Phenothiazine–rhodamine-based colorimetric and fluorogenic ‘turn-on' sensor for Zn2+ and bioimaging studies in live cells

Article abstract of DOI:10.1002/bio.3701

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528?nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608?nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89?×?10^?8?M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.

Full text of DOI:10.1002/bio.3701

Received: 20 March 2019
DOI: 10.1002/bio.3701
Revised: 18 June 2019
Accepted: 3 August 2019
R E S E A R C H A R T I C L E
Phenothiazine–rhodamine‐based colorimetric and fluorogenic
‘turn‐on’ sensor for Zn2+ and bioimaging studies in live cells
Muthu Vengaian Karmegam1 Sekar Karuppannan1
Denzil Britto Christopher Leslee1
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Singaravadivel Subramanian2 Sivaraman Gandhi3
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1 Department of Chemistry, Anna University,
Abstract
University College of Engineering, Dindigul,
Tamilnadu, India
A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its
ability to selectively sense Zn2+ ions in solution and in in vitro cell lines was tested
using various techniques. When compared with other competing metal ions, the
PTRH probe showed the high selectivity for Zn2+ ions that was supported by
electronic and emission spectral analyses. The emission band at 528 nm for the
PTRH probe indicated the ring closed form of PTRH, as for Zn2+ ion binding to
PTRH, the λem get shift to 608 nm was accompanied by a pale yellow to pink
colour (under visible light) and green to pinkish red fluorescence emission (under
UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spec-
tral overlap of the donor emission band and the absorption band of the ring
opened form of the acceptor moiety contributed towards the fluorescence reso-
nance energy transfer ON mechanism for Zn2+ ion detection. The PTRH sensor
had the lowest detection limit for Zn2+, found to be 2.89 × 10−8 M. The sensor also
demonstrated good sensing application with minimum toxicity for in vitro analyses
using HeLa cells.
2 Department of Chemistry, SSM Institute of
Engineering and Technology, Dindigul,
Tamilnadu, India
3 Department of Chemistry, Gandhigram Rural
Institute (Deemed to be University),
Gandhigram, Dindigul, Tamilnadu, India
Correspondence
S. Karuppannan, Department of Chemistry,
Anna University – University College of
Engineering. Dindigul – 624622, India.
Email: karuppannansekar@gmail.com
Funding information
University Grants Commission ‐ Major
Research Project, Grant/Award Number:
43‐186/2014(SR)
KEYWORDS
Fluorescent chemosensor, FRET, Phenothiazine, Rhodamine, Zinc ion
enzyme regulation.[1–7] Conversely, zinc deficiency leads to many dis-
eases such as infection susceptibility, sickle cell disease, diarrhoea, dia-
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1
INTRODUCTION
Zinc is the most inexhaustible element on the Earth's crust, and plays a
very important role in several physiological and pathological processes
such as apoptosis, catalytic function of protein, gene transcription, and
betes, chronic renal disease, and chronic liver disease.[8–10] Excess
accumulated levels of zinc arrest copper and iron absorption in
humans and lead to a series of health problems.[11] Therefore, a more
accurate and sensitive fluorescence sensor is required to detect Zn2+
ions which interference from Cd2+ ions.[12–14]
Due to increased levels of Zn2+ in water systems, several analytical
techniques such as atomic absorption spectroscopy (AAS), atomic
emission spectroscopy (AES), inductively coupled plasma mass spec-
trometry (ICP‐MS), X‐ray fluorescence (XRF), and electrochemical
methods have been used to monitor Zn2+ ion levels,[15–19] however
these types of techniques are highly time consuming and expensive.
Therefore, the development of a new method for Zn2+ ion detection
Abbreviations used: CDCl3, deuterated chloroform; DFT, density functional theory; DMSO,
dimethyl sulfoxide; EDAX, energy dispersive X‐ray spectroscopy; EET, excitation energy
transfer (also known as FRET); ESI‐MS, electrospray ionization mass spectrometry; FBS,
fetal bovine serum; FRET, fluorescence resonance energy transfer; FT‐IR, Fourier transform
infrared spectroscopy; HEPES, 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid; HOMO,
higher occupied molecular orbital; LOD, limit of detection; LUMO, lower unoccupied
molecular orbital; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; NMR,
nuclear magnetic resonance spectroscopy; PTRH, phenothiazine–rhodamine‐based sensor
[(10‐hexyl‐10h‐phenothiazin‐3‐yl)methylene
dihydrospiro(isoindole‐1,9′‐xanthene)‐3‐one]; PTRH+Zn2+
amino‐3′,6′‐bis(diethylamino)‐2,3‐
phenothiazine–rhodamine–zinc
,
complex; SEM, scanning electron microscopy; UV, ultraviolet
Luminescence. 2019;1–8.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

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VENGAIAN ET AL.
in environmental and biological systems is a critical need. In recent
studies, processes using fluorescence chemosensors were found to
deliver the best analytical techniques for monitoring metal ions in
solution, due to their effective features such as simplicity, high sensi-
tivity, ease of function, quick response to fluorogenic and chromo-
genic colorimetric changes, and biocompatibility in cell imaging, plus
generating inexpensive detection systems that delivered considerable
advantages over other methods.[20–23]
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2.1
Synthesis
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2.1.1
General procedure for synthesis of
10‐hexyl‐10H‐phenothiazine (1) and
10‐hexyl‐10H‐phenothiazine‐3‐carbaldehyde (2)
The precursor compounds 1 and 2 were synthesized in accordance
with previously reported methods (see Supporting information).[28,29]
In recent years, several fluorescent probes for Zn2+ ion have been
developed, some of which, for example, were turn‐on rhodamine‐based
fluorescent sensors containing bis‐triazolyl moieties for Zn2+ detection
among various other metal ions and that had a detection limit of 1 M for
application in in vitro HeLa cell imaging.[24] A fluorescent, colorimetric
sensor based on 8‐aminoquinoline for Zn2+ detection in living cells
through internal charge transfer (ICT) and chelation‐enhanced fluores-
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2.1.2
Synthesis of PTRH
Rhodamine B hydrazine (1.28 mmol) and compound 2 (1.28 mmol)
were mixed with ethanol (30 ml) and acetic acid (3–5 drops) under a
nitrogen atmosphere and the reaction mixture was refluxed overnight.
After the reaction was completed, the precipitate was filtered, washed
with methanol and dried in a vacuum. Finally, the crude product was
purified by column chromatography using ethyl acetate:hexane (2:8,
v/v) to furnish pure compounds of the PTRH sensor as a greenish yel-
low powder. Yield: 81%. Melting point: 180°C. FT‐IR (cm−1): 3056
(=C–H, str), 2965–2862 (C–H, str), 1719 (C=O, str), 1461 (C–H, bend),
1306–1114 (C–N, str), 1233 (C–O, str). 1H‐NMR: (300 MHz, CDCl3), δ
(ppm) 8.22 (s, 1H), 7.98 (d, 1H), 7.45 (t, 1H), 7.40–7.37 (d, 1H), 7.11 (m,
2H), 6.87 (m, 4H), 6.81 (t, 1H), 6.73 (t, 1H), 6.43 (d, 1H), 6.23 (d, 2H),
6.21 (d, 2H), 3.80–3.75 (t, 2H), 3.35–3.28 (q, 8H), 2.71 (s, 1H), 2.26–
2.14 (d, 2H), 1.73 (m, 6H), 1.19–1.14 (t, 12H), 0.85 (t, 3H). 13C‐NMR:
(100 MHz, CDCl3), δ (ppm) 164.92, 152.97, 152.04, 148.92, 133.22,
129.11, 128.19, 127.94, 127.19, 126.72, 123.71, 123.31, 115.39,
114.90, 108.01, 105.96, 97.90, 65.87, 44.33, 31.42, 26.56, 22.57,
13.98, 12.65. Mass (ESI‐MS): C47H51N5O2S [M + H+]: 749.48.
cence mechanisms has been described previously.[25]
A pyrenyl
appended hexahomotrioxacalix[3]arene was found to exhibit high
selectivity towards Zn2+ in mixed aqueous medium[26] and using two
simple compounds based on conjugated Schiff bases from indole‐
derived p‐phenylenediamine for Zn2+ ions.[27]
Here, we describe a phenothiazine‐linked rhodamine‐based fluo-
rescent sensor, (10‐hexyl‐10H‐phenothiazin‐3‐yl)methyleneamino‐
3′,6′‐bis(diethylamino)2,3dihydrospiro(isoindole‐1,9′‐xanthene)‐3‐one
(PTRH), for the selective sensing of Zn2+ in water:acetonitrile (8:2, v/v)
through fluorescence resonance energy transfer (FRET) (also called
excitation energy transfer, EET). This sensor exhibited a ‘turn‐on’ fluo-
rescence sensing behaviour during continuous addition of Zn2+ ions.
Zn2+ ion binding brought about spirolactam ring opening of the recep-
tor unit due to the EET from phenothiazine to the rhodamine moiety.
The sensing process involved a red shift in absorbance and a fluores-
cence spectrum with fluorescence emission from a green to pinkish
red colour. The PTRH sensor was used to image Zn2+ ions in live cells,
with good biocompatibility.
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3
RESULTS AND DISCUSSION
Synthesis of PTRH
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3.1
The PTRH sensor was successfully synthesized through condensation
of rhodamine B hydrazine and compound 2 with drops of acetic acid in
ethanol (Scheme 1). The purity of the ligand PTRH was established
from FT‐IR, 1H‐NMR, 13C‐NMR, and ESI‐iass (see Supporting informa-
tion Figures S1–S4).
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2
EXPERIMENTAL
All reagents and solvents were purchased from Central Drug House
Ltd, India; phenothiazine and rhodamine were obtained from Sigma‐
Aldrich Co Ltd, India; and metal chloride salts were purchased from
Merck & Co., India.
All samples for characterization were prepared at room tempera-
ture. FT‐IR spectra were recorded using a JASCO‐460 plus model
spectrometer. 1H‐NMR (300 MHz) and 13C‐NMR (100 MHz) spectra
were obtained on Bruker Avance spectrometer. ESI‐Mass spectra
were analyzed using an LCQ Fleet mass spectrometer. Absorption
and fluorescence measurements were recorded on an AN‐UV‐7000
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3.2
UV–visible spectral response to metal ions
Influence of various heavy metal ions such as Hg2+, Mg2+, Co2+, Cu2+
,
Ba2+, Al3+, Mn2+, Pb2+, Cd2+, Fe2+, Fe3+, Ni2+, Ca2+, Na+, and K+
(10 μM) on the absorbance of the PTRH ligand (10 μM) was investi-
gated using an electronic spectral method (Figure 1). The spectrum
of the free PTRH ligand showed four absorption peaks at 235 nm,
276 nm, 308 nm, and 390 nm. None of the metal ions presumably
had made any changes in the absorbance of the ligand PTRH, except
that addition of Zn2+ ions produced the formation of new band at
559 nm, i.e. a red shift that demonstrated strong n–π* transition. This
red shift occurred due to spirolactam ring opening of the rhodamine
spectrophotometer and
a JASCO FP‐8200 spectrofluorometer,
respectively. Surface morphology was imaged using a Vega3 Tescan
model scanning electron microscope. EDAX analysis was carried out
using a Bruker Nano GMBH model energy dispersive X‐ray analyzer.
Fluorescent bioimaging was undertaken in living cells and visualized
using a Nikon fluorescence microscope.

VENGAIAN ET AL.
3
SCHEME 1 Synthesis of PTRH
intense emission band at 608 nm was observed with simultaneous
quenching of the phenothiazine peak at 528 nm. The new band at
608 nm represented the spirolactam ring‐opening effect of rhodamine
due to binding with Zn2+ ion, thereby extending conjugation of the
system and a green to pinkish red emission transformation (Figure
S9). Fluorescence changes in PTRH following addition of other metal
ions was not significantly discriminative; this finding showed the selec-
tive nature of the PTRH probe for Zn2+ ions in solution.[32,33]
Fluorescence titration of the PTRH ligand (10 μM) with Zn2+ ions
(0–1.1 equiv.) was performed. The titration plot (Figure 2) indicated
that fluorescence of the phenothiazine moiety was gradually
quenched at 528 nm with simultaneous enhancement at 608 nm that
indicated the spirolactam ring‐opening process and increasing conju-
gation characteristics of the ligand. A clear isoemissive point was
observed around 575 nm that reflected the point of equilibrium, i.e.
the existence of the two forms of the ligand (the closed ring and the
opened ring forms of the lactam unit). Increase in intensity at
608 nm on addition of the Zn2+ ion solution was due Zn2+ ion binding
brought about by formation of a conjugative bridge between the
donor and acceptor that favoured EET. A large volume of unstable
energy from the donor (phenothiazine) in the excited state was
released to excite the receptor (rhodamine moiety) from its ground
FIGURE 1 UV–visible spectrum of PTRH (10 μM) in the presence of
various competitive metal ions in a water:acetonitrile (8:2, v/v) mixture
moiety, induced by energy transfer from the donor (phenothiazine) in
the excitation state that was absorbed by the acceptor (rhodamine)
unit at the ground state. This excitation resulted in excitation energy
transfer that drove the sensing process. Therefore, the electronic
spectrum revealed selectivity of PTRH for Zn2+ ions.
Energy transformation of the PTRH probe (10 μM) in the presence
of Zn2+ ions (10 μM) was studied using UV–visible titration studies with
varying concentration of Zn2+ ions (0–2 equiv.) in a water:acetonitrile
(8:2, v/v) system. Figure S5 showed that, on increasing the concentra-
tion of the Zn2+ ions, ligand absorbance intensity was enhanced linearly
and indicated the sensitivity of the probe towards Zn2+ ions. This was
also observed by the naked eye through colour change from pale yellow
to pink under visible light, as shown in Figures S6 and S7.[30,31]
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3.3
Fluorescence titration and selectivity
Fluorescence changes of the PTRH probe (10 μM) on addition of dif-
ferent metal ions (10 μM) were investigated. Compared with observa-
tions from absorbance studies, Zn2+ alone contributed a major
influence on ligand fluorescence. As seen from the spectrum (Figure
S8), the free PTRH probe displayed fluorescence at 528 nm following
excitation at λem 559 nm, the emission at 528 nm was characteristic of
phenothiazine units. On addition of the Zn2+ ion solution, a new
FIGURE 2 Concentration‐dependent fluorescence enhancement of
PTRH (10 μM) on the addition of various amounts of Zn2+ (0–1.1
equiv.) in a water:acetonitrile (8:2, v/v) mixture (λex = 559 nm)

4
VENGAIAN ET AL.
state. This energy transfer was notable from the titration spectrum in
Figure 2, showing a regular and spontaneous increase in emission from
the rhodamine moiety and decrease in phenothiazine emission. There-
fore, the FRET process was highly amenable based on direct evidence
from the large spectral overlap of the donor (phenothiazine) emission
and acceptor (ring opened rhodamine unit) absorption band depicted
in Figure 3. Emission analysis also demonstrated that the PTRH ligand
was highly selective and sensitive towards Zn2+ ions (Figure S9).[34,35]
The stoichiometric binding ratio was established using the continu-
ous variation method (Job's plot). Figure S10 shows the variation in
fluorescence with increasing mole fractions of Zn2+ ions to PTRH
ligand. The figure shows a maximum at 0.5 mole fraction of Zn2+ ions
concentration (Figure S12). Using the slope of the linear fit, LOD was
calculated from the expression LOD = K × σ/S, and found to be
2.89 × 10−8 M, where K = 3.[37]
To investigate the effect of pH on fluorescence, an emission spec-
trum of PTRH with Zn2+ ion at different pH, ranging from 1 to 14, was
generated at λem = 608 nm (Figure S13). Fluorescence intensity of
PTRH in the presence of Zn2+ did not show any significant change
between pH 5 and 14; this result indicated that the spirolactam ring
of the rhodamine moiety was in its closed form. When pH was
adjusted to between 1 and 5, the fluorescence intensity of the PTRH
was reputedly enhanced due to spirolactam ring opening in the amide
form of rhodamine. These results clearly indicated that the PTRH sen-
sor was insensitive to pH ranging from 5 to 14 and may work under
approximately physiological conditions with very low background
fluorescence.[38,39]
that indicated the probability of a 1:1 stoichiometry of PTRH to Zn2+
.
This result was also in agreement with the ESI‐MS peak of the complex
at 813.3125 m/z (Figure S11).[36] The association constant of ligand–
metal was calculated from fluorescence titration data using the
Benesi–Hildebrand equation, and was found to be 1.26 × 106 M−1. A
plausible binding mode for PTRH with Zn2+ is shown in Scheme 2.
The lowest detection limit of PTRH for Zn2+ ion was obtained
from a linear fit by plotting fluorescence intensity against the titration
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3.4
FT‐IR titration analysis
To confirm the nature of the complex between PTRH and Zn2+, a
Fourier transform infrared (FT‐IR) spectrum for PTRH was recorded
in the absence and presence of Zn2+ ions (Figure 4). The characteristic
amide carbonyl (C=O) stretching vibrations at 1719 cm−1 shifted to
1684 cm−1 in the presence of Zn2+, indicating that the amide spiro‐
carbonyl O atom (CO) of the rhodamine B unit was involved in recog-
nition of Zn2+. A shift in the carbonyl absorption band in the infrared
(IR) region proved to be an effective way to confirm this structural
change;[40,41] this finding was also consistent with that of previous
studies. These results strongly supported the involvement of oxygen
atoms in the spirolactam amide in Zn2+ ion binding.
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3.5
Proton NMR titration analysis
A 1H‐NMR titration was performed by addition of different concentra-
tions (0.0 equiv., 0.5 equiv., and 1.0 equiv.) of Zn2+ with PTRH (1.0
equiv.) in CDCl3 (Figure 5). Upon addition of 0.5 equiv. Zn2+, the imine
proton Hg in PTRH shifted downfield from 8.22 ppm to 8.29 ppm.
FIGURE 3 Spectral overlap between donor emission and absorption
of Zn2+–rhodamine
SCHEME 2 Plausible binding mode of PTRH with Zn2+ ions

VENGAIAN ET AL.
5
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3.6
SEM and EDAX analysis
Surface morphology of PTRH and PTRH+Zn2+ sensors was character-
ized by SEM .[43] The PTRH sensor exhibited a sandstone‐like mor-
phology and the PTRH+Zn2+ complex appeared as an agglomerated‐
like structure, as shown in Figure S14. Changes observed in the PTRH
morphology in the PTRH–Zn2+ complex indicated the involvement of
Zn2+ ions in altering the physical properties of PTRH. The EDAX peak
at 1.0 kev and 8.6 keV in the PTRH–Zn2+ complex, along with C, N, O,
S in PTRH, clearly indicated the presence of Zn2+ ions in the PTRH–
Zn2+ complex (Figure S15).[44]
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3.7
Application in living cells
PTRH cytotoxicity was evaluated by adding various concentrations
from 0 μM to 20 μM to HeLa cells. More than 98% of the cells
remained viable, therefore this sensor could be used for live cell imag-
ing. HeLa cells were incubated with PTRH (20 μM) for 30 min at 37°C,
and showed an obvious green fluorescence. After addition of 20 μM
Zn2+, cells were incubated for another 30 min, then washed with
HEPES buffer three times, following which bright red fluorescence
was observed (Figure 6). This effect demonstrated that PTRH was
capable of sensing Zn2+ ions in living cells.[45]
FIGURE 4 Comparison between the FT‐IR spectra of PTRH and the
PTRH+Zn2+ complex
Subsequently, following addition of another 0.5 equiv. Zn2+, the Hg
proton further shifted downfield to 8.37 ppm, accompanied by broad-
ened and reduced peak intensities. Protons Ha, Hb, Hc, Hd, He and Hf
are also shifted downfield upon interaction with Zn2+ ions, and indi-
cated that Zn2+ underwent ring‐opening.[42]
FIGURE 5 Partial 1H‐NMR (300 MHz) spectra of: (a) PTRH, (b) PTRH + 0.5 equivalents of Zn2+, and (c) PTRH +1 equivalent of Zn2+ in CDCl3

6
VENGAIAN ET AL.
FIGURE 6 Bright field image and fluorescence images of HeLa cells. (a) Bright field image of HeLa cells. (b) Fluorescence imaging of PTRH
(20 μM). (c) Fluorescence imaging of PTRH after addition of 20 μM Zn2+ ion. (d) Overlap imaging of [a] HeLa cell and [b] PTRH. (e) Overlap
imaging of (a) HeLa cell and the (c) PTRH+Zn2+ complex. (f) Overlap imaging of (d) and (e)
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geometries (shown in Figure S16). In PTRH, the HOMO was expanded
on phenothiazine, while the LUMO was centred on the spirocycle of
the rhodamine moiety and more restricted. The π electrons in the
HOMO of the PTRH+Zn2+ complex were primarily positioned in rho-
damine, while the LUMO was finely positioned on the guest Zn2+
ion. The energy gaps of the LUMO and the HOMO in the probe and
the complex were calculated, and found to be PTRH = 3.5244 eV
3.8
DFT studies
To exploit the knowledge of spatial distributions and orbital energies
for the LUMO and HOMO in PTRH and PTRH+Zn2+, DFT calculations
were carried out with B3LYP and 6‐311G/LANL2DZ basis sets using
the Gaussian 09 program (Figure 7). Frontier molecular orbitals in
PTRH and the PTRH+Zn2+ complex were derived from optimized
FIGURE 7 Frontier molecular orbitals of PTRH and the PTRH+Zn2+ complex obtained from DFT calculations using the Gaussian 09 program

VENGAIAN ET AL.
7
and PTRH+Zn2+ = 2.3074 eV, respectively. This result demonstrated
that the energy gaps of the LUMO and the HOMO in the Zn2+ com-
plex were small when compared with that of the PTRH sensor.[46]
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4
CONCLUSION
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We successfully synthesized a new fluorescence sensor, PTRH, com-
posed of phenothiazine (the energy donor) coupled to a rhodamine
(the energy acceptor) moiety that showed specific colorimetric,
fluorogenic, and ratiometric fluorescence responses towards Zn2+ ions
through the FRET process. Upon binding to Zn2+ ions, the spirolactam
ring in rhodamine opened and induced an excitation energy transfer
from phenothiazine to rhodamine, evident from the large spectral
overlap of the donor emission band and the acceptor absorption band.
Selective binding of Zn2+ ions was highly spontaneous, with a fluores-
cence colour change from yellowish green to pink. The PTRH sensor
provides a reasonable lowest detection limit over the nanomolar range
for Zn2+ ions. Nontoxicity, permeability and fluorescence transparency
of the sensor make it useful as an imaging agent for detecting Zn2+
ions in living cells.
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ACKNOWLEDGEMENTS
K. Sekar thanks the University Grants Commission, New Delhi, India
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[30] Y. Xua, H. Lib, X. Menga, J. Liu, L. Suna, X. Fanb, L. Shi, New J. Chem.
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ORCID
Sekar Karuppannan https://orcid.org/0000-0003-4294-4264
[31] K. Muthu Vengaian, C. Denzil Britto, K. Sekar, G. Sivaraman, S.
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SUPPORTING INFORMATION
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Supporting Information section at the end of the article.
How to cite this article: Karmegam MV, Karuppannan S,
Christopher Leslee DB, Subramanian S, Gandhi S. Phenothia-
zine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on’
sensor for Zn2+ and bioimaging studies in live cells. Lumines-
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