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Efficient fluorescence energy transfer system
between fluorescein isothiocyanate and CdTe
quantum dots for the detection of silver ions
Yueshu Feng, Liwei Liu,* Siyi Hu, Peng Zou, Jiaqi Zhang, Chen Huang,
Yue Wang, Sihan Wang and Xihe Zhang
ABSTRACT: We report a fluorescence resonance energy transfer (FRET) system in which the fluorescent donor is fluorescein iso-
thiocyanate (FITC) dye and the fluorescent acceptor is CdTe quantum dot (QDs). Based on FRET quenching theory, wedesigned a
method to detect the concentration of silver ions (Ag
+
). The results revealed a good linear trend over Ag
+
concentrations in the
range 0.01–8.96 nmol/L, a range that was larger than with other methods; the quenching coefficient is 0.442. The FRET mecha-
nism and physical mechanisms responsible for dynamic quenching are also discussed. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: quantum dots; fluorescence resonance energy transfer; fluorescence spectrum; energy-transfer efficiency; quenching method
Introduction
Fluorescence resonance energy transfer (FRET) is a non-radiative
process in which energy transfers from a donor fluorophore to a
nearby acceptor chromophore via dipole interactions between
molecules. This specific performance manifests itself through a de-
crease in or quenching of the fluorescence intensity of the donor
and an increase in the fluorescence intensity of the acceptor. Be-
cause of its advantages of high detection speed and sensitivity,
FRET technology has been used in a wide range of chemical and
biological fields, such as molecular binding events, protein and
DNA conformation analysis, and acceptor–ligand interactions
(1–4). The application ofFRET technology to the detection ofmetal
ions has also attracted considerable attention. In recent years, a
large amount of effort has been devoted to developing sensitive
FRET-based sensing strategies for metal ions. One of the most
important issues to be considered is how to improve the energy-
transfer efficiency of the FRET system. This depends on many
factors, such as the extent of the spectral overlap, the relative
orientation of the transition dipoles, and the distance between
the donor and acceptor. Therefore, the construction of a suitable
donor and acceptor FRET system will play a decisive role in achiev-
ing high energy-transfer efficiency for FRET performance.
The unique photophysical properties of CdTe quantum dots
(QDs), such as their high photostability and fluorescence quantum
yields, broad excitation bands, and sharp and tunable emission
spectrum, offer significant advantages and QDs are frequently
used as a FRET probe (5–7). Although many studies have shown
that QDs can function effectively as energy acceptors in
FRET-based assays, inorganic fluorophores may also be candidate
energy donors. QDs have large extinction coefficients that extend
over a broad range of wavelengths, and inorganic dyes have rela-
tively high photoluminescence quantum yields. Based on Förster
formalism predictions, we conclude that dyes are able to donate
energy to QD acceptors, and efficient FRET should be achieved in
adye–QD system. Although several types of fluorescent materials
can be used together with CdTe QDs to form the FRET system, a
system based on fluorescein isothiocyanate (FITC) and CdTe QDs
is rarely reported. FITC is a type of fluorescent dye used in fluores-
cent antibody techniques. It can combine with a variety of
antibody proteins without losing the ability to bind with the
antigen and is often used in medicine and agriculture.
As a heavy metal, Ag
+
has toxicity in a living system, and a sen-
sitive and selective heavy metal ion detection method that can
provide a simple, practical and high-throughput routine determi-
nation of heavy metals is urgently required. There have been many
attempts to design sensing systems for metal ions, e.g. UV/vis spec-
trometry, atomic fluorescence spectroscopy, high-performance
liquid chromatography (HPLC) inductively coupled plasma/mass
spectra (ICP/MS), and sensors based on organic chromophores or
fluorophores, gold nanoparticles, QDs and proteins. All of the
above methods has made significant progress because of its ad-
vantages of a low detection limit and high sensitivity. However,
the above-mentioned methods are complex and expensive, waste
reagent and manpower, and some do not meet the detection
requirements. A new method to detect ultra-trace amounts of
Ag
+
inwaterthatismoresensitive,fasterandcheaperisrequired.
Because of its high sensitivity, fluorescence spectroscopy has
become a powerful tool for detecting ultra-trace quantities of
heavy metal ions. Moore first put forward a method for detecting
metal ions using QDs (8); the fluorescence spectroscopy method.
It was thought that an interaction might occur between the QDs
surface and metal ions, which would lead to a significant change
in the surface charge and structure, resulting in a change in the
* Correspondence to: Liwei Liu, School of Science, Changchun University of
Science and Technology, International Joint Research Center for
Nanophotonics and Biophotonics, Changchun 130000, People’sRepublic
of China. Tel: +86-0431-85582524, E-mail: liulw@cust.edu.cn
School of Science, Changchun University of Science and Technology,
Changchun, People’sRepublicofChina
Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd.
Research article
Received: 16 October 2014, Revised: 6 May 2015, Accepted: 9 June 2015 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2967
1
luminescent properties of QDs.Most sensors developed for the de-
tection of heavy metal ions are based on conventional fluores-
cence emission by either quenching (turn off ) or enhancement
(turn on) of the fluorescence intensity; few are based on FRET.
When the FRET process occurs, energy is transferred from a donor
to an acceptor. However, the FRET process is destroyed on
injectingmetal ions, resulting in changes in the fluorescence inten-
sity of the system. In this study, we used FITC–CdTe to construct a
FRET system and apply this proposed system in the analysis of Ag
+
.
Such a FRET strategy has not been reported previously.
This paper reported a novel platform for effectively measuring
and determining Ag
+
using FRET from FITC dye to CdTe QDs. We
discussed the energy-transfer mechanism under different experi-
mental conditions; the fluorescence resonance energy transfer
parameters were also calculated. Upon the introduction of Ag
+
,
the fluorescence of CdTe QDs became weaker, with a linear rela-
tionship between the degree of quenching and the Ag
+
concen-
tration; thus, the change in fluorescence intensity provided a
novel method for the detection of ultra-trace amounts Ag
+
.The
energy-transfer mechanism was studied in detail. Ultra-sensitive
quantitative detection could be realized because of the high fluo-
rescence quantum yield of FITC and high extinction coefficient of
CdTe QDs. The proposed method was used to determine Ag
+
,
giving a satisfactory result.
Experimental
Instrumentation
The following instrumentation was used: multifunctional micro-
plate reader (TECAN Company, Männedorf, Switzerland), FLS980
fluorescence spectrometer (Edinburgh Instruments, Livingston,
UK), semiconductor lasers (447 nm), QE65000 fiber optic spectrom-
eter (Ocean Optics, Shanghai, China), UB-7 pH meter (Denver
Instrument Company, Arvada, USA) and a HS
4
type heating
magnetic stirrer (IKA, Guangzhou, China).
Materials
All chemicals used in the experiments were of analytical grade.
Cadmium chloride, anhydrous (CdCl
2
,≥99.0%); tellurium powder
(Te, ≥99.5%), sodium borohydride (NaBH
4
,≥98.0%) and 3-
mercaptopropionic acid (MPA, ≥99%) were purchased from Alfa
Aesar (MA, USA). FITC (≥95%) was purchased from TCI (Shanghai,
China). Phosphate-buffered saline (PBS, ≥98%) was purchased
from Thermo (Massachusetts, USA) and sodium hydroxide (NaOH,
≥99%) was from Sigma-Aldrich (ST.LOUIS, MO, USA). Ultrapure wa-
ter used in the experiments.
Synthesis of CdTe QDs/poly(allylamine hydrochloride)
CdTe QDs were prepared as described previously (9), Te precursor
was prepared by reducing 90 mg of tellurium powder with 140 mg
of sodium borohydride (NaBH
4
) in 5 mL of ultrapure water at room
temperature. The mixture was stirred for 1–2 h until it became col-
orless. CdCl
2
(183 mg), MPA (220 μL) and ultrapure water (20 mL)
were loaded into a three-necked flask under stirring. The pH was
adjusted to 9 by dropwise addition of NaOH solution. The flask
was sealed and the Te precursor was injected into the mixture un-
der a nitrogen atmosphere. The reaction mixture was slowly
heated under a nitrogen atmosphere to 98°C. CdTe QDs with
different emission wavelengths were collected every 2 h.
Poly(allylamine hydrochloride) (PAH) was purchased from
Sigma-Aldrich. A 50-μL aliquot of QDs stock solution was washed
with ethanol and centrifuged at 7800 r.p.m. Next, 250 μLofPAH
solution (0.5 mg/mL) was added to redisperse the QDs precipitate,
followed by sonication for 10 min. The QDs/PAH particles were
then collected by centrifugation at 15 000 r.p.m. for 10min to re-
move the non-adsorbed PAH. The resulting QDs/PAH particles
were dispersed in water and stored at 4 °C in the dark.
Construction of the FRET system
The FRET system was constructed as follows (2): 0.5 mL of FITC solu-
tion (6.25 × 10
-4
mg/mL) and 0.5 mL of CdTe QDs/PAH solution
(6 mg/mL) were ad ded to 1.2 × 1.2 cm cuvettes in turn. PBS (pH 7.2)
was then injected into the mixture to give a volume of 2 mL; the
solution was kept at room temperature for 30 min before use.
Results and discussion
Characterization of CdTe QDs
CdTe QDs were collected at different reaction times. The particle
size of the QDs can be calculated using the empirical formula (10):
D¼9:8127107
λ31:7147103
λ2þ1:0064λ194:84
(1)
where λis the wavelength of the first excitonic absorption peak of
the QDs and Dis the diameter of the QDs. The diameters of CdTe
QDs were calculated to be 2.80, 2.86, 2.93 and 3.00nm for named
samples 1–4. The absorption and emission spectra were obtained
for samples 1–4. As shown in Fig. 1(a,b), as the crystal size was in-
creased, the absorption spectrum showed a red shift from 517 to
541 nm and the emission spectrum showed a red shift from 573
to 610 nm. The emission peaks of samples 1–4 were located at
573, 586, 591 and 610 nm, respectively.
FITC–CdTe FRET system
FITC was selected as the energy donor and CdTe QDs as the energy
acceptor. The overlap between the emission spectrum of FITC and
the absorption spectrum of samples 1–4 is shown in Fig. 2. The
emission wavelength range for FITC was 480–650 nm. The absorp-
tion peaks of all the CdTe QDs samples were covered by the emis-
sion spectrum of FITC to different extents. The zeta potential was
measured using a Malvern Zetasizer Nano-ZS (Malvern Instruments
Ltd., Worcestershire, UK) in three series of 30 measurements. As
illustrated in Fig. 3, both FITC and QDs have a negative zeta poten-
tial, –36.2 mV for FITC and –51 mV for CdTe QDs when dispersed in
water. Under these conditions, it may be difficult to construct a
FRET system only by mixing. To construct the FRET system, the cat-
ionic polymer PAH was used to change the surface potential of
QDs. The surface of CdTe QDs was modified with PAH to form a
composite structure. As shown in Fig. 3, CdTe QDs/PAH has a
positive zeta potential (30.1 mV). The surfaces of CdTe QDs are
positive, and can combine with FITC molecules via an electrostatic
interaction to form FITC–CdTe. This indicates that the Förster energy
transfer can take place from FITC to CdTe QDs/PAH (Scheme 1).
Significant Förster energy transfer occurs when the distance
between the donor and the acceptor is reduced in PBS.
Formation of the FITC–CdTe QDs/PAH FRET system was
demonstrated by the fluorescence spectrum. Figure 4 shows the
emission spectra of pure FITC, FITC–CdTe QDs/PAH and pure CdTe
Y. Feng et al.
Luminescence 2015Copyright © 2015 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/luminescence
2
QDs/PAH. Compared with pure FITC, the fluorescence intensity of
FITC in FITC–CdTe QDs/PAH decreased by ~ 31%, indicating that
FRET had occurred. The Förster energy-transfer efficiency of two
independent molecules can be expressed as (11):
E¼kDA
kDAþτ1
D
¼R6
0
R6
0þr6(2)
where k
DA
is the energy transfer rate of two independent
single molecules and τ
D
is the lifetime of the donor excited
states. R
0
is the Förster distance and is constant for a parti-
cular system, representing the critical distance between
donor and acceptor when the energy transfer rate is 50%.
The Förster distance depends on not only the spectrum
properties of the donor and acceptor, but also on relative
direction. ris the distance between the donor and the
acceptor.
Figure 1. (a) Absorption and (b) emission spectra of CdTe QDs.
Figure 2. Normalized absorption and emission spectra of FITC and CdTe QDs.
Figure 3. Zeta potential of FITC and CdTe QDs.
Scheme 1. Schematic representation of the FRET donor-acceptor assembly of posi-
tively charged PAH-CdTe-QDs and negatively charged FITC.
Figure 4. FRET spectrum of FITC and CdTe QDs.
FRET system between FITC and CdTe QDs for silver ion detection
Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
3
When there are multiple acceptors simultaneously around one
QD donor, eqn (2) can be modified to eqn (3).
E¼nR0
nR0þr6(3)
where nis the average number of acceptor molecules interacting
with donor molecules. We can clearly see that the energy-transfer
efficiency increases with n.
In order to obtain the energy-transfer efficiency of the system
more directly, Styrer and Haugland proposed a new method to ob-
tain the energy-transfer efficiency (12). The energy-transfer efficiency
can be calculated quantitativelybythefollowingformula(13):
E¼1IDA
ID
(4)
where I
DA
,I
D
are the fluorescence intensity of the donor with and
without acceptor, respectively. It is known that the FRET efficiency
is related to the area of the spectrum overlap and the particle size
of QDs. Table 1 shows the area of overlap, particle size and energy-
transfer efficiency for each FITC–CdTe QDs/PAH. The results
suggest that as the area of overlap increases by 0.64 (±0.06)
(arbitrary unit;a.u.) the particle size increases by 0.01 nm, leading
to an increase in the energy-transfer efficiency. The relationships
between energy-transfer efficiency, particle size and area of over-
lap are shown in Fig. 5(a,b). In Fig. 5, both the area of overlap
and the size of the QDs show an approximately linear relationship
with the energy-transfer efficiency in the range of 0.24–0.33.
Influence of pH on FRET
The FRET system (0.5 mL FITC–0.5 mL CdTe) was added to PBS so-
lutions at different pH values, and the effect of pH on the FRETsys-
tem was investigated. As shown in Fig. 6, the fluorescence intensity
of CdTe QDs/PAH was quite weak at low pH. According to a
previous study by our group (14), an aqueous solution at low pH
has numerous hydrogen ions (H
+
), which aides the protonation
of amino groups while suppressing the dissociation of carboxyl
groups. By contrast, a high pH solution is rich in hydroxide ions
(OH
–
), which facilitate the dissociation of carboxyl groups, but
make it difficult for amino groups to acquire hydrogen and be
ionized (15,16). So the fluorescence intensity of CdTe QDs/PAH
was increased when the FRET system was in a neutral or alkaline
environment. An alkalescent environment is the most suitable for
a living organism, and from the results we can see that FRET from
the donor to the acceptor exhibits strong pH dependency. There-
fore, we used alkalescent (pH 7.2) PBS for the remainder of the
experimentsinthispaper.
The impact of FITC concentration on FRET
The FITC concentration might influence the energy-transfer
efficiency. In the system used (pH 7.2), FITC solutions of different
concentrations were added to 5 mg/mL QDs solution. As shown
in Fig. 7, the fluorescence intensity increased gradually with in-
creasing FITC concentration. The energy-transfer efficiency values
were calculated quantitatively, and the results are shown in Table 2.
The energy-transfer efficiency decreased as the concentration of
the donor increased. The energy-transfer efficiency was highest
at a FITC concentration of 6.25 × 10
-4
mL, and this concentration
was chosen as ideal in the remaining experiments in this study.
The energy transfer mechanism in the system might be as follows:
Table 1. Overlapping area, quantum dot size and energy-
transfer efficiency for each FITC–CdTe QDs/PAH conjugates
Sample Particle size
(nm)
Overlapping
area
Energy-transfer
efficiency (%)
1 2.80 49.74 24.80
2 2.86 52.61 27.80
3 2.93 57.24 30.90
4 3.00 62.73 32.80
Figure 5. The relationship between (a) overlapping area and energy transfer and (b) particle size and energy-transfer efficiency.
Figure 6. Effect of pH on FRET.
Y. Feng et al.
Luminescence 2015Copyright © 2015 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/luminescence
4
the surfaces of CdTe QDs were positive, and can combine with FITC
moleculesvia an electrostatic interaction to form FITC–CdTe. Then,
the distance between the donor and the accepter was close
enough to ensure that FRET occurred. FITC–CdTe may comprise
one FITC molecule (donor) and more CdTe QDs/PAH (acceptor)
when the concentration of FITC is decreased. If the concentration
of FITC increased, the number of acceptors in FITC–CdTe showed
a relative reduction, which resulted in a decrease in the energy-
transfer efficiency (17). This result corresponded with the conclu-
sions deduced from eqn (3).
Impact of CdTe QDs/PAH concentration on FRET
The effect of CdTe QDs/PAH in the FRET process was also studied.
Figure 8 shows the fluorescence spectrum for different concentra-
tions of CdTeQDs/PAH in the FRET system(pH 7.2); with increasing
QDs concentrations, the fluorescence intensity of FITC decreased
and the fluorescence intensity of QDs increased gradually. Because
the number of CdTe QDs around the FITC molecules increased, the
energy-transfer efficiency of FITC–CdTe was higher than before.
The results correspond with the conclusions deduced from eqn
(2), which means the energy-transfer efficiency is proportional to
the absorption capacity of the acceptor in the FRET system. We
can see from Table 3, that the energy-transfer efficiency is almost
stable when the concentration of QDs was ~ 5.0 to 7 mg/mL, indi-
cating that the concentration was close to the limits of the FRET
system. In this work, we choose 6 mg/mL as the most suitable con-
centration CdTe QDs in the remaining detection experiments.
Response of FRET-based QDs emission to Ag
+
concentration
As is mentioned above, oppositely charged FITC and CdTe
QDs/PAH can form FRET donor–acceptor assemblies due to elec-
trostatic interactions, which effectively enhance the photolumi-
nescence intensity of the QDs. It is reasonable to expect that the
efficiency of FRET between FITC and CdTe QDs/PAH would be
affected by Ag
+
; the FRET process will be suppressed by the
addition of Ag
+
, and the FRET-based QDs emission would respond
to Ag
+
concentration (Scheme 2).
The FITC–CdTe FRET system was used as a fluorescence probe to
detect Ag
+
.TheAg
+
solution (2.93 × 10
–5
mol/L) was mixed with
FITC–CdTe, and the relative optical intensities were studied from
the absorption and fluorescence spectra.
In Fig. 9, compared with pure FITC and CdTe QDs/PAH, the ab-
sorption intensities of the donor and acceptor in the FITC–CdTe
FRET system were both decreased. The absorption peak of FITC
did not change significantly, but the absorption peak of the CdTe
QDs/PAH shows an obvious red shift from 525 to 537 nm. This is
due to the combination of donor and acceptor which leads to an
increase in the overall dimension of QDs, the energy gap becomes
narrower and there is a shift in the absorption wavelength. The ab-
sorption spectrum of FRET system did not change significantly on
addition of Ag
+
solution.
Figure 10(a) shows the fluorescence resonance spectrum of the
FITC–CdTe system at different concentrations of Ag
+
over
the range of 0.01–8.96 × 10
–5
mol/L. The fluorescence intensity of
the FITC–CdTe system was successively depressed by increasing
concentrations of Ag
+
. When the concentration of Ag
+
reached
Table 2. Relation between concentration of donor and
energy-transfer efficiency
Sample Concentration
(mg/mL)
Energy-transfer
efficiency (%)
1 2.65 × 10
–4
63.4
2 6.25 × 10
–4
65.7
3 8.15 × 10
–4
64.2
4 10.6 × 10
–4
61.4
5 13.9 × 10
–4
60.8
616×10
–4
59.3
Figure 8. Effect of CdTe QDs concentrationon FRET (0–6, CdTe QDs concentration of
0, 2.3, 3.0, 4.2, 5.0, 6.1 and 7.0 mg/mL, respectively).
Table 3. Relation between concentration of acceptor and
energy-transfer efficiency
Sample Concentration
(mg/mL)
Energy-transfer
efficiency (%)
1 2.3 55.8
2 3.0 59.4
3 4.2 63.2
4 5.0 65.4
5 6.1 66.1
6 7.0 65.7
Figure 7. Effect of FITC concentration on FRET (0–6: FITC concentration of 0, 2.65,
6.25, 8.15, 10.6, 13.9 and 16 × 10
–4
mg/mL, respectively).
FRET system between FITC and CdTe QDs for silver ion detection
Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
5
1×10
–4
mol/L, the fluorescent intensity of QDs decreased sharply.
That concentration is outside the detection range of the resonance
system. In order to confirm whether pure QDs could complete the
detection, we used pure QDs under the same experimental condi-
tions. The results are showed in Fig. 10(b), and show that the fluo-
rescence intensity of CdTe QDs was almost unchanged when the
concentration of Ag+ gradually approached 5.3 mol/L. The results
show that CdTe QDs were not able to successfully detect low
concentrations of silver ions.
Quenching mechanism of the FITC–CdTe-Ag
+
system
Ag
+
is a common heavy metal with unique properties. If Ag
+
and
fluorescence nanoparticles are mixed in an aqueous solution,
metal-enhanced fluorescence is produces when the distance
between the silver ionsand the QDs is within a certain range. How-
ever, when the QDs stay close to Ag
+
, they show a quenching
phenomenon. Therefore, fluorescence may occur via two oppos-
ing phenomena (quenching or enhancement) when silver ions
were added to the solution. Similarly, the size of the QDs is also a
main factor affecting quenching. Therefore, the metal-enhanced
fluorescence effect of Ag
+
and the quantum size effect of QDs
results in the different phenomenon seen with Ag
+
compared with
other heavy metal ions during the detection process.
When Ag
+
is mixed with QDs of small size, two types of competi-
tive reaction occur in this system: more defects exist on the surface
of small QDs, resulting in obvious enhanced fluorescence when com-
pared with large QDs, and this will affect the detection to a great
extent. Therefore, we selected large CdTe QDs (emission wavelength
is 580 nm) in this study; the injection of Ag
+
led to significant
quenching, greatly improving the sensitivity of the detection.
The quenching mechanism is divided into three conditions:
dynamic quenching, static quenching and combined dynamic
and static quenching (18). The quenching mechanism for the
FITC–CdTe–Ag
+
system might be dynamic quenching, and the
specific analysis is described below.
Static quenching refers to a complex-forming reaction between
the fluorescent molecule and the quencher, which results in the
generation of a non-fluorescent substance or the fluorescent
body being dissolved by the complexes. The dissociation process
is very slow, and the complexes decay to the ground state by
non-radiative transition. The performances of static quenching
are that the fluorescence intensity is decreased and the absorption
spectrum changes significantly, which is in contrast to the lifetime
after addition of fluorescent quencher. In Fig. 9 (UV absorption
spectrum of FITC–CdTe system), when the Ag
+
solution is added,
the UV absorption of the resonance system remained almost
unchanged, indicating that nocomplex was formed through inter-
action between the resonance system and the Ag
+
solution, which
showed that the process is not static quenching. The lifetime of the
Scheme 2. Schematic representation of the detection method for Ag
+
determina-
tion in FRET efficiency between FITC and QDs.
Figure 9.AbsorptionspectrumofFRETsystem.
Figure 10. Fluorescence spectrum of (a) the FITC–CdTe system and (b) CdTe in the presence of Ag
+
(1–8: Ag
+
concentration 0.01, 2.02, 2.93, 3.53, 4.09, 5.3, 8.96 and 10 mol/L).
Y. Feng et al.
Luminescence 2015Copyright © 2015 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/luminescence
6
fluorescent substance remains unchanged in static quenching,
and can be expressed as τ
0
/τ=1.
Dynamic quenching involves an interaction between the
quencher and the excited state or ground state molecules of the
fluorescent substance. When the excited state molecule interacts
with a quencher, it loses its excitation energy and returns to the
ground state because the energy or charge is transferred. The pres-
ence of quencher will shorten the lifetime of the fluorescent sub-
stance, and can be expressed as τ
0
/τ=F
0
/F. We measured the
lifetime of the FRET system before and after addition of Ag
+
.The
results are shown in Fig. 11.
Dynamic quenching can be described by the Stern–Volmer
equation (19):
F0=F¼1þKsv Q½¼
τ0
τ(5)
where F
0
and Fare the fluorescence intensity of the system in the
absence and presence of quencher, respectively; τ
0
and τare the
lifetimes in the absence and presence of quencher, respectively;
K
SV
is the dynamic quenching constant and [Q] is the concentra-
tion of the quencher. The above equation illustrates the important
characteristics of quenching, which led to a decrease in the fluores-
cence intensity and lifetime. A linear Stern–Volmer quenching
curve of F
0
/Fvs. [Q] is shown in Fig. 12, with the concentration of
Ag
+
[Q] as the abscissa and the fluorescence intensity F
0
/Fas the
ordinate. From the slope of the plot, K
SV
was found to be 0.442.
A decrease in the fluorescence lifetime of the QDs is equivalent
to F
0
/F.
The Stern–Volmer curve in Fig. 12 indicates that the quenching
process for the FITC–CdTe–Ag
+
system is dynamic quenching; it
will affect the excited state of the dynamic fluorescence quenching
molecule and change the lifetime, but it will not change the ab-
sorption spectrum of the substance. Based on the above results,
the quenching mechanism of FITC–CdTe–Ag
+
system is dynamic
quenching.
Interference experiment
Interference by ions (relative error within ± 5) potentially coexis-
tent within the proposed system was also investigated. Figure 13
shows the intensity of the FRET systems when different types of
metal ions were injected. The results shows that the fluorescence
intensity of CdTe was barely influenced in the presence of a 500
times excess of Na
+
,K
+
,Ca
2+
,Ba
2+
,Cl
-
and OH
-
, 300 times excess
of Zn
2+
, 100 times of excess Cu
2+
and Cd
2+
, or 50 times excess of
Mn
2+
. These results indicated that CdTe might recognize Ag
+
with
high selectivity in the proposed FRET system.
Comparison of methods for the determination of Ag
+
The limit of detection for Ag
+
in previous reports and this work
were compared and are summarized in Table 4, which highlights
the superiority of some of the reported studies, although the linear
Figure 11. The lifetime of FITC–CdTe and FITC–CdTe–Ag
+
. The excitation wave-
length was 377 nm. The lifetime values were 55.44 and 30.71 ns, respectively.
Figure 12.Stern–Volmer curve of the FITC–CdTe–Ag
+
system.
Figure 13. Effects of the metal ions on the FRET system.
Table 4. Comparison of the proposed FRET method with
other assays for the determination of Ag
+
Methods Reference Linear range
(μmol)
Detection
limit (nmol)
FRET This study 0.01–8.96 4.2
Quantum dots [20] 0.01–51.6
Fluorescence probe [21] 0.02–15.0
Fluorescence
quenching
[22] 0–0.74 7.4
Fluorescence-
labeled DNA
[23] 0.5–35
Circular dichroism [24] 1 × 10
5
–0.01 2 × 10
–3
FRET system between FITC and CdTe QDs for silver ion detection
Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
7
range and detection limit in this work were better than in other re-
search. The method used here has a greater linear range than
other previous methods. Therefore, based on the above results, it
is possible to determine Ag
+
using the FITC–CdTe FRET system.
Conclusion
We developed an efficient FRET system, and the results revealed
energy transfer between the donor and CdTe QDs/PAH with differ-
ent emission wavelengths. It was also demonstrated that the en-
ergy transfer in this system followed a fluorescence resonance
energy transfer pattern. We also found that due to interaction be-
tween the positive and negative charges, the energy-transfer effi-
ciency decreased as FITC (donor) concentration increased,
whereas the energy-transfer efficiency increase gradually as CdTe
QDs (acceptor) concentration increased. We have designed an ef-
fective method for the detection of silver ions. The limit of detection
is 4.2 nmol/L, the linear range is greater than that of other methods,
and the quenching mechanism is proved to be dynamic quenching.
Such a FRET system between FITC and CdTe QDs/PAH might be the
best candidate for designing sensors to detect metal ions.
Acknowledgements
Project supported by the National Natural Science Foundation of
China (Grant No. 11204020). International Cooperation Project of
Jilin Province Science and Technology Department (Grant No.
20110748).
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