Accepted Manuscript

2+
A highly selective dual-channel fluorescent probe for the detection of Zn ion and
pyrophosphate in micelle

Min Jung Chang, Min Hee Lee

PII: S0143-7208(17)32107-1
DOI: 10.1016/j.dyepig.2017.11.057
Reference: DYPI 6403

To appear in: Dyes and Pigments

Received Date: 10 October 2017

Revised Date: 8 November 2017
Accepted Date: 29 November 2017

Please cite this article as: Chang MJ, Lee MH, A highly selective dual-channel fluorescent probe for
2+
the detection of Zn ion and pyrophosphate in micelle, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2017.11.057.

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 ACCEPTED MANUSCRIPT

A highly selective dual-channel fluorescent probe for the

detection of Zn2+ ion and pyrophosphate in micelle

Min Jung Chang, Min Hee Lee*

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Department of Chemistry, Sookmyung Women’s University, Seoul, 04310, Korea

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*Corresponding author: minheelee@sookmyung.ac.kr (M. H. Lee)

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Abstract

We presented a micellar probe 1 as a highly selective dual-channel fluorescent probe for Zn2+

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and pyrophosphate (PPi) ions. Probe 1 was comprised of tetraphenylethene (TPE) as an
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AIEgen and naphthalimide-dipicolylamine (DPA) scaffold as a Zn2+-chelating fluorescent

reporter, and it was developed as a micellar 1 in the presence of sodium dodecyl sulfate
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(SDS). The micellar probe 1 showed a blue fluorescence at 482 nm ascribed to the
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aggregation-induced emission (AIE) of TPE moiety. However, in the presence of Zn2+ ion,
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the micellar probe exhibited a strong green fluorescence at 530 nm by a suppression of photo-

induced electron transfer (PET) occurring from DPA to naphthalimide. This fluorescence
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enhancement was found to be highly selective for Zn2+ ion over other metal ions. In addition,

the Zn2+-chelated probe 1 in micelle displayed a selective ratiometric change in fluorescence

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intensities at 530 and 474 nm for PPi over other anions. The detection limits (LOD) of 1 to
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Zn2+ and PPi also turned out to be at 46 and 88 nM, respectively.

Keywords

Zinc detection, Pyrophosphate (PPi) detection, Ratiometric fluorescent probe, Aggregation-

induced emission (AIE), Photo-induced electron transfer (PET)

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Highlights

• A TPE-appended naphthalimide-dipicolylamine was synthesized.

• This was developed for the ratiometric detection of Zn2+ and PPi.

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• This showed the detection limits to Zn2+ and PPi with 46 and 88 nM, respectively.
• This probe readily responded to the Zn2+ and PPi ions in a wide range of pH 5-10.

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1. Introduction

Zinc ion (Zn2+) is one of the essential cationic element in living system due to its biological

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significance. The Zn2+ serves as catalytic center and structural cofactor of many enzymes
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involved in a variety of physiological processes such as cellular metabolism, gene expression,

cell apoptosis, regulation of metalloenzymes and neural signal transmission.1-3 Meanwhile,

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pyrophosphate (P2O74-, PPi) is one of the important biological anions due to its significant
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roles in hydrolysis of ATP, polymerization of DNA, and other metabolic reactions.4,5 Thus,
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abnormal levels6,7 of Zn2+ and PPi are associated with cellular dysfunctions and pathological

states, e.g., Alzheimer’s disease8 and cancers.9 Although the biological roles of individual
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Zn2+ and PPi are well established, the study with respect to the correlated physiological

responses of the ions still remains unrevealed. Therefore, it is required to exploit the
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analytical tools that can detect Zn2+ and PPi concurrently in both physiological and
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pathological conditions.

Currently, a number of fluorescent probes for detection of Zn2+ and PPi are developed.10-12

However, most of Zn2+ sensing probes suffer from a poor selectivity due to interferences by

other heavy and transition metal ions.13 For example, some metal chelating ligands, e.g.,

dipicolylamine (DPA), N,N-di-(2-picolyl)ethylenediamine (DPEN), N,N,N'-tris(pyridin-2-

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ylmethyl)ethylenediamine (TRPEN), and etc., are usually conjugated with fluorophores to

detect Zn2+ ion, whereas these ligands also have an affinity to Cd2+, Pb2+, Hg2+, Ni2+, Cu2+

and Fe2+ ions. On the other hand, despite the biological significance of PPi, it is still urgent

need to develop PPi-selective fluorescent probes that can operate in aqueous solution. In

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recent years, several metal (e.g., Zn2+, Cd2+, and Cu2+) complexes-based fluorescent probes

were exploited to detect PPi in physiological condition.14-16 However, these probes often

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display a relatively poor sensitivity to PPi with a fluorescent Off-On manner. Thus, the

development of fluorescent probes that can detect both Zn2+ and PPi ions selectively in

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physiological condition is crucial.

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Herein, we presented a dual-channel fluorescent micellar probe 1 for detecting Zn2+ and
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PPi ions in physiological condition. The probe 1 was comprised of dipicolylamine (DPA)-

linked naphthalimide as a Zn2+-sensitive fluorophore and tetraphenylethene (TPE) as an

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AIEgen that can display an aggregation-induced emission (AIE). In addition, to improve

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water solubility, this probe was mixed with surfactants resulting in a micellar probe 1.
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Besides, only a few fluorescent probes for Zn2+ and PPi based on aggregation induced

emission (AIE) effect have been reported.17,18

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Briefly, as illustrated in scheme 1, probe 1 was mixed with sodium dodecyl sulfate (SDS)
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forming a micellar probe 1. Upon excitation at 341 nm corresponding to the absorption of

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TPE part, the micellar probe 1 displayed a blue emission at 482 nm due to an AIE effect from

the TPE moiety19-21 and its naphthalimide showed almost no fluorescence by a photo-induced

electron transfer (PET) occurring from DPA to naphthalimide.22 However, upon binding of

Zn2+ ions, the micellar 1 showed a strong green fluorescence at 530 nm from the

naphthalimide through a suppression of the PET effect.11,22 In addition, the Zn2+-chelated

probe 1 in micelle exhibited a ratiometric fluorescence change in the fluorescence intensities

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at 530 and 450 nm towards PPi ion presumably due to a rearrangement of Zn2+ binding.11,15

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Scheme 1. The illustration of proposed sensing mechanism of 1 to Zn2+ and PPi ions.
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2. Experimental

2.1. Materials and instrumentation

All UV/Vis absorption and fluorescence spectroscopic data were collected using UV-2600
(Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) and RF-6000 (Shimadzu
Corporation, Kyoto, Kyoto Prefecture, Japan) spectrophotometers, respectively. 1H and 13
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NMR spectra were collected in CDCl3 (Cambridge Isotope Laboratories, Cambridge, MA) on
Brucker 500 MHz spectrometers. HR-ESI-MS data were obtained using liquid

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chromatography mass spectrometer (LC/MS) at the Korea Basic Science Institute (Seoul).
Field emission scanning electron microscopy (FE-SEM) images were conducted on a JEOL
JSM-7600F instrument at an accelerating voltage of 3 kV. The specimens for SEM images
were prepared by depositing several drops of micellar solutions onto stubs attached with
carbon tapes then dried under freeze-drying conditions with freeze dryer of Operon for 3 days.
Silica gel 60 (Merck, 0.063-0.2 mm) was used for column chromatography. Analytical thin

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layer chromatography (TLC) was performed using Merck60 F254 silica gel (precoated sheets,
0.25 mm thick). All reactions were carried out under nitrogen atmosphere. All reagents

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including dipicolylamine, dimethylformamide (DMF), chloride salts of metal ions,
tetrabutylammonium (TBA) salts of anions, other chemicals for synthesis and analysis were

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purchased from Aldrich, TCI, Alfa and used as received. All solvents were analytical reagents.
DMSO for spectroscopic experiments was HPLC reagent without fluorescent impurity and

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water was HPLC grade.
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2.2. UV/Vis absorption and fluorescence spectroscopic methods
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Stock solutions of synthetic compounds were prepared in DMSO. Stock solutions of TBA
salts of anions and chloride salts of metal ions were prepared in CH3CN and HPLC grade
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water, respectively. Different pH buffer solutions were prepared by using literature

procedures.23 In order to prepare the micellar probe, the 30 µL of probe (1 mM of stock
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solution) was added into 2.970 mL of HEPES buffer (10 mM) solution containing 1 mM of
SDS under agitation at room temperature. Samples for absorption and emission
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measurements were contained in quartz cuvettes (4 mL volume). Excitation was provided at

341 nm with excitation and emission slit widths being set at 5/10 nm or 5/3 nm.
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2.3. Synthesis

2.3.1. Synthesis of 2 and TPE-NH2

Precursors 2 and TPE-NH2 were prepared according to the previously published

procedures.22,24

2.3.2. Synthesis of Probe 1

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Compound 2 (0.3 g, 0.6 mmol) was stirred with the solution of trifluoroacetic acid (TFA)
(5 mL) and dichloromethane (DCM) (10 mL) for 2 h at room temperature. After evaporation
of the solution in vacuo, the resulting product was treated with TPE-NH2 (0.6 g, 1.8 mmol)
in the presence of EDCI (0.5 g, 2.7 mmol) and DMAP (0.4 g, 3.7 mmol) in DMF (10 mL)
solvent and stirred for overnight under nitrogen atmosphere. The solvent was then evaporated
under vacuum. The crude product was dissolved in ethyl acetate (EA) (100 mL), thoroughly

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washed with brine (100 mL), dried over Na2SO4. After evaporating solvents, the crude
product was purified by silica gel column chromatography using EA and hexanes (v/v, 1:2) as

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an eluent, giving product 1. In addition, the 1 was further purified by a recrystallization with
DCM and diethyl ether. This gave product 1 as a pale yellow solid in 27% yield (0.14 g).

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ESI-MS m/z [M+H]+ calc 853.3502, obs 853.3493. 1H NMR (CDCl3, 500 MHz): δ 2.87 (t,
2H, J = 6.8 Hz); 3.63 (s, 2H); 4.08 (s, 4H); 4.56 (t, 2H, J = 6.8 Hz); 6.96-7.16 (m, 19H); 7.29

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(d, 2H, J = 7.5 Hz); 7.34 (d, 2H, J = 8.5 Hz); 7.61 (t, 2H, J = 7.5 Hz); 7.85 (t, 1H, J = 7.8 Hz);
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8.22 (s, 1H); 8.43 (d, 2H, J = 4.0 Hz); 8.57 (d, 2H, J = 8.5 Hz); 8.66-8.70 (m, 2H); 9.13 (d,
1H, J = 8.5 Hz); 11.80 (s, 1H). 13C NMR (CDCl3, 125 MHz): δ 36.6, 36.8, 59.2, 60.7, 117.1,
118.9, 122.7, 123.4, 123.6, 126.2, 126.3, 126.4, 126.4, 127.6, 127.6, 127.8, 128.8, 129.2,
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131.3, 131.3, 131.4, 131.5, 132.0, 133.3, 136.7, 140.4, 143.8, 149.6, 157.7, 164.1, 164.6,
168.5, 170.9 ppm.
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2.3.2. Synthesis of TPE-amide

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To a mixture butyric acid (0.03 g, 0.3 mmol), EDCI (0.3 g, 1.6 mmol) and DMAP (0.3 g,
2.0 mmol) in 10 mL of DMF, the solution of TPE-NH2 (0.1 g, 1.8 mmol) dissolved in DMF
(1 mL) was added. The resulting solution was stirred overnight under nitrogen atmosphere.
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Then, the solvent was removed under vacuum. The crude product was dissolved in ethyl
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acetate (EA) (100 mL), thoroughly washed with brine (100 mL), dried over Na2SO4.
Evaporation of the solvents provided TPE-amide as a pale yellow solid in 98% yield (0.15 g).
ESI-MS m/z [M+H]+ calc 418.20926, obs 418.21723. 1H NMR (CDCl3, 500 MHz): δ 0.94 (t,
3H, J = 7.5 Hz); 1.66-1.70 (m, 2H); 2.24 (t, 2H, J = 7.5 Hz); 6.93 (d, 2H, J = 8.5 Hz); 6.99-
7.08 (m, 15H); 7.24 (d, 2H, J = 8.4 Hz); 7.50 (s, 1H). 13C NMR (CDCl3, 125 MHz): δ 13.9,
19.2, 39.9, 118.9, 126.5, 126.6, 126.6, 127.2, 127.8, 127.8, 127.9, 128.0, 128.6, 131.5, 131.5,
132.1, 136.4, 139.7, 140.4, 140.5, 140.9, 143.8, 143.9, 171.2 ppm.

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3. Results and discussion

Probe 1 was synthesized by the synthetic routes depicted in Scheme 2a. Precursors 2-4 and

TPE-NH2 were prepared according to the previously published procedures.22,24 The 2

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underwent de-tert-butylation in TFA and DCM, which then reacted with TPE-NH2 in the

presence of EDCI, DMAP in DMF yielding probe 1 in 27% yield. In addition, TPE-amide,

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lacking the naphthalimide-DPA moiety, was prepared as a control compound using a similar

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condensation reaction using EDCI, DMAP and DMF (Scheme 2b). The chemical structures
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of 1 and TPE-amide were confirmed by H NMR, C NMR, and HR-ESI mass

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spectroscopies (Figs. S19-S24). In order to prepare micellar probe 1, a solution of 1 in DMSO
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was added into HEPES buffer containing 1 mM of surfactants (e.g., CTAB;

cetyltrimethylammonium bromide or SDS; sodium dodecyl sulfate), which was then agitated
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at room temperature. The micellar probe 1 was also identified by SEM image (Fig. S15).
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Scheme 2. Synthetic routes for probe 1 (a) and TPE-amide (b).

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First of all, optical properties of probe 1 were investigated in HEPES buffer (pH 7.4, 10
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mM) containing 1% DMSO (v/v). In Fig. 1a, probe 1 displayed a shouldered absorption with

two maxima at 341 and 371 nm derived from the TPE and naphthalimide moieties,

respectively. In addition, upon excitation at 341 nm, the probe exhibited a shouldered

emission with maxima at 482 and 520 nm ascribed to the TPE and naphthalimide moieties,

respectively (Fig. 1b). The absorption and emission spectra of analogues 2 and TPE-amide,

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lacking TPE and naphthalimide-DPA moieties, respectively, were also checked under similar

conditions for the comparison.

The fluorescence spectra of micellar probe 1 were monitored in presence of cationic

(CTAB) and anionic (SDS) surfactants in HEPES buffer (pH 7.4, 10 mM) containing 1%

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DMSO (v/v). In Fig. 1c, probe 1 showed a green fluorescence cantered at 520 nm. However,

micellar probe 1 composed of SDS displayed a blue fluorescence cantered at 482 nm. In case

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of micellar 1 composed of CTAB, a green fluorescence at 530 nm was seen (see also insets of

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Fig. 1c). In Fig. 1d, the fluorescence spectra of 2 and TPE-amide were also monitored in

presence of SDS. It was revealed that TPE-amide, lacking naphthalimide-DPA moiety,

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displayed a blue fluorescence at 482 nm in presence of SDS (see also insets of Fig. 1d). In
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contrast, 2 lacking TPE moiety exhibited a very weak emission around 500 nm under similar

condition. This implied that micellar probe 1 composed of SDS gave a blue fluorescence at
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482 nm ascribed to the AIE effect from the TPE moiety, and it would be utilized for detecting
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Zn2+ and PPi in a ratiometric fluorescence manner.

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In addition, to check if probe 1 itself displays an aggregation-induced emission, the

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changes in the fluorescence intensity were tested at different water fractions of the mixed

solvents (e.g., water-DMSO and water-THF mixtures) (Fig. S1). However, probe 1 has the
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AIE phenomenon in neither water-DMSO nor water-THF mixtures. Analogue 2, lacking TPE
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moiety, also exhibited no AIE phenomenon (Fig. S2). In contrast, TPE-amide, lacking

naphthalimide, displayed a strong increase in the fluorescence intensity at 482 nm as

increasing water fraction (fw) upon excitation at 341 nm (Fig. S3). These results revealed that

probe 1 showed no aggregation-induced emission (AIE) in the mixed solvents, whereas in the

SDS micellar condition, probe 1 became aggregated giving an AIE signal from the TPE

moiety.

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Fig. 1. (a) UV/Vis absorption and (b) fluorescence spectra of 1, 2 and TPE-amide in HEPES
buffer (pH 7.4, 10 mM) containing 1% DMSO (v/v). (c) Fluorescence spectra of 1 with and
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without surfactants (CTAB or SDS). Insets show the fluorescent color changes of 1 at
different surfactants. (d) Fluorescence spectra of 1, 2 and TPE-amide with SDS. Excitation
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was affected at 341 nm. Insets show the fluorescent color changes of 1, 2 and TPE-amide
with SDS.
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Sensing ability of nonmicellar probe 1 for Zn2+ ion was then tested in HEPES buffer (pH
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7.4, 10 mM) containing 1% DMSO (v/v). In Fig. S4a, probe 1 exhibited a large fluorescence
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enhancement at 530 nm upon adding 1.0 equiv. of Zn2+ ion. In addition, in case of Cd2+ and

Pb2+, the distinct fluorescence enhancements at 460 and 550 nm were observed. However, a

negligible fluorescence change was seen in the presence of other metal ions such as K+, Na+,

Ca2+, Mg2+, Ba2+, Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+ ions. The large

fluorescence enhancement observed for 1 in presence of Zn2+ presumably due to a prohibition

of PET effect occurring from the naphthalimide-DPA. To investigate the proposed PET

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mechanism, we monitored the fluorescence changes of 2 and TPE-amide to various metal

ions in similar condition. In Fig. S4, 2 lacking TPE moiety exhibited a similar fluorescence

enhancement at 530 nm to Zn2+ ion. However, TPE-amide lacking naphthalimide-DPA

moiety displayed a negligible change to Zn2+ ion. These results validated that the increase of

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fluorescence intensity at 530 nm observed for 1 was ascribe to a suppression of the PET

occurred from DPA to naphthalimide upon the Zn2+ binding.

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Next, sensing ability of micellar probe 1 to Zn2+ ion was evaluated (Figs. 2b and S5).

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Probe 1 was formulated with 1 mM of SDS in HEPES buffer (pH 7.4, 10 mM) containing 1%

DMSO (v/v), resulting in micellar probe 1. Unlike probe 1, micellar probe 1 exhibited a

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highly selective fluorescence enhancement at 530 nm to Zn2+ over other metal ions including
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Cd2+ and Pb2+ ions. The fluorescent color was also changed from blue to green in presence of

Zn2+ ions (Fig. S6). In addition, parallel experiments were carried out using 2 and TPE-
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amide formulated with SDS. The micellar 2 exhibited fluorescence changes for several metal
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ions such as Zn2+, Pb2+, Cd2+ and Mg2+ ions (Fig. S7). In contrast, the micellar TPE-amide
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displayed no fluorescence change to various metal ions (Fig. S8). On this basis, we believed

that probe 1 possessing a hydrophobic TPE moiety became highly aggregated with SDS,
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which grant a selective binding affinity to Zn2+ over other metal ions as well as a ratiometric

fluorescent color change based on the combination of AIE and PET phenomenon.
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The absorption and fluorescence titrations of micellar probe 1 were then carried out with

different concentrations of Zn2+ ions. In Fig. 2c, the micellar probe 1 displayed a blue

fluorescence at 482 nm ascribed to AIE effect of the TPE moiety. However, upon adding the

increasing concentrations of Zn2+ ion, the fluorescence intensity at 530 nm was gradually

enhanced (see also Fig. S9). In Fig. 2d, the fluorescence enhancement was found to be

saturated at 1.0 equiv. of Zn2+ ion. The gradual color change from blue to green was also seen

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in the insets of Fig. 2d. As seen in Fig. S10, the fluorescence intensity at 530 nm was linearly

increased in a dynamic range of 0-1.0 µM of Zn2+ ion (R2 = 0.969). The detection limit of

micellar 1 to Zn2+ was calculated to be 46 nM (3σ/s, LOD = limit of detection).25 In addition,

Job’s plot analysis indicated that micellar 1 bound to Zn2+ with a 1:1 stoichiometry (Fig.

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S11). On this basis, Benesi-Hildebrand plot was created and the binding constant (Ka) of

micellar 1 to Zn2+ ion was found to be 1.28 × 104 M-1 (Fig. S12). These results suggested that

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micellar 1 could detect nanomolar levels of Zn2+ ion in a ratiometric fluorescence change

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manner without interfering from other metal ions.

To evaluate the sensing ability of micellar probe 1 to Zn2+ ion in presence of other

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competitive metal ions, the enhancement in the fluorescence intensity (FI) at 530 nm was
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monitored. In Fig. 2d, micellar 1 displayed a significant enhancement in FI at 530 nm in

presence of Zn2+ ion, while a negligible fluorescence change was seen in the case of other
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metal ions (see gray bars in Fig. 2d). In contrast, upon the subsequent addition of Zn2+ ions to
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1 in the presence of other metal ions, a noticeable fluorescent enhancement was observed (see
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blue bars in Fig. 2d). This tell us that micellar probe 1 can detect Zn2+ ions even in the

presence of other competitive metals.

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Fig. 2. (a) Fluorescence spectra of 1 (10 µM) in the presence of various metal ions (K+, Na+,
Ca2+, Mg2+, Ba2+, Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+; 1.0 equiv.,
respectively). (b) Fluorescence changes of 1 (10 µM) at different concentrations of Zn2+ ion
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(0-50 equiv.). (c) Plot of fluorescence intensity (FI) at 530 nm vs [Zn2+]; insets display the
fluorescent color change of 1 at different concentrations of Zn2+ (0-1.0 equiv.). (d)
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Fluorescence changes of 1 to Zn2+ in the presence of various metal ions. Gray bars represent
fluorescence intensity (FI) at 530 nm in the presence of metal ions (1.0 equiv., respectively).
Blue bars represent FI at 530 nm upon the subsequent addition of Zn2+ (1.0 equiv.). All data
were obtained in HEPES buffer (pH 7.4, 10 mM) containing 1% DMSO (v/v) and 1 mM of
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SDS. Excitation wavelength was affected at 341 nm.

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The sensing ability of the Zn2+-chelated fluorescent probe 1 (1-Zn) for PPi was
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investigated in HEPES buffer (pH 7.4, 10 mM) containing 1% DMSO (v/v) and 1 mM of

SDS. In Fig. 3a, upon the addition of PPi to the solution of micellar 1-Zn, the fluorescent

emission was blue-shifted from 530 to 474 nm (∆λ = 56 nm). A distinct color change was

also monitored from green to blue. However, a negligible fluorescence change was seen upon

the addition of other anions such as HPO4-, ClO4-, AcO-, CN-, OH-, F-, Cl- and I- ions (Fig.

S13). In addition, in the absence of Zn2+, the micellar 1 showed no fluorescence response to
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PPi (Fig. S14). Also, the micellar structure of 1 in presence of Zn2+ and PPi ions was

identified by SEM images (Fig. S15). These results inferred that the Zn2+-chelating

fluorescent micellar 1 can be used as a ratiometric fluorescent probe for PPi in physiological

condition.

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The absorption and fluorescence titrations of 1-Zn were then carried out with increasing

concentrations of PPi (Figs. 3b and S16). The micellar 1-Zn exhibited a strong green

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fluorescence at 530 nm. However, upon increasing concentrations of PPi from 0 to 200 equiv.,

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the fluorescent emission was gradually blue-shifted from 530 to 474 nm. This could be

explained by a rearrangement of the Zn2+ ion of 1-Zn in the presence of PPi as illustrated in

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Scheme 1. This presumption was reflected the facts that the amide linker of naphthalimide-
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DPA moiety undergoes tautomerization forming an imidic tautomer upon binding of some

metal ions.10 In the case of imidic tautomer, the naphthalimide-DPA displays a green
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fluorescent emission, while a blue fluorescence is observed in its amide tautomer. On the
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basis, we could assume that probe 1 bind to Zn2+ in an imidic tautomer giving a green
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fluorescence, while in the presence of PPi the imidic tautomer of 1-Zn might be changed to

the amide tautomer through a rearrangement of Zn2+ binding, resulting in the fluorescent
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color change from green to blue.15

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Moreover, the precise and quantitative detection of 1-Zn to PPi was achieved by measuring
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the ratiometric change between fluorescence intensities at 450 (I450) and 530 nm (I530). In Fig.

3c, with an increase of PPi equivalent, the fluorescence ratio (I450/I530) was gradually

increased. The gradual change was also visualized by a fluorescent color change from green

to blue (see insets of Fig. 3c). In addition, the fluorescence ratios of 1-Zn displayed a good

linearity in the dynamic range of 0-20 µM of PPi (R2 = 0.959) (Fig. S17). The detection limit

of 1-Zn to PPi turned out to be 88 nM. The binding constant (Ka) of 1-Zn to PPi was also

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found to be 1.83 × 104 M-1 by Benesi-Hildebrand plot (Fig. S18). Therefore, we demonstrated

that micellar probe 1 could be used as a ratiometric fluorescent probe for Zn2+ and PPi ions in

physiological conditions, and it would permit a dual-channel imaging of Zn2+ (green channel)

and PPi (blue channel) ions. Further, micellar probe 1 would serve as a promising fluorescent

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indicator for studying the Zn2+/PPi-related physiological responses.

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Fig. 3. (a) Fluorescence change of micellar 1 (10 µM) in the absence and presence of Zn2+
(1.0 equiv.) and PPi (100 equiv.). (b) Fluorescence spectral change of the solution of micellar
1 (10 µM) containing Zn2+ (1 equiv.) at different concentrations of PPi (0-200 equiv.). (c) Plot
of the fluorescence ratio (I450/I530) vs [PPi]; the insets display the fluorescent color change of
1-Zn at different concentrations of PPi (0-100 equiv.). All data were obtained in HEPES
buffer (pH 7.4, 10 mM) containing 1% DMSO (v/v) and 1 mM of SDS. Excitation
wavelength was affected at 341 nm.

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Furthermore, pH effect on the fluorescence change of micellar 1 was investigated in the

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absence and presence of Zn2+ and PPi ions. In the aspect of Zn2+ sensing, micellar probe 1

showed a very weak fluorescence at 530 nm, but in the presence of Zn2+ ion (1.0 equiv.) the

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fluorescence intensity was readily enhanced in pH range from 6 to 11 (Fig. 4a). On the other

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hand, in the case of PPi sensing, the Zn2+-chelated micellar probe 1-Zn exhibited a gradual
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increase in the ratio of fluorescence intensities at 450 and 530 nm (I450/I530) upon acidifying

pH value (Fig. 4b). However, in the presence of PPi (100 equiv.), a clear enhancement in the
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fluorescence ratio (I450/I530) was readily monitored in pH ranging 5-10. These results

indicated that micellar probe 1 provided a fluorescence change to Zn2+ and PPi ions in a wide
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pH range from 5 to 10, and thus it would be applicable for imaging biological Zn2+ and PPi
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ions in physiological condition.

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Fig. 4. Fluorescence changes of 1 (10 µM) to Zn2+ (1.0 equiv.) and PPi (100 equiv.) ions at
different pH values. All data were obtained in HEPES buffer (pH 7.4, 10 mM) containing 1%
DMSO (v/v) and 1 mM of SDS. Excitation wavelength was affected at 341 nm.
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4. Conclusions

We developed a highly selective ratiometric fluorescent probe 1 for the detection of Zn2+

and PPi ions in physiological condition. This probe was composed of TPE as an AIEgen and

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naphthalimide-linked DPA as a Zn2+ sensitive unit. Micellar probe 1 was prepared using SDS

in HEPES buffer at pH 7.4. Interestingly, we found that the micellar 1 displayed a blue

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fluorescence at 482 nm ascribed to the AIE effect caused by an aggregation of the TPE

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moiety. In addition, micellar 1 showed a highly selective enhancement in the fluorescence

intensity at 530 nm for Zn2+ over other metal ions. The detection limit of 1 to Zn2+ also turned

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out to be 46 nM. Moreover, we demonstrated that upon binding with Zn2+ ion the micellar
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probe 1 has a strong affinity to PPi, providing a ratiometric change in the fluorescence

intensities at 530 and 474 nm with a distinct color change from green to blue. The detection
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limit was revealed to be 88 nM. Furthermore, micellar probe 1 readily responded to the Zn2+
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and PPi ions in a wide range of pH 5-10. As a result, we suggested that micellar probe 1
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could detect Zn2+ and PPi ions in a ratiometric manner allowing for the precise qualitative

and quantitative analyses. Moreover, this probe would serve as a promising ratiometric
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fluorescent probe for imaging Zn2+/PPi-related physiological responses.

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5. Acknowledgments

This study was supported by the National Research Foundation of Korea (NRF)

(2015R1C1A2A01054496, MHL).

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6. Supplementary data

Supplementary data related to this article can be found http:////////////

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7. References

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9. Xu S, He M, Yu H, Cai X, Tan X, Lu B, Shu B. A quantitative method to measure

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11. Xu Z, Baek KH, Kim HN, Cui J, Qian X, Spring DR, Shin I, Yoon J. Zn2+-triggered

amide tautomerization produces a highly Zn2+-Selective, cell-permeable, and

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ratiometric fluorescent sensor. J Am Chem Soc 2009;132:601–10.

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12. Zhang JF, Kim S, Han JH, Lee SJ, Pradhan T, Cao QY, Lee SJ, Kang C, Kim JS.
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Pyrophosphate-selective fluorescent chemosensor based on 1,8-naphthalimide–DPA–

Zn(II) complex and its application for cell imaging. Org Lett 2011;13:5294−7.
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13. Xu Z, Yoon J, Spring DR. Fluorescent chemosensors for Zn2+. Chem Soc Rev
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14. Ngo HT, Liu X, Jolliffe KA. Anion recognition and sensing with Zn(II)–

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15. Kurishita Y, Kohira T, Ojida A, Hamachi I. Rational design of FRET-based

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ratiometric chemosensors for in vitro and in cell fluorescence analyses of nucleoside

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polyphosphates. J Am Chem Soc 2010;132:13290–9.

16. Lee S, Yuen KKY, Jolliffe KA, Yoon J. Fluorescent and colorimetric chemosensors

for pyrophosphate. Chem Soc Rev 2015;44:1749−62.

17. Chao D, Ni S. Nanomolar pyrophosphate detection and nucleus staining in living

cells with simple terpyridine–Zn(II) complexes. Sci Rep 2016;6:26477−84.

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18. Park C, Hong JI. A new fluorescent sensor for the detection of pyrophosphate based

on a tetraphenylethylene moiety. Tetrahedron Lett 2010;51:1960-2.

19. Shin WS, Park SK, Verwilst P, Koo S, Lee JH, Chi SG, Kim JS. Targeted

combinational therapy inducing mitochondrial dysfunction. Chem Commun

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aggregation induced emission theranostics: crucial importance of in situ activation.

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5361–88.
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22. Kim KT, Yoon SA, Ahn J, Choi Y, Lee MH, Jung JH, Park J. Synthesis of fluorescent

naphthalimide-functionalized Fe3O4 nanoparticles and their application for the

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luminogen based on cyclohexanehexone substituted with AIE active

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tetraphenylethene functionality. RSC Adv 2015;5:56270–3.

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Graphical Abstract

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Highlights

• A TPE-appended naphthalimide-dipicolylamine was synthesized.

• This was developed for the ratiometric detection of Zn2+ and PPi.
• This showed the detection limits to Zn2+ and PPi with 46 and 88 nM, respectively.
• This probe readily responded to the Zn2+ and PPi ions in a wide range of pH 5-10.

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Supporting Information

A highly selective dual-channel fluorescent probe for the

detection of Zn2+ ion and pyrophosphate in micelle

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Min Jung Chang, Min Hee Lee*

Department of Chemistry, Sookmyung Women’s University, Seoul, 04310, Korea

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*Corresponding author: minheelee@sookmyung.ac.kr (M. H. Lee)

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Contents

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1. Additional spectroscopic data
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2. 1H, 13C NMR, and ESI-MS spectroscopies
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1. Additional spectroscopic data

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Figure S1. (a) Absorption and (b) fluorescence spectra of 1 (10 µM) in water-DMSO mixtures with different
fractions of water (fw). (c) Plot of fluorescence intensities at 450 (black) and 530 nm (red) vs fw of water-DMSO
mixtures. (d) Absorption and (e) fluorescence spectra of 1 in water-THF mixtures with different fractions of
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water (fw). (f) Plot of fluorescence intensities at 450 (black) and 530 nm (red) vs fw of water-THF mixtures. All
data were obtained using an excitation wavelength of 341 nm (slit widths = 5/3).
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Figure S2. (a) Absorption and (b) fluorescence spectra of 2 (10 µM) in water-DMSO mixtures with different
fractions of water (fw). (c) Plot of fluorescence intensities at 450 (black) and 530 nm (red) vs fw of water-DMSO
mixtures. All data were obtained using an excitation wavelength of 341 nm (slit widths = 5/3).
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Figure S3. (a) Absorption and (b) fluorescence spectra of TPE-amide (10 µM) in water-DMSO mixtures with
different fractions of water (fw). (c) Plots of fluorescence intensity (FI) at 482 nm vs fw of water-DMSO mixtures.

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All data were obtained using an excitation wavelength of 341 nm (slit widths = 5/3).

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Figure S4. Fluorescence spectra of nonmicellar (a) 1 (b) 2 and (c) TPE-amide (10 µM for both) in the presence
of various metal ions (K+, Na+, Ca2+, Mg2+, Ba2+, Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+, 1.0
equiv., respectively). All data were obtained using an excitation wavelength of 341 nm in HEPES buffer (pH 7.4,
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10 mM) solution containing 1% DMSO (v/v) (slit widths = 5/3).

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Figure S5. Absorption spectra of 1 (10 µM) in the presence of various metal ions (K+, Na+, Ca2+, Mg2+, Ba2+,
Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+ ,1.0 equiv., respectively) in HEPES buffer (pH 7.4, 10
mM) solution containing 1% DMSO (v/v) and 1 mM of SDS.
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Figure S6. Fluorescence changes of 1 (10 µM) in the absence (black) and presence of Zn2+ (1.0 equiv.) (red) in
(a) HEPES buffer (pH 7.4, 10 mM) solution containing 1% DMSO (v/v), (b) HEPES buffer (pH 7.4, 10 mM)

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solution containing 1% DMSO (v/v) and 1 mM of CTAB, and (c) HEPES buffer (pH 7.4, 10 mM) solution
containing 1% DMSO (v/v) and 1 mM of SDS. All data were obtained using an excitation wavelength of 341
nm (slit widths = 5/10). Note that CTAB and SDS indicates cetyltrimethylammonium bromide and sodium

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dodecyl sulfate, and used for preparation of micellar probe, respectively.

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Figure S7. (a) Absorption and (b) fluorescence spectra of 2 (10 µM) in the presence of various metal ions (K+,
Na+, Ca2+, Mg2+, Ba2+, Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+; 1.0 equiv., respectively). All data
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were obtained using an excitation wavelength of 341 nm in HEPES buffer (pH 7.4, 10 mM) solution containing
1% DMSO (v/v) and 1 mM of SDS (slit widths = 5/10).
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Figure S8. (a) Absorption and (b) fluorescence spectra of TPE-amide (10 µM) in the presence of various metal
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ions (K+, Na+, Ca2+, Mg2+, Ba2+, Cd2+, Co2+, Pb2+, Hg2+, Ni2+, Cu2+, Zn2+, Fe2+ and Fe3+, 1.0 equiv., respectively).
All data were obtained using an excitation wavelength of 341 nm in HEPES buffer (pH 7.4, 10 mM) solution
containing 1% DMSO (v/v) and 1 mM of SDS (slit widths = 5/10).

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Figure S9. Absorption spectra of micellar 1 (10 µM) upon the addition of different concentrations of Zn2+ (0-50
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equiv.) in HEPES buffer (pH 7.4, 10 mM) solution containing 1% DMSO (v/v) and 1 mM of SDS.
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Figure S10. Plot of fluorescence intensity of micellar 1 (10 µM) at 530 nm vs [Zn2+]. The limit of detection
(LOD) of 1 to Zn2+ turned out to be 46 nM.
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Figure S11. Job’s plot of the complexation of micellar 1 (50 µM) with Zn2+ (50 µM) was plotted as function of
the different molar ratios [1]/[1 + Zn2+] by fluorescence method. All data were obtained in HEPES buffer
solution (10 mM, pH 7.4) containing 1% DMSO (v/v) and 1 mM of SDS using an excitation wavelength of 341

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nm (slit widths = 5/10).
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Figure S12. Benesi-Hildebrand plot of micellar 1 (10 µM) with Zn2+ ions obtained from fluorescence titration
data. The binding constant (Ka) of 1 to Zn2+ turned out to be 1.28 × 104 M-1.
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Figure S13. (a) Absorption and (b) fluorescence spectra of 1 (10 µM) and Zn2+ (1.0 equiv.) upon the addition of

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various anions (PPi, HPO4-, ClO4-, AcO-, CN-, OH-, F-, Cl- and I-, 1000 µM, respectively). All data were
obtained using an excitation wavelength of 341 nm in HEPES buffer (pH 7.4, 10 mM) solution containing 1%
DMSO (v/v) and 1 mM of SDS (slit width = 5/10).

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Figure S14. (a) Absorption and (b) fluorescence spectra of 1 (10 µM) upon the addition of various anions (PPi,
HPO4-, ClO4-, AcO-, CN-, OH-, F-, Cl- and I-, 1000 µM, respectively). All data were obtained using an excitation
wavelength of 341 nm in HEPES buffer (pH 7.4, 10 mM) solution containing 1% DMSO (v/v) and 1 mM of
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SDS (slit width = 5/10).

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Figure S15. SEM images of the micellar probe 1 (10 µM) (a) before and (b) after addition of Zn2+ (1.0 equiv.)

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and PPi (100 equiv.). Scale bar: 100 nm.

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Figure S16. Absorption spectra of the solution of 1 (10 µM) and Zn2+ (1.0 equiv.) upon the addition of different
concentrations of PPi (0–200 equiv.) in HEPES buffer (pH 7.4, 10 mM) solution containing 1% DMSO (v/v)
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and 1 mM of SDS.
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Figure S17. Plot of fluorescence intensity of 1-Zn (10 µM) at 450 nm vs [PPi]. The limit of detection (LOD) of
1-Zn to PPi turned out to be 88 nM.
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Figure S18. Benesi-Hildebrand plot of 1 (10 µM) and Zn2+ (1.0 equiv.) with PPi ions obtained from

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fluorescence titration data. The binding constant (Ka) of 1-Zn to PPi turned out to be 1.83 × 104 M-1.

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2. 1H, 13C NMR and ESI-MS spectroscopies

20170420-TPEamide

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N
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TPE-amide

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9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
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f1 (ppm)

Figure S19. 1H NMR spectrum of TPE-amide in CDCl3.

20170808-TPEref
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TPE-amide
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180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10

f1 (ppm)

Figure S20. 13C NMR spectrum of TPE-amide in CDCl3.

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O
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TPE-amide

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Figure S21. HR-ESI-MS spectrum of TPE-amide.

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20170405-H

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13.5 12.5 11.5 10.5 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
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Figure S22. 1H NMR spectrum of 1 in CDCl3.

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20170405-C

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180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

Figure S23. 13C NMR spectrum of 1 in CDCl3.

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Figure S24. HR-ESI-MS spectrum of 1.