A selective triarylmethine-based spectroscopic probe for Zn2+ ion monitoring

Article abstract of DOI:10.1016/j.dyepig.2019.107721

We report the synthesis and application of a novel probe of the di-2-picolylamine (DPA)-conjugated triarylmethine (TAM) dye which has aimed at detecting Zn²⁺ ions. The 1:2 binding stoichiometry is estimated between Zn²⁺ and DPA-TAM a

Full text of DOI:10.1016/j.dyepig.2019.107721

DyesandPigments171(2019)107721
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A selective triarylmethine-based spectroscopic probe for Zn2+ ion
monitoring
Naru Leea, Nguyen Hoang Lyb, Jong Seung Kimc,∗∗, Hyo Sung Junga,∗, Sang-Woo Joob,∗∗∗
a Department of Biological Sciences, Hyupsung University, Hwaseong, 18330, South Korea
b Department of Information Communications, Materials, and Chemistry Convergence Technology, Soongsil University, Seoul, 06978, South Korea
c Department of Chemistry, Korea University, Seoul, 02842, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synthetic probe
We report the synthesis and application of a novel probe of the di-2-picolylamine (DPA)-conjugated triar-
ylmethine (TAM) dye which has aimed at detecting Zn2+ ions. The 1:2 binding stoichiometry is estimated
between Zn2+ and DPA-TAM according to Job's plot diagram and density functional theory (DFT) calculations.
The increment of Zn2+ ion concentrations leads to the elevated SERS intensity of DPA-TAM on gold nano-
particles (AuNPs) due to the intermolecular chelation between the Zn2+ ion and DPA-TAM. Our synthetic
spectroscopic probe shows [Zn2+]-correlated SERS intensities with an estimated detection limit of 50 μM. This is
due to a plasmonic enhancement effect from the well-dispersed metal nanoparticles to DPA-TAM-AuNP ag-
gregates, which is activated by Zn2+ ion. The prepared DPA-TAM-AuNP system can also be successfully applied
to monitoring of intracellular Zn2+ ions both in human cervical carcinoma HeLa cells in vitro and daphnia in vivo
through dark-field microscopy (DFM)-equipped Raman cellular imaging. Our DPA-TAM-AuNP system provides a
new insight in potential applications of bioanalytical Zn2+ ion detection methodology.
Intracellular Zn2+ ion detection
Surface enhanced Raman scattering
Dark-field microscopy
In vitro
In vivo
1. Introduction
introducing nanostructures to enhance sensitivities [20]. Several types
of SERS dyes have been introduced for better applications of in vitro and
Zinc, the second most plentiful transition metal ions in the human
body, is present at its largest amounts in the brain [1]. High in-
tracellular zinc levels are known to promote cellular death by inhibiting
energy production [2], as is found in Alzheimer's disease [3]. Therefore,
the ability to precisely sense intracellular Zn2+ ion is important for
understanding Zn2+ homeostasis and human pathophysiology in bio-
logical processes [4]. Fluorescence sensors have gained considerable
attention for this reason [5]. Di-2-picolylamine (DPA) has been known
as one of the most commonly used Zn2+ receptor because it binds Zn2+
ions specifically over biologically relevant metals in cellular systems. In
this regard, since the first fluorescent Zn2+ probe with DPA [6], a
number of DPA-based fluorescent Zn2+ probes have been developed
[7–13]. Furthermore, the DPA-Zn2+ complex appears to be a tool for
the detection of anions, such as phosphate ions [14–17].
in vivo molecular imaging [21]. Such an effective strategy of attaching
organic materials onto gold particles has emerged as an attractive
sensing method as an amplification of Raman signaling. The triar-
ylmethine dye can be one of most suitable candidates as excellent
molecular markers for SERS detection, because of its huge Raman
scattering cross-section and efficient adsorption on noble AuNPs [22].
In this study, herein we report the synthesis of a new type of SERS
dye DPA-TAM with both di-2-picolylamine and
a triarylmethine
moiety, which can sensitively detect biological Zn2+ ions in aqueous
environment. The probe exhibits Raman intensites correlated with
[Zn2+] due to an effect attributed to the strong interparticle plasmonic
coupling produced by the Zn2+ ion-prompted aggregation of DPA-
TAM-AuNPs. To prove the potential applications for this response, DPA-
TAM has been tested with biological Zn2+ ion in HeLa cancer cells and
daphnia as both in vitro and in vivo examination. Hence, we herein
present feasibility of DPA-TAM for selective monitoring of Zn2+ among
several other metal ions and quantification of the entrapped Zn2+ ions
by virtue of the chelation enhanced SERS intensity.
However, the probes could suffer from photobleaching, which could
prevent long-time monitoring [18]. Nanoparticles have been used to
detect heavy metal ions [19]. Surface-enhanced Raman scattering
(SERS) provides a complementary way to detect ionic species by
∗ Corresponding author.
∗∗ Corresponding author.
∗∗∗ Corresponding author.
E-mail addresses: jongskim@korea.ac.kr (J.S. Kim), hs0101j@uhs.ac.kr (H.S. Jung), sjoo@ssu.ac.kr (S.-W. Joo).
https://doi.org/10.1016/j.dyepig.2019.107721
Received 1 June 2019; Received in revised form 14 July 2019; Accepted 15 July 2019
Availableonline16July2019
0143-7208/©2019PublishedbyElsevierLtd.

N. Lee, et al.
DyesandPigments171(2019)107721
2. Experimental
2.1. Materials
All chemicals except for p-chloranil (TCI) and N,N′-dimethylaniline
(Alfa Aeser) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The metal nitrate salts except NaCl and KNO3 were used for all analy-
tical experiments obtained from Merck (St. Louis, MO, USA). The syn-
thetic mixtures were purified by using thin layer chromatography and
column chromatography on 70–230 mesh silica gel 60 as a stationary
phase.
2.2. Preparation of DPA-TAM
In this work, we prepare a new organic Raman reporter, DPA-TAM,
which has a triarylmethine dye for SERS imaging (TAM) and a di-2-
picolylamine moiety (DPA) as a receptor that has a high binding affinity
for Zn2+. As shown in Scheme 1, compounds 2 and 3 were synthesized
according to reported procedures [23].
A mixture of 2 (1.52 g,
5.00 mmol), N,N′-dimethylaniline (1.82 g, 15.0 mmol), and NbCl5
(0.135 g, 0.500 mmol) was heated at 120 °C. After stirring for 2 h, it was
washed with water and extracted with ethyl acetate (EA). The EA layer
was dried with magnesium sulfate. After removal of the solvent, the
next step was progressed using the remaining product, presumed to be
compound 1, without further purification. Compound 1 (0.264 mg,
0.500 mmol) was dissolved in EA (5 mL). p-Chloranil (0.184 g,
0.750 mmol) was then treated to the solution and the mixed solution
was stirred at 25 °C for 2 h. The mixed solution was washed with dis-
tilled water and extracted with EA. The EA layer was dried with mag-
nesium sulfate. After removal of the solvents, the remaining product
Scheme 1. Synthetic schemes of resonance Raman dye (DPA-TAM) are as fol-
lows: (i) 2-(Chloromethyl)pyridine hydrochloride, K2HPO4, MeCN, reflux, 24 h,
90%; (ii) POCl3, DMF, 90 °C, 2 h, 92%; (iii) N,N′-dimethylaniline, NbCl5 120 °C,
2 h, 92%; (iv) p-chloranil, r.t., 2 h, 86%. The detection strategy of Zn2+ ions
uses DPA-TAM. The gold structures are used to yield high resonance Raman
intensities.
Fig. 1. (A) Colorimetric photo after binding with the metal ions (all at ions concentrations of 1 mM) and (B) UV–Visible absorbance spectrum of pristine AuNPs, DPA-
TAM-AuNPs, initial DPA-TAM (10 μM) before and after binding with Zn2+ (10 mM), and {Zn2+-DPA-TAM}-AuNPs, respectively. (C) UV–Visible absorption spectra
and (D) the photo of Zn2+-DPA-TAM complex corresponding to various concentrations of the Zn2+ ion.
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DyesandPigments171(2019)107721
Fig. 2. (A) Concentration-dependent SERS spectra of DPA-TAM-AuNPs in the range of 0–100 μM of Zn2+. (B) A calibration curve of the vibrational band intensities at
1174 cm−1 and a linear fitting result for the 0–100 μM regions. (C) The SERS spectra of DPA-TAM on AuNPs in the presence of various metals. (D) Histogram of the
SERS relative intensity at 1174 cm−1. The concentrations of DPA-TAM and metal ions are 200 nM and 60 μM, respectively.
was purified over silica gel using a mobile phase (n-Hexane/EA (v/v,
1:2) as the eluent to yield DPA-TAM (0.241 g, 86%). Mp 78 °C. IR
(neat): νmax 3364.2, 2927.4, 1579.4, 1470.5, 1357.6, 1295.0, 1238.1,
1174.4, 1060.7, 941.1, 828.3, 795.5, 755.0, 714.5, 656.6 cm−1 1H
NMR (CDCl3): δ 8.64 (d, J = 4.69 Hz, 2H), 7.75 (t, J = 7.90 Hz, 2H),
7.39-7.23 (m, 10H), 6.96 (d, J = 9.21 Hz, 2H), 6.87 (d, J = 9.21 Hz,
4H), 5.05 (s, 4H), 3.30 (s, 12H). 13C NMR (MeOH-d6): δ 158.3, 157.7,
155.9, 150.5, 141.3, 140.3, 139.1, 129.3, 128.0, 124.3, 123.0, 114.2,
113.8, 57.7, 40.7. ESI-MS calc. for C35H36ClN5 [M-Cl]+ 526.30, found
526.40. HRMS (FAB) calc. for C35H36ClN5 [M-Cl]+ 526.2971, found
526.2973.
final concentration range between 0 and 3.5 mM was added into the
DPA-TAM solution (10 μM, in ethanol). The mixtures were stabilized for
15 min at 25 °C and then checked by the UV–Visible spectrometer.
For the SERS measurements, 1 μL of the Zn2+ solution (6 mM in
water) was added to 99 μL of the resultant mixture (DPA-TAM-AuNPs).
Subsequently, the 100 μL mixture of DPA-TAM-AuNPs (containing
200 nM of DPA-TAM) and Zn2+ (containing 60 μM final concentration)
was sonicated for 30 s and remained stablized for 30 min at room
temperature, and then the SERS spectra of the {Zn2+-DPA-TAM}-
AuNPs complex with a volume of 100 μL were obtained using a Raman
spectrometer.
2.5. DFM and confocal Raman intracellular imaging of Zn2+
2.3. Preparation of AuNPs and DPA-TAM-AuNPs
SERS spectra were examined using AuNPs prepared according to
Lee and Meisel scheme [24]. The DPA-TAM solution (1 μL, 20 μM in
ethanol) and the AuNP solution (98 μL) were mixed into a 1.5 mL tube,
subsequently sonicated for 30 s, and remained stable for 15 min at
25 °C. The DPA-TAM-coated AuNPs should be immediately prepared
before each of the experiments. The interfacial behavior of DPA-TAM
on AuNPs were examined by SERS with respect to [Zn2+].
Human cervical cancer HeLa cells were obtained from the Korean
cell line bank (KCLB No. 10002). Daphnia magna were cultured in M4
media and HeLa cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) treated with fetal bovine serum (FBS, 10% (v/v))
purchased from WelGene (Seoul, Korea) and penicillin-streptomycin
(1% (v/v)) at 37 °C in a 5% CO2 atmosphere incubator.
HeLa cells were seeded onto the glass bottom in confocal dishes at a
density of 5 × 104 cells in 2 mL of DMEM medium and incubated
24 h at 37 °C. The cells were washed with pH 7.4 PBS solution, and then
added 2 mL of fresh DMEM (containing 100 μM final concentration of
Zn2+) to the confocal dish, incubated overnight. HeLa cells were
2.4. UV–Visible and SERS detection for Zn2+
For the monitoring of UV–Visible spectra, the Zn2+ solution in the
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N. Lee, et al.
DyesandPigments171(2019)107721
Fig. 3. In vitro test of HeLa cells. (A) The SERS spectra of DPA-TAM-coated AuNPs (500 nM) inside HeLa cells, showing SERS signals of DPA-TAM peaks corre-
sponding to various concentrations of Zn2+ ion intracellular. The DFM images of HeLa cells with different Zn2+ concentration treatments such as (B) 0 μM, (C) 1 μM,
(D) 100 μM.
washed with PBS solution, suspended in 2 mL of fresh DMEM (con-
taining DPA-TAM-AuNPs with 500 nM final concentration of DPA-
TAM) and then DFM Raman imaging was performed.
(DPA-TAM) was synthesized as depicted in Scheme 1. Compound 3 was
synthesized by treatment of aniline with 2-(chloromethyl)-pyridine
hydrochloride and dipotassium phosphate in acetonitrile (MeCN). The
Vilsmeier-Haack reaction of 3 yielded compound 2. Condensation re-
actions of two equivalents of dimethylaniline with DPA derivative 2 at
120 °C for 2 h in a catalytic amount of NbCl5 produced compound 1.
Extension of the π-conjugate systems of 1 with p-chloranil resulted in
DPA-TAM. The DPA-TAM was confirmed by 1H NMR, 13C NMR, FT-IR,
ESI-MS, and HR-MS results (Figs. S1–S4). AuNPs were prepared ac-
cording to Lee and Meisel scheme [24]. Morphological change of the
DPA-TAM-AuNP was observed by SERS and FT-IR spectrometer. De-
tailed characterizations of the prepared particles are given in ESI as
well.
2.6. Physicochemical characterization
The size distributions and morphologies of AuNPs were examined
using a JEOL-3010 transmission electron microscope (TEM) (Fig. S5).
NMR spectra were collected on Varian 400 MHz NMR spectrometer.
ESI-MS and HR-FAB-MS were measured on Shimadzu LCMS-2020 and
Jeol LTD JMS-HX 110/110A mass spectrometers, respectively. FT-IR
spectra were obtained by using a FT/IR-4100 spectrometer (Jasco).
UV–Visible absorption spectral changes of DPA-TAM, before and after
the presence of the Zn2+ solution, were monitored using a spectro-
photometer (Optizen 3220UV, Mecasys, Daejeon, Korea). Confocal
Raman microspectroscopy was performed using a Renishaw RM 1000
microscope (Wotton-under-Edge, UK) equipped with a cytoviva high-
resolution adaptor for DFM at the excitation wavelength of 632.8 nm
(HeNe excitation laser). The integration time for the SERS anaylsis was
set to 10 s per wave spectrum and the wavenumber range was
200–3200 cm−1. Prior to the experiment, the spectral positions were
3.2. UV–Visible sensing of Zn2+ based on the DPA-TAM
Addition of 1 mM Zn2+ to DPA-TAM shows an approximately 20 nm
bathochromic shift of the absorption maximum (from 590 nm to
610 nm), resulting in a perceived color change from dark blue to cyan,
whereas no change was observed with various ions, such as Zn2+
Ca2+, Pb2+, Mn2+, Mg2+, Co2+, NH4+, Hg2+, Cd2+, Fe3+, Cr3+
,
,
corrected based on the Si peak at wavenumber 520 cm−1
.
Fe2+, K+, and Na+ (all at ions concentrations of 1 mM). The results
indicated that DPA-TAM not only exhibits a colorimetric method but
also changes UV–Visible absorbance spectra before and after binding
with Zn2+. As Fig. 1B shows UV–Visible absorbance spectra of initial
DPA-TAM, Zn2+-DPA-TAM complex, pristine AuNPs, DPA-TAM-AuNPs,
and Zn2+ complex with DPA-TAM-AuNPs. The addition of DPA-TAM
into the AuNP solution produces a slightly broadened absorption band
3. Results and discussion
3.1. Preparation and characterization of DPA-TAM & DPA-TAM-AuNPs
The putative Zn2+ Raman dye of di-2-picolylamine triarylmethine
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N. Lee, et al.
DyesandPigments171(2019)107721
Fig. 4. In vivo test of Daphnia magna. Bright images of Daphnia magna (A) with and (B) without Zn2+ (50 μM) treatments. The points of P1, P2, and P3 where
nanoparticles appeared to be accumulated, whereas P1′, P2′, and P3′ are the positions where the transparent tissues existed. The overall SERS intensities of DPA-TAM
on AuNPs (500 nM) (C) with and (D) without Zn2+ (50 μM) treatments became increased upon binding to Zn2+
.
at 520 nm. Subsequently, upon the addition of the Zn2+ ion, the ap-
pearance of a new peak at around 700 nm can be attributed to the
Zn2+-induced aggregation of DPA-TAM-AuNPs. Based on UV–Visible
spectra, we have to admit that the tested ion concentrations using the
colorimetric and UV–Visible absorption spectroscopy are too high
(∼1 mM) to be implemented for the practical purposes of sensors
(Fig. 1C–D).
the binding schemes and spectral behavior of DPA-TAM with the Zn2+
ion.
After performing the physical characterization of AuNPs, we as-
sembled DPA-TAM on the gold nanostructures and added Zn2+ ions
into the DPA-TAM-AuNPs. Fig. 1B also shows UV–Visible absorption
maxima of AuNPs became changed slightly before and after binding the
DPA-TAM. Considering that the aggregated DPA-TAM-AuNPs in the
presence of Zn2+ ions have absorption at ∼700 nm, this shifted
plasmon maximum would be helpful to enhance the Raman signals via
resonance effects, since the absorption band can be coincident with the
laser excitation wavelength at 633 nm. We have found that the SERS
intensities of DPA-TAM-AuNPs increased as the aggregation of the DPA-
TAM-AuNPs by adding different concentrations of Zn2+. As shown in
Fig. 2A, the SERS spectra of DPA-TAM-AuNPs have been successfully
investigated after the addition of the Zn2+ ion, exhibiting the enhanced
SERS intensity.
DPA-TAM-AuNP aggregate solutions exhibit the strongest Raman
peak at 1174 cm−1. Vibrational band analysis of DPA-TAM is sum-
marized in Table S1. Our results illustrate that the SERS intensity of
DPA-TAM-AuNPs becomes more aggregated, as the concentration of
Zn2+ increases due to the formation of “hot spots” via interparticle
aggregation. As shown in Fig. 2B, the I1174 values increases almost
linearly in the concentration range of Zn2+ between 1 and 100 μM.
SERS spectra of DPA-TAM-AuNPs could be quantified at different
concentrations of Zn2+ of 1–100 μM. According to the present SERS
study, the limit of detection (LOD) can be estimated to be around
50 μM. The determined value is found to be lower than that of U.S.
3.3. SERS sensing of Zn2+ based on the DPA-TAM-AuNPs
Spectroscopic probes have been widely applied in many fields due
to give a direct and convenient tool for the Zn2+ ion. Recently, nano-
particles [25], luminescence [26], aggregation induced fluorescence
properties [27] were used to detect the Zn2+ ions. Since metal nanos-
tructures with the surface plasmon resonance have been introduced for
intracellular ion monitoring by combining vibrational spectroscopy, we
have tried to investigate the Zn2+-induced spectral changes of DPA-
TAM on AuNPs by means of SERS spectroscopy.
According to the density functional theory (DFT) calculations
[28,29], a dimeric form of DPA-TAM with Zn2+ appears to have higher
binding energy values than those of the monomeric cases, as summar-
ized in Table S2. The HOMO-LUMO transition for the monomeric
binding case occurs around 476 and 478 nm at the level of B3LYP/6-
31G/SMD, before and after binding the Zn2+ ion, respectively, whereas
the dimeric binding with the Zn2+ ion the exhibits the value of 479 nm
(Fig. S7). Although these values are somewhat lower than the experi-
mental values, the DFT calculations can provide a helpful guideline of
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N. Lee, et al.
DyesandPigments171(2019)107721
Environmental Protection Agency-recommended maximum allowable
Appendix A. Supplementary data
Zn2+ concentration in drinking water at 76 μM.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.dyepig.2019.107721.
3.4. Possible mechanism of SERS response of DPA-TAM-AuNPs towards
Zn2+
References
As proposed in Scheme 1, the sandwiched binding of Zn2+ and
subsequent interparticle aggregation of AuNPs can result in strong SERS
intensity to detect the Zn2+ ion. We have found that the SERS in-
tensities significantly increased in the presence of Zn2+. This behavior
was not observed for Cd2+, Fe3+, Mg2+, Ca2+, Mn2+, Na+, K, and
Co2+. To examine the mechanism of Zn2+ ion-triggered SERS en-
hancement, we introduced the Job's continuous variation method to
estimate the stoichiometry between the Zn2+ and DPA-TAM [30]. As
can be estimated from our Job's method, the maximum value was found
at the mole fraction 0.3 (Fig. S6), which should indicate the 1:2 binding
stoichiometry between Zn2+ and DPA-TAM. In the case of the free
solution without AuNPs, the ion such as Cu2+ with the 1:1 binding
[1] Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev
Neurobiol 1989;31:145–238.
[2] Dineley KE, Votyakova TV, Reynolds IJ. Zinc inhibition of cellular energy produc-
tion: implications for mitochondria and neurodegeneration. J Neurochem
2003;85:563–70.
[3] Schrag M, Crofton A, Zabel M, Jiffry A, Kirsch D, Dickson A, Mao XW, Vinters HV,
Domaille DW, Chang CJ, Kirsch W. Effect of cerebral amyloid angiopathy on brain
iron, copper, and zinc in Alzheimer's disease. J Alzheimer's Dis 2011;24:137–49.
[4] Que EL, Domaille DW, Chang CJ. Metals in neurobiology: probing their chemistry
and biology with molecular imaging. Chem Rev 2008;108:1517–49.
[5] Wu D, Sedgwick AC, Gunnlaugsson T, Akkaya EU, Yoon J, James TD. Fluorescent
chemosensors: the past, present and future. Chem Soc Rev 2017;46:7105–23.
[6] Walkup GK, Burdette SC, Lippard SJ, Tsien RY. A new cell-permeable fluorescent
probe for Zn2+. J Am Chem Soc 2000;122:5644–5.
[7] Komatsu K, Kikuchi K, Kojima H, Urano Y, Nagano T. Selective zinc sensor mole-
cules with various affinities for Zn2+, revealing dynamics and regional distribution
of synaptically released Zn2+ in hippocampal slices. J Am Chem Soc
2005;127:10197–204.
appeared to bind DPA-TAM as efficiently as Zn2+
.
[8] Maruyama S, Kikuchi K, Hirano T, Urano Y, Nagano T. A novel, cell-permeable,
fluorescent probe for ratiometric imaging of zinc ion. J Am Chem Soc
2002;124:10650–1.
3.5. DFM Raman intracellular imaging of Zn2+
[9] Shirbhate ME, Jeong Y, Ko G, Baek G, Kim G, Kwon Y-U, Kim KM, Yoon J. Selective
fluorescent recognition of Zn2+ by using a binaphthol-pyrene probes. Dyes
Pigments 2019;167:29–35.
Dark-field microscopy (DFM) is mainly applied to optical imaging
and spectroscopic analysis of nanoscale materials [31]. To further
confirm that DPA-TAM-coated AuNPs can sense intracellular Zn2+ ions,
DFM experiments were performed. Fig. 3A shows SERS signals of the
HeLa cells, a human cervical cancer cell line, regarding the presence of
Zn2+ ions. The SERS intensities ascribed to the DPA-TAM were pro-
portionally stronger with increasing intracellular Zn2+ concentration
(Fig. 3B–D). We also accomplished an in vivo study on Zn2+ imaging of
Daphnia magna, which can be considered to be an ideal in vivo model,
since they are highly sensitive to dissolved pollutants, such as metal
ions [32]. It was also found that DPA-TAM-AuNPs exhibited higher
Raman signals with Zn2+ (50 μM) treatments compared to those of no-
treatment groups (Fig. 4). We thus conclude that DPA-TAM-AuNPs can
be applied for detecting Zn2+ ions in living biological systems.
[10] Woodroofe CC, Lippard SJ. A novel two-fluorophore approach to ratiometric sen-
sing of Zn2+. J Am Chem Soc 2003;125:11458–9.
[11] Hu Z, Yang G, Hu J, Wang H, Eriksson P, Zhang R, Zhang Z, Uvdal K. Real-time
visualizing the regulation of reactive oxygen species on Zn2+ release in cellular
lysosome by a specific fluorescent probe. Sens Actuators B Chem 2018;264:419–25.
[12] Chen X-Y, Shi J, Li Y-M, Wang F-L, Wu X, Guo Q-X, Liu L. Two-photon fluorescent
probes of biological Zn(II) derived from 7-hydroxyquinoline. Org Lett
2009;11:4426–9.
[13] Masanta G, Lim CS, Kim HJ, Han JH, Kim HM, Cho BR. A mitochondrial-targeted
two-photon probe for zinc ion. J Am Chem Soc 2011;133:5698–700.
[14] Ngo HT, Liu X, Jolliffe KA. Anion recognition and sensing with Zn
(II)–dipicolylamine complexes. Chem Soc Rev 2012;41:4928–65.
[15] Moro AJ, Cywinski PJ, Körstena S, Mohr GJ. An ATP fluorescent chemosensor based
on a Zn(II)-complexed dipicolylamine receptor coupled with a naphthalimide
chromophore. Chem Commun 2010;46:1085–7.
[16] Cooley CM, Hettie KS, Klockow JL, Garrison S, Glass TE. A selective fluorescent
chemosensor for phosphoserine. Org Biomol Chem 2013;11:7387–92.
[17] Lee S, Yuen KKY, Jolliffe KA, Yoon J. Fluorescent and colorimetric chemosensors for
pyrophosphate. Chem Soc Rev 2015;44:1749–62.
[18] Montalti M, Cantelli A, Battistelli G. Nanodiamonds and silicon quantum dots: ul-
trastable and biocompatible luminescent nanoprobes for long-term bioimaging.
Chem Soc Rev 2015;44:4853–921.
[19] Bi L, Wang Y, Yang Y, Li Y, Mo S, Zheng Q, Chen L. Highly sensitive and re-
producible SERS sensor for biological pH detection based on a uniform gold na-
norod array platform. ACS Appl Mater Interfaces 2018;10:15381–7.
[20] Samanta A, Maiti KK, Soh K-S, Liao X, Vendrell M, Dinish US, Yun S-W,
Bhuvaneswari R, Kim H, Rautela S, Chung J, Olivo M, Chang Y-T. Ultrasensitive
near‐infrared Raman reporters for SERS‐based in vivo cancer detection. Angew
Chem Int Ed 2011;50:6089–92.
[21] Vendrell M, Maiti KK, Dhaliwal K, Chang Y-T. Surface-enhanced Raman scattering
in cancer detection and imaging. Trends Biotechnol 2013;31:249–57.
[22] Qian X, Emory SR, Nie S. Anchoring molecular chromophores to colloidal gold
nanocrystals: surface-enhanced Raman evidence for strong electronic coupling and
irreversible structural locking. J Am Chem Soc 2012;134. 2000–3.
[23] Kumari N, Jha S, Misra SK, Bhattacharya S. A probe for the selective and part-
s‐per‐billion‐level detection of copper(II) and mercury(II) using a micellar medium
and its utility in cell imaging. ChemPlusChem 2014;79:1059–64.
[24] Lee PC, Meisel D. Adsorption and surface-enhanced Raman of dyes on silver and
gold sols. J Phys Chem 1982;86:3391–5.
4. Conclusions
We have developed a new sensing method of Zn2+ ions sandwiched
by the newly synthesized DPA-TAM dye and the interparticle ag-
gregation of AuNPs. The DPA unit to bind the Zn2+ ion is conjugated
onto a TAM SERS dye. Zn2+ ion can trigger the interparticle aggrega-
tion of AuNPs and result in the increase of SERS intensities of DPA-
TAM. DFM-equipped Raman technology was successfully performed for
the observation of the Zn2+ concentrations under intracellular condi-
tions as an in vitro nanosensor at a single cell level. The advantage of
SERS strategy in the implementation of DPA-TAM-AuNPs for the de-
tection of Zn2+ affords a new method showing potential for a variety of
successful Zn2+ ion-sensing. DPA-TAM can also be applied to Zn2+ ion
detection of in vivo imaging of Daphnia magna. This finding can be a
promising guideline in designing Raman spectroscopic probes in the
future.
[25] Kaur P, Singh R, Kaur V, Talwar D. Reusable Schiff base functionalized silica as a
multi-purpose nanoprobe for fluorogenic recognition, quantification and extraction
of Zn2+ ions. Sens Actuators B Chem 2018;254:533–41.
Acknowledgment
[26] Ksenofontov AA, Guseva GB, Antinɑ EV, Khodov IA, Vyugin AI. Zinc(II) bis(di-
pyrromethenate)s crystal solvates with dimethyl sulfoxide Composition, stability
and spectral-luminescent properties. Sens Actuators B Chem 2017;251:858–68.
[27] He X, Wang X, Zhang L, Fang G, Liu J, Wang S. Sensing and intracellular imaging of
Zn2+ based on affinity peptide using an aggregation induced emission fluorescence
“switch-on” probe. Sens Actuators B Chem 2018;271:289–99.
We would like to thank Ngo Dinh Nghi, Thanh Danh Nguyen, Prof.
Kyungho Choi, Le Quang Trung and Ho Ngoc Thanh for helping ex-
periments and DFT calculations. This work was supported in part by a
Creative Research Initiatives project (grant no. 2018R1A3B1052702,
[28] Erdemir S, Kocyigit O. Dual recognition of Zn2+ and Al3+ ions by a novel probe
containing two fluorophore through different signaling mechanisms. Sens Actuators
B Chem 2018;273:56–61.
J.S.K.)
and
the
Basic
Science
Research
Programs
(2015R1A6A3A04058789, HSJ) from the National Research
Foundation of Korea (NRF) funded by the Ministry of Education.
[29] Wang Y, Mao P-D, Wu W-N, Mao X-J, Fan Y-C, Zhao X-L, Xu Z-Q, Xu Z-H. New
pyrrole-based single-molecule multianalyte sensor for Cu2+, Zn2+, and Hg2+ and
6

N. Lee, et al.
DyesandPigments171(2019)107721
its AIE activity. Sens Actuators B Chem 2018;255:3085–92.
2014;1:725–31.
[30] Li D, Ma Y, Duan H, Jiang F, Deng W, Ren X. Fluorescent/SERS dual-sensing and
[32] Galdiero E, Falanga A, Siciliano A, Maselli V, Guida M, Carotenuto R, Tussellino M,
Lombardi L, Benvenuto G, Galdiero S. Daphnia magna and Xenopus laevis as in vivo
models to probe toxicity and uptake of quantum dots functionalized with gH625.
Int J Nanomed 2017;12:2717–31.
imaging of intracellular Zn2+. Anal Chim Acta 2018;1038:148–56.
[31] Hill DJ, Pinion CW, Christesen JD, Cahoon JF. Waveguide scattering microscopy for
dark-field imaging and spectroscopy of photonic nanostructures. ACS Photonics
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