An amidochlorin-based colorimetric fluorescent probe for selective Cu2+ detection

Article abstract of DOI:10.3390/molecules21010107

The design and synthesis of selective and sensitive chemosensors for the quantification of environmentally and biologically important ionic species has attracted widespread attention. Amidochlorin p6 (ACP); an effective colorimetric and fluorescent probe for copper ions (Cu²⁺) in aqueous solution derived from methyl pheophorbide-a (MPa) was designed and synthesized. A remarkable color change from pale yellow to blue was easily observed by the naked eye upon addition of Cu²⁺; and a fluorescence quenching was also determined. The research of fluorescent quenching of ACP-Cu²⁺ complexation showed the detection limit was 7.5 ×10^-8 mol/L; which suggested that ACP can act as a high sensitive probe for Cu²⁺ and can be used to quantitatively detect low levels of Cu²⁺ in aqueous solution. In aqueous solution the probe exhibits excellent selectivity and sensitivity toward Cu²⁺ ions over other metal ions (M = Zn²⁺; Ni²⁺; Ba²⁺; Ag⁺; Co²⁺; Na⁺; K⁺; Mg²⁺; Cd²⁺; Pb²⁺; Mn²⁺; Fe³⁺; and Ca²⁺). The obvious change from pale yellow to blue upon the addition of Cu²⁺ could make it a suitable "naked eye" indicator for Cu²⁺.

Full text of DOI:10.3390/molecules21010107

molecules
Article
An Amidochlorin-Based Colorimetric Fluorescent
Probe for Selective Cu2+ Detection
Wenting Li †, Guohua Zhu †, Jinghua Li †, Zhiqiang Wang * and Yingxue Jin *
Received: 5 December 2015 ; Accepted: 14 January 2016 ; Published: 18 January 2016
Academic Editors: Scott Reed and Marino Resendiz
Key Laboratory for Photonic and Electronic Bandgap Materials, Ministry of Education,
College of Chemistry & Chemical Engineering, Harbin Normal University, Harbin 150025, China;
m13039864430@163.com (W.L.); zhuguohua201@163.com (G.Z.); jyxprof@163.com (J.L.)
* Correspondence: wzq70402@163.com (Z.W.); tgh.123@163.com (Y.J.); Tel.: +86-451-8806-0570 (Z.W.);
+86-451-8806-0569 (Y.J.)
† These authors contributed equally to this work.
Abstract: The design and synthesis of selective and sensitive chemosensors for the quantification
of environmentally and biologically important ionic species has attracted widespread attention.
Amidochlorin p6 (ACP); an effective colorimetric and fluorescent probe for copper ions (Cu2+)
in aqueous solution derived from methyl pheophorbide-a (MPa) was designed and synthesized.
A remarkable color change from pale yellow to blue was easily observed by the naked eye upon
addition of Cu2+; and a fluorescence quenching was also determined. The research of fluorescent
quenching of ACP-Cu2+ complexation showed the detection limit was 7.5
ˆ
10´8 mol/L; which
suggested that ACP can act as a high sensitive probe for Cu2+ and can be used to quantitatively detect
low levels of Cu2+ in aqueous solution. In aqueous solution the probe exhibits excellent selectivity
and sensitivity toward Cu2+ ions over other metal ions (M = Zn2+; Ni2+; Ba2+; Ag+; Co2+; Na+; K+;
Mg2+; Cd2+; Pb2+; Mn2+; Fe3+; and Ca2+). The obvious change from pale yellow to blue upon the
addition of Cu2+ could make it a suitable “naked eye” indicator for Cu2+
.
Keywords: fluorescent probe; copper ions; chlorophyll
1. Introduction
The design and synthesis of selective and sensitive chemosensors for the quantification of
environmentally and biologically important ionic species has attracted widespread attention [ ].
Among ionic species, copper is one of the important pollution sources [ ]. As a common heavy
1
2
metal existing widely in Nature and all living organisms, an appropriate amount of copper ion
is essential to living organisms because it is a key constituent of the respiratory enzyme complex
cytochrome c oxidase [
3]. However, excess copper ion may cause physical discomfort and sometimes
life-threatening illness [
4–7
]. Therefore the determination of heavy metal content in living organisms
and the environment is particularly important.
Because chromo- or fluoroionophores are highly effective for these determinations, given their
easy handling and the simple equipment required, effort has been expended to develop optical
chemosensors that selectively respond to the Cu2+ ion. Various methods have been developed in
the past decades to determine Cu2+ ion content, such as spectrophotometric [
(EM) [11
absorption spectroscopic (AAS) [18
8
–
10], electrochemical
13], inductively coupled plasma atomic emission spectrometric (ICP-AES) [14 17], atomic
19] and fluorescence methods [20 22]. Among them, the
–
–
,
–
fluorescence method utilizes a specific chemical reaction between dosimeter molecules and the
target species to form a fluorescent or colored product. Thus, high selectivity toward the probe
is an advantage of chemodosimeters, making them useful for detecting Cu2+ ions. Meanwhile, as
Molecules 2016, 21, 107; doi:10.3390/molecules21010107
www.mdpi.com/journal/molecules

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paramagnetic Cu(II) ion has a strong ability to quench fluorescence, recent years have seen a growing
interest in the development of fluorescent probes for Cu2+ with different chemical transducers, such
as rhodamine and semiconductor quantum dot-based probes [23–27]. Although rhodamine dyes are
widely used as fluorescent probes owing to their high photostabilities, high extinction coefficients, and
high fluorescent quantum yields, their structural instability in strong acid or base media (pH < 4 or
pH > 9) has limited their applications [28]. Semiconductor quantum dots (QDs) have also emerged as
an important class of inorganic nanomaterial that affords promising potential in the ion-detection field,
yet QDs probes cannot be applied under alkaline conditions, while the morphology, size and surface
defects of the nanocrystals could influence the detection sensitivity [29].
Therefore, the search for new fluorescence probes with sufficient high sensitivity and a wide
application range is still an active field as well as a challenge for the analytical chemistry community.
Recently, porphyrins have gained widely attention for their good photophysical properties with
large Stokes shifts and relatively long excitation (>400 nm) and emission (>600 nm) wavelengths that
minimize the effects of the background fluorescence [30]. A newly reported pyro-pheophorbide-a
methyl ester (PPME) could selectively complex with Cu2+ ions, leading to a distinct change in its
absorption spectrum as well as efficient fluorescence quenching [31]. However, the association rate
between PPME and Cu2+ is very slow, and no systematic research on quantitative detection of Cu2+ has
been performed. Moreover, the poor water-solubility of PPME had limited its application in sensing
Cu2+ in aqueous solution, hence improving the water-solubility is necessary and desirable. In this
paper, a new chlorophyll-based Cu2+ fluorescent probe, amidochlorin p6 (ACP), was designed and
synthesized. (Scheme 1) Two flexible side chains with hydrophilic hydroxyl groups were introduced
to improve the water-solubility of the designed molecule, while the hydroxyl groups may also serve
as ligand binding sites to chelate heavy metals. ACP has large absorption, strong fluorescence and
a relatively long emission wavelength in visible region, displaying high selectivity for Cu2+ in aqueous
solution among the metal ions examined, with a low detection limit in a wide pH range of 1 to 12.
Moreover, ACP exhibited marked fluorescence quenching upon the binding of Cu2+ ion, thus it has
potential applied value for rapid detection of Cu2+ in aqueous solution.
Scheme 1. The proposed synthesis of amidochlorin p6.
2. Results and Discussion
2.1. Chemistry
Methyl pyropheophorbide a (Mpa) was synthesized according to the literature procedure [32].
Then propanolamine (1 mL) was introduced to Mpa through a aminolysis reaction of the methyl ester
to give the title compound ACP.
2.2. Recognition of Metal Ion
To verify its metal ion sensing abilities, ACP was titrated over a wide range of metal ions, such
as Cu2+, Zn2+, Ni2+, Ba2+, Ag+, Co2+, Na+, K+, Mg2+, Cd2+, Pd2+, Mn2+, Fe3+, and Ca2+. Stock

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1 mM solutions of metallic ions were prepared by dissolving the appropriate salts in doubly distilled
water, respectively, and then diluting to a lower concentration of 10 M. Meanwhile, a stock 1 mM
solution of ACP was also prepared in ethanol, and then diluted to a lower concentration of 10 M.
In brief, to a 10 mL volumetric flask, 100 L of the stock solution (1 mM) of ACP was added, followed
by addition of 100 L of different metal ions stock solutions, the mixtures were diluted to lower
concentrations by addition of 50% ethanol (v/v) solution. As a control, the same procedure was
performed but in the absence of Cu2+
µ
µ
µ
µ
.
2.3. Spectral Titration of ACP with Cu2+
Copper is a quenching metal ion and the coordination of ACP with Cu2+ would quench the
fluorescence of ACP. The UV-Vis and fluorescence titration experiments of ACP with Cu2+ were
performed in 50% ethanol (v/v) solution. Figure 1a shows the UV-visible absorption spectrum of ACP
ACP absorbs throughout the ultraviolet region into the visible region between about 400 and 800 nm
with four peaks: a strong Soret absorption peak at 399 nm, two weak absorption peaks at 499 nm and
605.5 nm, and a Qy peak at 660.5 nm. The absorption of ACP is highly affected by the presence of Cu2+
ions. Upon addition of Cu2+ ions, the absorption intensity of Soret peak at 399 nm decreased with
a little red shift, and the peak at 499 nm also decreased, with no peak shift. Meanwhile, the Qy peak
gradually reduced in intensity with the formation of a new absorption peak at about 632 nm and with
the formation of an isosbestic point at 652 nm. When the concentration of Cu2+ increased to the same
level as ACP, the Qy peak disappeared yet the absorption intensity at 632 nm reached a maximum.
The change of ACP absorption spectra demonstrated the complexation between ACP and Cu2+. The
value of the shift is indicative of the degree of the interaction between the fluorophore and the bound
Cu2+. To study the binding stoichiometry of ACP and Cu2+, a Job’s plot experiment was carried out
by using the UV-Vis absorbance spectrum at 632 nm. Keeping the sum of the initial concentration of
.
Cu2+ and ACP at 10
µ
M, increasing the concentration of Cu2+ from 0 to 1. The maximum absorbance
occurred when the [Cu2+]/{[ACP]+[Cu2+]} reached at 0.5.
Figure 1.
added Cu2+; (
ACP and Cu2+ is 10
ratio between ACP (10 µM each) and Cu2+
(
a
) The absorption spectrum of ACP in water/ethanol (v/v = 50/50) solution (10
) Job’s plot according to the method for continuous variations (the total concentration of
M. The absorbance was measured at 632 nm; ( ) Mole ratio plot for stoichiometric
µM) with
b
µ
c
.

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This observation indicates that ACP and Cu2+ formed at 1:1 ratio complex. In order to verify this,
the mole ratio plot for stoichiometric ratio between ACP (10 µ
M each) and Cu2+ was measured. As can
be seen from Figure 1c, the molar ratio of ACP to Cu2+ was 1:1.
The fluorescence titration of Cu2+ was carried out using a solution of 10
µM ACP in ethanol,
using 412 nm as excitation wavelength. As illustrated in Figure 2a, the fluorescence intensity of ACP
decreases with increasing concentration of Cu2+, which constitutes the basis for the determination
of Cu2+ with the fluorescent probe proposed in this paper. Moreover, it can be seen from Figure 2b
that the fluorescence intensity at 632 nm showed a linear quenching with the increasing addition
of Cu2+. The fluorescent response of ACP toward Cu2+ was calculated to cover a linear range from
1 to 10
intensity at 668 nm measured at a given Cu2+ concentration and x is the concentration of Cu2+ added.
The detection limit of Cu2+ is 7.5 10´8 mol/L, which is lower than the limit of Cu2+ in drinking
water (~20 M) demanded by U.S. Environmental Protection Agency. This result showed that ACP is
sensitive enough to monitor the concentration of Cu2+ in drinking water.
µM. The linear equation was y =
´
45.66x + 546.27 (R2 = 0.999), where y is the fluorescence
ˆ
µ
Figure 2. (a b) The relation
) Fluorescence spectra of [ACP-Cu2+] with emission wavelength 668 nm; (
between ACP fluorescence intensity and the concentration of Cu2+
.
2.4. Selectivity and Interference Studies
Selectivity is a very important parameter to evaluate the performance of a probe. Development
of chemosensors with “naked eye” capability has an advantage over traditional fluorescence sensors
because they do not need cumbersome labor and a sophisticated instruments [33]. The selectivity of
ACP toward Cu2+ and the interference of a number of common ions with the determination of Cu2+
were investigated. The experiments were carried out by fixing the concentration of Cu2+ at 10
then recording the change of the UV-Vis absorbance and fluorescence intensity before and after adding
µM and
the interferent into the Cu2+ solution (Figure 3). In the presence of other tested metal ions (Zn2+, Ni2+
Ba2+, Ag+, Co2+, Na+, K+, Mg2+, Cd2+, Pd2+, Mn2+, Fe3+, and Ca2+), the UV-Vis absorbance spectra
showed almost no obvious change relative to the free ligand ACP, and the absorbance of ACP was
only slightly influenced by the addition of other ions (Figure 3a). When 1 equiv. of Cu2+ and selected
metal ions (10 µM) was added into the solution of ACP (10 µM), many of the investigated metal ions
do not interfere with detection of Cu2+. The data in Figure 4 clearly reveals that the addition of other
common metal ions can hardly affect the fluorescence response of ACP towards Cu2+. There are only
slight interfering effects of Mg2+ and Cd2+. These results clearly suggest that the probe ACP shows
a high anti-interference ability against other potentially coexisting metal ions.
,
Furthermore, upon addition of the same amount of the various metal ions, respectively, only Cu2+
induced a striking color change from pale yellow to blue, as observed by the naked eye (Figure 3a).
Those observations indicate that ACP has a high selectivity to Cu2+ and can be a good colorimetric
sensor for Cu2+ ions. Moreover, upon addition of Cu2+ and selected metal ions (10 M), only Cu2+
µ
showed distinct quenching (Figure 3a,b), which suggested that ACP can be a selective fluorescent
sensor for Cu2+ ions. It’s worth mentioning that upon addition of Cu2+, the color of ACP changed

Molecules 2016, 21, 107
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much faster than PPME that previously reported in the literature [31]. In brief, our proposed probe
shows extraordinary selectivity to Cu2+ and could meet the selectivity requirements for biomedical
and environmental applications.
Figure 3.
of various metal ions (10
(10 M) upon addition 1 equiv. of various metal ions under natural light and UV-Vis are also displayed.
(
a) UV-Vis absorption and (b) fluorescence emission spectra of ACP (10 µM) upon addition
µM) in water/ ethanol (v/v = 50/50) solution. The color changes of ACP
µ
Figure 4. The relative fluorescence intensity diagram of ACP (10 µM) to different metal ions (1 equiv.).
Excitation was at 412 nm, and emission was at 668 nm.
2.5. Spike and Recovery Test
Spike and recovery test was conducted in tap water to examine whether there is any positive or
negative interference in real drinking water samples. We first examined the effect of tap water on the
fluorescence stability and found no quenching effect. The local tap water was filtered first through filter
paper to remove any insoluble suspended solids. The recovery study was carried out on a mixture of
water and ethanol (1:1, v/v) which was spiked with 2, 5 and 8 µ
M Cu2+. Each experiment was done in
quintuplicate and the average was presented with relative standard deviation. The contents of Cu2+
were recovered using the linear equation obtained in Figure 3. The analysis results for the sample
with spiked Cu2+ were given in Table 1. The result showed that the method had a good recovery
at the concentration test, suggesting no serious positive or negative interferences for selectively and
sensitively determining copper(II) ion in real water samples.
Table 1. Recovery test of Cu2+ in tap water 1.
Tap Water Sample
Cu2+ Added (µM)
Cu2+ Found (µM)
RSD (%, n = 5)
Recovery (%)
Sample 1
Sample 2
Sample 3
2
5
8
2.242
5.198
8.404
3.75
1.90
1.43
112.1
104.0
105.1
1
Values shown were the calculated mean Cu2+ for each sample.

Molecules 2016, 21, 107
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2.6. Effect of pH
The spectroscopic characters of the probe were studied in the pH range 2–13 in sodium
acetate-acetic acid buffer solution. Figure 5 shows the fluorescence response of ACP toward Cu2+
in the pH range. The fluorescence intensities of the mixture were very high in the pH range 2–4,
yet the fluorescence emission (
λ /em = 561/580 nm) drastically decreases in pH up to 5 and varies
ex
slightly until 11. This may be attributed to the fact that H+ and Cu2+ competitively bind to ACP in
acid solutions, consequently the formation of Cu2+-ACP complexes are inhibited, thus the mixture
displayed high fluorescence intensities in the pH range 2–4. Moreover, in the pH range 11–13 the
mixture possess very high fluorescence emission, which is most probably due to the fact that in strongly
´
alkaline solutions OH and ACP competitively bind to Cu2+. The more alkaline of the buffer solution
is, the more liable it is to form Cu(OH)42´, and the more difficult it is to form Cu2+-ACP complexes.
Therefore, the Cu2+-ACP complexes are stable only in the pH range 6–11.
Figure 5. The effect of different pH values on the spectroscopic characteristics of Cu2+-ACP.
2.7. Binding Mechanism
As a new kind of porphyrin, ACP is endowed with a cyclic π-aromatic system and exhibits
unique coordination chemistry. Owing to the four pyrrole units of ACP, Cu2+ would coordinate with
pyrrole N atoms in a square planar shape. We have simulated the ACP-Cu2+ complex through density
functional theory (DFT) calculations with the Becke-3-Lee-Yang-Parr (B3LYP) exchange function using
the Gaussian 09 package [34]. The 6-31G (d, p) basis sets were used except for Cu2+, where a LANL2DZ
effective core potential (ECP) was employed. Figure 6 represents the molecular geometry optimization
according to the 1:1 binding stoichiometry of ACP with the Cu2+ ion. The atom distances of N1-N3
and N2-N4 in ACP were 4.272 and 4.115 Å (Figure 6a), respectively, yet in ACP-Cu2+ complex their
distances decreased to 3.999 and 3.776 Å, respectively, which can be attributed to the fact that the
electron-donating N atom of pyrrole rings have high affinity to bind to Cu2+ with short bond lengths
(shown in Figure 6c). Moreover, the four pyrrole rings in ACP are almost planar, yet during the
formation of ACP-Cu2+ complex, the Cu2+ ions occupy the coordination center of ACP and the
molecular plane was slightly contorted and metamorphosed due to the formation of coordination
bonds and steric strain (Figure 6d). According to the experimental results, the ACP-Cu2+ complex
exhibits an absorption at 632 nm compared with the absorption at 660.5 for ACP. This can be easily
explained by the above mentioned phenomena: the introduction of Cu2+ distorts the conjugate plane
of ACP molecule, and to some extent destroys the coniugated
π-bond of the four pyrrole rings, thus
leading to a blue-shift in the UV-visible absorption spectrum of ACP-Cu2+
.
In addition, we analysed the frontier molecular orbitals (FMO’s) of bare ACP and the ACP-Cu2+
complex. This will help us to understand the quenching phenomenon upon addition of Cu2+ ions. The
calculated FMO’s are shown in Figure 7.

Molecules 2016, 21, 107
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Figure 6. Energy-minimized structures by DFT calculations: (
) viewed from the side for ACP; (
) viewed from the front for ACP-Cu2+ complex; (
the side for ACP-Cu2+ complex.
a
) viewed from the front for ACP;
(
b
c
d
) viewed from
Figure 7. Frontier molecular orbitals of ACP and ACP-Cu2+ complex (1:1) obtained at B3LYP/6-31G(d,
p) and B3LYP/LANL2DZ level, respectively.
In the absence of Cu2+, the solution of ACP is pale yellow and fluorescent, and from the figure it
is seen that the electron density in HOMO and LUMO are both localized on the pyrrole rings, mainly
involving the π-π* electronic transitions of conjugated π
-bonds. Upon the addition of Cu2+ into the
ACP solution, the HOMO of ACP-Cu2+ is distributed over the pyrrole rings, Cu2+ and amide in the
side chain, while in the LUMO the electron density is mainly localized on the pyrrole rings. This is
mainly involved in the charge-transfer (CT) from Cu2+ and amide in the side chain to the pyrrole rings.
Therefore, the quenching phenomenon may be explained by two factors: one is that the coordination
of Cu2+ to ACP decreases the electron-donating ability of the nitrogen atoms of ACP, and to some
extent the conjugated-π bond, whereby the most important ultraviolet absorption and fluorescence
were destroyed, resulting in a colour change and fluorescence quenching; the other because of the
paramagnetic nature of Cu2+. These results support our expectation that ACP could serve as a sensitive
fluorescent probe as well as a naked-eye probe for Cu2+
.

Molecules 2016, 21, 107
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3. Experimental Section
3.1. General Information
All chemicals used in this paper were obtained from commercial suppliers and used without
further purification. Ultrapure water was used for aqueous solution preparation. All samples were
prepared at room temperature and promptly used for UV-Vis and fluorescence determination. Zinc
chloride (98%), copper(II) chloride dihydrate (99%), nickel(II) chloride hexahydrate (98%), barium
chloride (99.5%), silver nitrate (99.8%), cobalt(II) chloride hexahydrate (90%), sodium chloride (99.5%),
potassium chloride (99.5%), magnesium chloride hexahydrate (98%), cadmium chloride (99%), lead(II)
nitrate (99%), ferric chloride hexahydrate (99%) and calcium chloride anhydrous (96%) were obtained
from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the chemical reactions were
1
performed under argon protection and away from sunshine. H-NMR and 13C-NMR spectra were
recorded at 400 and 100 MHz, respectively, on an AMX400 spectrometer (Bruker, Bremen, Germany)
with tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded with a VG-7070
spectrometer (Hitachi, Manchester, UK). UV-Vis absorption and emission spectra were recorded using
a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan) and spectrofluorophotometer with a 150 W
xenon lamp as a visible excitation light source (RF-5301PC, Shimadzu), respectively. All measurements
were made at room temperature (about 25 ˝C). All spectra were obtained in a quartz cuvette (path
length = 1 cm). The excitation and emission slit widths were both 10 nm, and PMT voltage of 700 V.
The fluorescence intensities/spectra were measured at λ
/
= 412/668 nm.
ex em
3.2. General Procedure for Synthesis of the title Compound
Methyl pyropheophorbide a (Mpa) was synthesized according to the literature procedure [32].
Then propanolamine (1 mL) was added to a solution of Mpa (66.73 mg, 0.11 mmol) in chloroform
and the reaction stirred under a nitrogen atmosphere for 24 h at rt. The reaction mixture was then
concentrated, and the residue was dispersed in dichloromethane (30 mL), and then washed by water
(30 mL) for three times. After drying and evaporation of the solvent, the residue was purified by
silica gel chromatography with methanol: dichloromethane (1:15) as the eluent to give pure ACP
(76%).1H-NMR (CDCl3)
δ
(ppm): 1.00~1.10 (m, 2H, 134-CH2), 1.26~1.28 (m, 2H, 155-CH2), 1.67 (t,
J = 7.6 Hz, 3H, 82-CH3), 1.68 (d, J = 7.2 Hz, 3H, 18-CH3),1.73~1.81 (m, 2H, 154-CH2), 1.86~1.91 (b, 2H,
133-CH2), 2.22~2.28 (m, 2H, 172-CH2),2.41~2.45 (m, 2H, 171-CH2), 3.27 (s, 3H, 7-CH3), 3.48 (s, 3H,
2-CH3), 3.51 (s, 3H, 12-CH3), 3.68 (s, 3H, 173-OCH3), 3.72 (q, J = 3.76 Hz, 2H, 81-CH2), 3.75~3.88 (m, 4H,
135-CH2. 156-CH2), 4.31 (b, 1H, 17-H), 4.35 (q, J = 7.2 Hz, 1H, 18-H), 4.46 (d, J = 7.2 Hz, 3H, 18-CH3),
5.29 (d, J = 18.5 Hz, 1H, 15-H), 5.39 (d, J = 18.5 Hz, 1H, 15-H), 6.13 (d, J = 2.8 Hz, 1H, 32-H (Z)), 6.33
(d, J = 2.8 Hz, 1H, 32-H (E)), 7.32 (bs, 1H, 132-NH), 8.09 (dd, J1 = 6.0Hz, J2 = 11.6Hz, 1H 31-H)), 8.81
(s, 1H, 20-H), 9.62 (s, 1H, 10-H), 9.64(s, 1H, 5-H); 13C-NMR (MeOD)
δ (ppm): 10.0, 11.5, 11.8, 17.8, 19.8,
23.5, 29.5, 31.8, 32.0, 33.2, 33.3, 33.9, 37.4, 38.6, 38.8, 50.4, 52.8, 54.4, 56.0, 60.4, 60.9, 70.5, 94.7, 98.9, 101.8,
103.5, 121.2, 129.5, 130.5, 130.9, 135.1, 135.6, 136.1, 136.8, 139.6, 145.3, 149.8, 154.8, 171.8, 175.1, 175.2.
Anal calcd for C41H52N6O6: C 67.93, H 7.23, N 11.59; found C 67.78, H 7.46, N 11.28.
4. Conclusions
In summary, we have prepared ACP, a simple but effective colorimetric and fluorescent probe
for Cu2+ detection, from methyl pheophorbide-a. It shows excellent sensitivity and selectivity for
Cu2+ over other common metal ions in aqueous media. More importantly, the color change upon
the addition of Cu2+ to ACP solutions could make it a suitable “naked eye” indicator for Cu2+.
Meanwhile, our study of the fluorescence quenching of ACP-Cu2+ complex showed the detection limit
´8
was 7.5
ˆ
10 mol/L, which suggested that ACP can act as a highly sensitive probe for Cu2+ and can
be used to quantitatively detect low levels of Cu2+ in aqueous solution.

Molecules 2016, 21, 107
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Acknowledgments: This work was supported by the National Nature Science Foundation of China (No. 20972036,
21272048) and the Program for Scientific Technological Innovation Team Construction in Universites of
Heilongjiang Province (No. 211TD010). The theoretical calculations were conducted on the ScGrid and
Deepcomp7000 the Supercomputing Center, Computer Network Information Center of Chinese Academy
of Sciences.
Author Contributions: Yingxue Jin and Zhiqiang Wang conceived and designed the experiments; Guohua Zhu
and Wenting Li performed the experiments; Guohua Zhu and Jinghua Li analyzed the data; Yingxue Jin
contributed reagents/materials/analysis tools; Zhiqiang Wang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compound ACP are available from the authors.
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