Article

pubs.acs.org/ac

Ultraviolet-Visible Light (UV−Vis)-Reversible but Fluorescence-

Irreversible Chemosensor for Copper in Water and Its Application in
Living Cells
Fang-Jun Huo,†,§ Cai-Xia Yin,*,‡,§ Yu-Tao Yang,†,‡ Jing Su,‡ Jian-Bin Chao,† and Dian-Sheng Liu‡
†
Research Institute of Applied Cheemistry and ‡Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, People’s Republic of China
*
S Supporting Information
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ABSTRACT: An ultraviolet−visible light (UV−Vis)-reversi-

ble but fluorescence-irreversible chemosensor was developed
for the detection of copper. Coordination between the probe,
2-pyridylaldehyde fluorescein hydrazone (FHP), and Cu2+
gave a reversible UV−Vis response, Storage of the probe−
Cu complex resulted in hydrolytic cleavage of the NC bond,
which released the fluorophore (ring-opened fluorescein
hydrazine) and gave irreversible fluorescence. Thus, FHP becomes a multifunctional chemosensor, and its reversibility can be
controlled by the reaction time. Cu2+ in living cells could be detected using FHP and general fluorescence methods.

A nions are ubiquitous in nature and in biological processes,

and they are also responsible for industrial and agricultural
pollution.1−9 While transition-metal ions are important in many
different fields, including catalysis, organometallic reactions,
and biochemistry, they can be toxic at high concentrations and
disrupt normal cell function.9−14 These ions can be detected
using several instrumental techniques.15,16 However, these
methods are time-consuming and require expensive instru-
mentation. Chemosensors are powerful molecular tools that
can be used to detect many target molecules, such as biological
markers and environmental pollutants.17−23 Chemosensors that
use color and/or fluorescence intensity have been developed to
detect various analytes.24−28 To design new chemosensors,
mechanisms for recognizing target analytes and the signal
reporting units must be investigated.29 Fluorescein derivatives
can be produced in short synthetic routes, and they have several
and useful properties for chemosensors, such as high water Figure 1. Structure and thermal ellipsoids of the probe, drawn at the
solubility, long excitation and emission wavelengths, and high 50% probability level.
fluorescence quantum efficiencies. Chemosensors30−45 using
fluorescein derivatives have been designed based on ion- data associated with it: 1H NMR (300 MHz, 25 °C, DMSO-
induced changes in the fluorescence intensity. These sensors d6): δ 10.0 (bs, 2H), 8.53 (s, NC−H, 1H), 8.49 (d, Ar−H,
are simple to produce and have high detection sensitivities.46−52 1H), 7.97 (t, pyridine-H, 1H), 7.79 (t, pyridine-H, 1H), 7.60−
In this study, we investigated 2-pyridylaldehyde fluorescein 7.68 (m, Ar−H, 3H), 7.34 (t, pyridine-H, 1H), 7.13 (d,
hydrazone (FHP) as a novel ultraviolet−visible light (UV− pyridine-H, 1H), 6.69 (d, xanthene-H, 2H, J = 2.4 Hz), 6.55 (d,
Vis)-reversible but fluorescence-irreversible chemosensor for xanthene-H, 2H, J = 8.8 Hz), 6.47 (dd, xanthene-H, 2H, J = 8.8
Cu2+, as well as its application in living cells. Hz, J = 2.4 Hz); 13C NMR (75 MHz, DMSO-d6): δ 164.22,
The FHP probe (Figure 1) was synthesized according to the 158.84, 153.17, 151.90, 151.21, 149.54, 146.49, 137.00, 134.56,
literature10 by a reaction between fluorescein hydrazine and 129. 25, 127.93, 127.80, 124.59, 123.52, 119.21, 112.65, 109.50,
picolinaldehyde in methanol containing acetic acid (see Figure 102.73, 64.95; ESI-MS m/z 436[FHP + H]+, 458[FHP + Na]+;
S1 in the Supporting Information). The product was Elemental analysis (calcd %) for C26H17N3O4: C, 71.72; H,
characterized by electrospray ionization mass spectrometry
(ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, Received: October 15, 2011
and X-ray crystallography (see Figures S2 and S3 in the Accepted: January 26, 2012
Supporting Information). The FHP molecule has the following Published: January 26, 2012

© 2012 American Chemical Society 2219 dx.doi.org/10.1021/ac202734m | Anal. Chem. 2012, 84, 2219−2223
Analytical Chemistry Article

3.94; N, 9.65; Found: C, 71.70; N, 9.68; H, 4.01. Crystal data

for C26H17N3O4·1/2(CH3OH): crystal size: 0.20 × 0.05 × 0.05,
monoclinic, space group P21/c (No. 14). a = 9.7947(8) Å, b =
26.813(2) Å, c = 16.8152(14) Å, β = 100.685°, V = 4339.5(6)
Å3, Z = 4, T = 296 K, θmax = 26.0°, 24570 reflections measured,
8501 unique (Rint = 0.0633). Final residual for 614 parameters
and 8501 reflections with I > 2σ(I): R1 = 0.0686, wR2 = 0.1738,
and goodness of fit (GOF) = 0.999. The ability of the probe to
detect metal ions was investigated by UV−Vis and fluorescence
spectroscopy.
The effects of a wide range of environmentally and
physiologically active metal ions (Cu+, Cu2+, Ca2+, Fe2+, Zn2+,
Ni2+, Bi3+, Co2+, VO2+, Mn2+, Ru3+, Cd2+, Pb2+, Ag+, La3+, Ce4+,
Yb3+, Cr2+, Er3+, Mg2+, Sn2+, Al3+, Nd3+, Zr4+, K+, Sm3+, Fe3+,
and Eu3+) on FHP were investigated. UV−Vis spectra were
recorded of solutions containing FHP (36 μmol/L) and each
metal (360 μmol/L) in the HEPES buffer (10 mmol/L) pH 7.0
aqueous buffer. Among the metal ions, only Cu2+ caused any
changes in the UV−Vis spectra. When Cu2+ was added to the
FHP solution, a strong absorption peaks appeared at 500 nm
(Figure 2), and the solution changed color from colorless to
yellow. (See Figure S4 in the Supporting Information.)

Figure 3. (a) Absorption spectral changes of FHP (36 μM) in 10 mM

HEPES at pH 7.0 as an aqueous buffer upon the addition of Cu2+;
Cu2+ was added gradually, with the divalent copper species range being
[Cu2+] = 0−108 μM. Each spectrum was recorded 10 s after the
addition of Cu2+. (b) Absorption spectral changes of FHP−Cu2+ in 10
mM HEPES at pH 7.0 as an aqueous buffer upon the addition of PPi/
Figure 2. Optical density three-dimensional graph of the FHP probe C2O42−; PPi/C2O42− was added gradually with [PPi/C2O42−] = 0−216
(36 μM) at 500 nm upon the addition of several metal ions. Inset μM. Each spectrum was recorded 10 s after the addition of PPi/
shows a color change photograph for Cu2+ and the other metal ions. C2O42−.

A detailed investigation on the FHP recognition of Cu2+ was neously, the solution changed color from yellow to colorless.
performed. Figure 3a shows the change in the UV−Vis (See Figure S7 in the Supporting Information.)
spectrum when the Cu2+ solution was added to the HEPES Anions with a physiological function, including fluoride (F−),
buffer (10 mmol/L, pH 7.0) containing the probe (36 μmol/ chloride (Cl−), bromide (Br−), iodide (I−), acetate (AcO−),
L). As the concentration of Cu2+ increased, so did the thiocyanate (SCN−), nitrate (NO3−), sulfate (SO42−), carbo-
absorbance at 500 nm. In the presence of several metal ions, we nate (CO32−), oxalate (−OOCCOO−), phosphate (PO43−), and
investigated the ability of FHP to detect Cu2+ in the presence of CN−, were also investigated; however, their effect on the UV−
other metal ions. The other ions did not interfere with the
vis spectra was not as good as that for PPi/XO.
detection of Cu2+ (see Figure S5 in the Supporting
A series of FHP−Cu2+ HEPES solutions with different
Information. It should be noted that FHP and FHP−Cu2+
amount-of-substance ratios were stored for a long time at room
did not fluoresce during the UV−VIS detection process. The
probe could be used to detect 1.5−120 μmol/L of Cu2+ by the temperature. Interestingly, an emission peak was observed at
UV−Vis spectra changes, and the detection limit is 1.5 μmol/L. 518 nm (λex = 325 nm) for all these solutions, and their
(See Figure S6 in the Supporting Information.) fluorescence intensities increased rapidly as the concentration
It is well-known that chemosensor reversibility is required for of FHP−Cu2+ increased (Figure 4a) from 6 × 10−7 μmol/L to
reuse. When P2O74− (PPi) or C2O42− (XO) is added to the 9 × 10−6 μmol/L. This results indicate that FHP can detect
FHP−Cu2+ complex in HEPES buffer (10 mmol/L, pH 7.0) Cu2+ at low micromolar levels and produce a fluorescence
the absorbance at 500 nm decreased (Figure 3b). As the PPi or signal after the complex has been stored for some time.
XO concentrations increased, A500 nm gradually decreased until The emission peak of FHP−Cu2+ at 518 nm (λex = 325 nm)
the original spectrum of free FHP was obtained. Simulta- did not decrease when large quantities of PPi or XO were
2220 dx.doi.org/10.1021/ac202734m | Anal. Chem. 2012, 84, 2219−2223
Analytical Chemistry Article

Figure S12a in the Supporting Information). The free-probe

absorption peaks at 500 nm were greatly affected by strong
acidity, or strongly alkaline solutions, which produced new
absorption peaks at 500 nm. This would affect the detection of
Cu2+. No peak was observed for the free probe at 500 nm when
pH was between 2.0 and 7.0. However, the absorption peaks at
500 nm for the FHP−Cu2+ complex was best at pH 7.0 (see
Figure S12b in the Supporting Information), so this pH was
selected as being optimal for recognition.
No fluorescence is detected from FHP when it is in its free
form with a closed ring. When Cu2+ is added to the FHP
solution, the Cu2+ coordinates to FHP and forms a complex
with a new absorption peak at 500 nm without fluorescence.
(See Figure S13 in the Supporting Information.) After the
addition of PPi/XO to this system, FHP is released as PPi/XO
coordinates to Cu2+ and the peak at 500 nm disappears.
Therefore, the probe is UV−Vis-reversible. However, after the
FHP−Cu2+ solution is stored for a few hours, the system shows
strong fluorescence. If PPi/XO is added to this system, neither
the UV−Vis spectra nor the fluorescence spectra change if the
solutions are allowed to sit for a few days. This is because Cu
causes hydrolytic cleavage of the NC bond and the release of
the fluorophore, which is fluorescein hydrazine with an open
ring. Therefore, the probe is fluorescence-irreversible (see
Scheme 1, as well as Figures S14−S16 in the Supporting
Information). The property has been observed with some other
types of chemosensor.53
The ability of FHP to detect Cu2+ within living cells was also
evaluated by laser confocal fluorescence imaging using an
Olympus Model FV1000 laser scanning microscope. The
optical window at the green channel (490−550 nm) was
Figure 4. (a) Fluorescence spectral changes for FHP (3 μM) upon the chosen as a signal output. Under selective excitation at 405 nm,
addition of Cu2+ (0−9 μM) (λex = 325 nm, λem = 517 nm, slit: 5 nm/5
HepG2 cells incubated with 50 μmol/L FHP for 48 h at 37 °C
nm) in 10 mM HEPES at pH 7.0 as an aqueous buffer. Each spectrum
was recorded 48 h after Cu2+ addition. Inset: color (left) and visual showed green fluorescence. (See Figure 5b.) When the cells
fluorescence (right) change photographs for FHP (10 μM) upon the were pretreated with a membrane-permeable copper chelator
addition of Cu2+ in a HEPES (pH 7.0) buffer solution under UV (ethylene diamine tetra(methylene phosphonic acid), EDTM-
illumination (365 nm). (b) Fluorescence spectral changes of FHP (10 PA), incubation with FHP showed no emission. Cells
μM)−Cu2+ upon the addition of plenty of PPi/C2O42− (λex = 325 nm, pretreated with EDTMPA and subsequently incubated with
λem = 518 nm, slit: 5 nm/5 nm) in 10 mM HEPES at pH 7.0 as an CuCl2 and FHP displayed enhanced green fluorescence (see
aqueous buffer. The spectrum was recorded 60 h after PPi/C2O42− Figure 5d). This indicates that the green fluorescence is caused
addition. Inset: color (left) and visual fluorescence (right) change by the FHP responding to external copper ions. These cell
photographs for FHP (10 μM)−Cu2+ upon the addition of PPi/ experiments show FHP can permeate through cell membranes.
C2O42− in a HEPES (pH 7.0) buffer solution under UV illumination
(365 nm).
Therefore, it could be used to detect Cu2+ within living cells.
In summary, in this study, a controllable, regenerating, and
added (Figure 4b), and no changes were observed even after a multifunctional chemosensor was developed. 2-Pyridylaldehyde
fluorescein hydrazone (FHP) can recognize Cu2+ with very
few days. high selectivity, both UV−Vis spectrophotometrically and
The UV−Vis absorption spectra of the free probe under visually, via a simple coordination action between FHP and
different pH conditions were recorded (pH 2.0−13.0) (see Cu2+. PPi/XO can coordinate with Cu2+ and remove it from the

Scheme 1. The Proposed Determination Mechanism

2221 dx.doi.org/10.1021/ac202734m | Anal. Chem. 2012, 84, 2219−2223

Analytical Chemistry Article

of Chemistry, at the Chinese Academy of Sciences (Beijing,

PRC) and Prof. Zongxiu Nie.

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