maser, M. Gierth and J. I. Schroeder, Plant Soil, 2002, 247, 43–54.
T. Hirata, T. Terai, T. Komatsu, K. Hanaoka and T. Nagano, Bioorg. Med. Chem.
Lett., 2011, 21, 6090−6093.
7
8
M. F. S. Teixeira, B. H. Freitas, P. M. Seraphim, L. O. Salmazo, M. A. Nobre and S.
Lanfredi, Proc. Chem., 2009,
B. T. T. Nguyen, J. Q. Ang and C.ꢀS. Toh, Electrochem. Commun., 2009, 11
861−1864.
1, 293−296.
,
1
9
1
H. Chen, Y. S. Gal and S. H. Kim, Sens. Actuators, B, 2008, 133, 577−581.
0 B. Yu, L. Nie and S. Yao, J. Chromatogr. B: Biomed. Sci. Appl., 1997, 693
,
4
3−49.
1 L. B. de Caland, E. L. C. Silveira and M. Tubino, Anal. Chim. Acta, 2012, 718
16−120.
1
,
1

RSC Advances
Page 16 of 28
View Article Online
DOI: 10.1039/C4RA10925B
1
2 T. Li, E. Wang and S. Dong, Anal. Chem., 2010, 82, 7576–7580.
1
3 S. Kim, H. Kim, K. H. Noh, S. H. Lee, S. K. Kim and J. S. Kim, Talanta, 2003,
, 709−716.
61
1
4 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P.
McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515−1566.
5 X. Chen, X. Tian, I. Shin and J. Yoon, Chem. Soc. Rev., 2011, 40, 4783−4804.
6 (a) B. Valeur, Molecular Fluorescence. Principles and Applications, WileyꢀVCH,
Weiheim, 2002; (b) F. He, Y. Tang, S. Wang, Y. Li and D. Zhu, J. Am. Chem.
Soc., 2005, 127, 12343–12346; (c) J. Kim, D. T. McQuade, S. K. McHugh and T.
M. Swager, Angew. Chem., Int. Ed., 2000, 39, 3868−3872; (d) H. He, M. A.
Mortellaro, M. J. P. Leiner, R. J. Fraatz and J. K. Tusa, J. Am. Chem. Soc., 2003,
1
1
125, 1468–1469.
1
7 (a) W. Wei, C. Xu, J. Ren, B. Xu and X. Qu, Chem. Commun., 2012, 48, 1284–
1
286; (b) J. Yin, C. Li, D. Wang and S. Liu, J. Phys. Chem. B, 2010, 114, 12213–
1
2220; (c) B. Sen, M. Mukherjee, S. Pal, K. Dhara, S. K. Mandal, A. R. Khudaꢀ
Bukhsh and P. Chattopadhyay, RSC Adv., 2014,
8 (a) S.ꢀY. Lin, S.ꢀW. Liu, C.ꢀM. Lin and C.ꢀH. Chen, Anal. Chem., 2002, 74
30−335; (b) M.ꢀL. Ho, J.ꢀM. Hsieh, C.ꢀW. Lai, H.ꢀC. Peng, C.ꢀC. Kang, IꢀC. Wu,
C.ꢀH. Lai, Y.ꢀC. Chen and P.ꢀT. Chou, J. Phys. Chem. C, 2009, 113, 1686–1693;
c) S. Flink, F. C. J. M. Van Veggel and D. N. Reinhoudt, J. Phys. Chem. B, 1999,
4, 14919–14927.
1
,
3
(
103, 6515–6520; (d) G. W. Gokel, W. M. Leevy and M. E. Weber, Chem. Rev.,
2
004, 104, 2723−2750; (e) A. Yamauchi, T. Hayashita, A. Kato, S. Nishizawa, M.
Watanabe and N. Teramae, Anal. Chem., 2000, 72, 5841−5846.
1
9 (a) A. Yamauchi, T. Hayashita, S. Nishizawa, M. Watanabe and N. Teramae, J.
Am. Chem. Soc., 1999, 121, 2319–2320; (b) A. P. Desilva, H. Q. N. Gunaratne and
K. R. A. S. Sandanayake, Tetrahedron Lett., 1990, 31, 5193–5196; (c) M. Baruah,
W. Qin, R. A. L. Vallee, D. Beljonne, T. Rohand, W. Dehaen and N. Boens, Org.

Page 17 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Lett., 2005,
997, 48, 1609−1614.
0 M. J. RuedasꢀRama, X. Wang and E. A. H. Hall, Chem. Commun., 2007,
544−1546.
7, 4377−4380; (d) K. H. Mühling and B. Sattelmacher, J. Exp. Bot.
1
2
2
1
1 CꢀY. Chen, CꢀT. Cheng, CꢀW. Lai, PꢀW. Wu, KꢀC. Wu, PꢀT. Chou, YꢀH. Chou
and HꢀT. Chiu, Chem. Commun., 2006, 263−265.
2
2
2 W. R. Algar and U. J. Krull, Anal. Bioanal. Chem. 2008, 391, 1609−1618.
3 G. Chen, F. Song, X. Xiong and X. Peng, Ind. Eng. Chem. Res. 2013, 52, 11228
−
1
1245.
4 D. Liu, T. Pang, K. Ma, W. Jiang and X. Bao, RSC Adv., 2014,
5 X. Zhang, Y. Shiraishi and T. Hirai, Tetrahedron Lett., 2008, 49, 4178−4181.
2
2
2
4, 2563−2567.
6 W. Yuanboonlim, W. Siripornnoppakhun, N. Niamnont, P. Rashatasakhon, T.
Vilaivan and M. Sukwattanasinitt, Biosens. Bioelectron., 2012, 33, 17−22.
7 C. Shi, H. Gu and C. Ma, Anal. Biochem., 2010, 400, 99−102.
2
2
2
8 Y. Liu, B. Li, D. Cheng and X. Duan, Microchem. J., 2011, 99, 503−507.
9 X. Fan, H. Li, J. Zhao, F. Lin, L. Zhang, Y. Zhang and S. Yao, Talanta, 2012, 89
7−62.
0 Z. Chen, J. Guo, H. Ma, T. Zhoua and X. Lia, Anal. Methods, 2014,
1 Z. Chen, Y. Huang, X. Li, T. Zhou, H. Ma, H. Qiang and Y. Liu, Anal. Chim. Acta,
013, 787, 189–192.
2 N. Gavande, H.ꢀL. Kim, M. R. Doddareddy, G. A. R. Johnston, M. Chebib and J.
R. Hanrahan, ACS Med. Chem. Lett., 2013, , 402−407.
,
5
3
3
6, 8018−8021.
2
3
3
3
4
3 H. Mattoussi, J. M. Mauro, E. R. Goldman, G. P. Anderson, V. C. Sundar, F. V.
Milkulec and M. G. Bawendi, J. Am. Chem. Soc., 2000, 122, 12142−12150.
4 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed; Springer: New
York, 2006.
3
5 E. Lee, C. Kim and J. Jang, Chem. Eur. J., 2013, 19, 10280−10286.
6 K. Rurack, W. Rettig,and U. ReschꢀGenger, Chem. Commun., 2000, 407–408.
3

RSC Advances
Page 18 of 28
View Article Online
DOI: 10.1039/C4RA10925B
3
3
3
7 C. Han and H. Li, Anal. Bioanal. Chem., 2010, 397, 1437–1444.
8 P. Job, Ann. Chem., 1928,
9 A. K. Katz, J. P. Glusker, S. A. Beebe and C. W. Bock, J. Am. Chem. Soc., 1996,
18, 5752–5763.
0 R. Freeman, T. Finder, L. Bahshi and I. Willner, Nano lett., 2009,
9, 113−203.
1
4
4
9, 2073−2076.
1 S. Nagatoishi, T. Nojima, E. Galezowska, A. Gluszynska, B. Juskowiak and S.
Takenaka, Anal. Chim. Acta, 2007, 581, 125–131.
4
2 J.ꢀL. Mergny and J.ꢀC. Maurizot, ChemBioChem, 2001,
3 B. Hu, L.ꢀL. Hu, M.ꢀL. Chen and J.ꢀH. Wang, Biosens. Bioelectron., 2013, 49
99–505.
2, 124–132.
4
,
,
4
4
4 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703−2707.
5 C.ꢀL. Chang, P.ꢀY. Tsai, Y.ꢀP. Chang and K.ꢀC. Lin, ChemPhysChem, 2012, 13
4
2
711–2720.

Page 19 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Figure and Scheme captions
Fig. 1 Fluorescence spectra of QDCE, RBCE and QDCE-RBCE conjugate in pH 8.3
buffer. λex = 375 nm. Inset: Spectral overlap between emission spectrum of QDCE
and absorption spectrum of RBCE.
Fig. 2 Fluorescence spectra of QDCE-RBCE conjugate with different concentration
+
-3
of K ion (0 to 2.5×10 M) in pH 8.3 buffer. λex = 375 nm.
+
Fig. 3 Job’s plot for the binding of K ion with QDCE-RBCE conjugate. Mole
+
fraction = [conjugate] / ([conjugate] + [K ion]).
Fig. 4 Metal ion selectivity plot of QDCE-RBCE conjugate upon addition of different
metal ions in pH 8.3 buffer.
Fig. 5 FRET efficiency of QDCE-RBCE conjugate with different metal ions in pH 8.3
buffer.
Table 1 Forster radius, rate constant and efficiency of energy transfer for QDCE-
RBCE conjugate with metal ion systems.
Scheme 1 The synthetic procedure of 15-Crown-5-ether attached rhodamine B.
Scheme 2 The synthetic procedure of 15-Crown-5-ether capped CdSe/ZnS QDs.
Scheme 3 Schematic representation of FRET between QDCE and RBCE.

RSC Advances
Page 20 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Fig. 1 Fluorescence spectra of QDCE, RBCE and QDCE-RBCE conjugate in pH 8.3
buffer. λex = 375 nm. Inset: Spectral overlap between emission spectrum of QDCE
and absorption spectrum of RBCE.

Page 21 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Fig. 2 Fluorescence spectra of QDCE-RBCE conjugate with different concentration
+
-3
of K ion (0 to 2.5×10 M) in pH 8.3 buffer. λex = 375 nm.

RSC Advances
Page 22 of 28
View Article Online
DOI: 10.1039/C4RA10925B
+
Fig. 3 Job’s plot for the binding of K ion with QDCE-RBCE conjugate. Mole
+
fraction = [conjugate] / ([conjugate] + [K ion]).

Page 23 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Fig. 4 Metal ion selectivity plot of QDCE-RBCE conjugate upon addition of different
metal ions in pH 8.3 buffer.

RSC Advances
Page 24 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Fig. 5 FRET efficiency of QDCE-RBCE conjugate with different metal ions in pH 8.3
buffer.

Page 25 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Table 1: Forster radius, rate constant and efficiency of energy transfer for QDCE-
RBCE conjugate with metal ion systems.
Metal
ions
R
0
r
k
ET (r)
E
7
-1
(Å)
(Å)
[×10 s ]
(%)
+
Li
45.06
44.19
44.34
46.06
44.03
44.75
46.43
46.26
62.46
53.46
46.12
65.8
0.99
2.24
5.55
0.82
3.67
2.50
0.40
0.13
12.36
24.21
44.13
10.53
34.30
26.32
5.40
+
Na
+
K
+
Rb
2
+
Mg
49.07
53.16
74.94
90.10
2
+
Ca
2+
Sr
2
+
Ba
1.80

RSC Advances
Page 26 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Scheme 1 The synthetic procedure of 15-Crown-5-ether attached rhodamine B.

Page 27 of 28
RSC Advances
View Article Online
DOI: 10.1039/C4RA10925B
Scheme 2 The synthetic procedure of 15-Crown-5-ether capped CdSe/ZnS QDs.

RSC Advances
Page 28 of 28
View Article Online
DOI: 10.1039/C4RA10925B
Scheme 3 Schematic representation of FRET between QDCE and RBCE.