A FRET based colorimetric and fluorescence probe for selective detection of Bi3+ ion and live cell imaging

Article abstract of DOI:10.1016/j.jphotochem.2018.04.005

A Rhodamine B- quinoline conjugate (RSQ) functions as a colorimetric and fluorescence probe for selective recognition of Bi³⁺ ion based on CHEF assisted FRET process. RSQ visibly changes its colour and fluorescence profile upon addition of Bi

Full text of DOI:10.1016/j.jphotochem.2018.04.005

Accepted Manuscript
Title: A FRET based colorimetric and fluorescence probe for
3
+
selective detection of Bi ion and live cell imaging
Authors: Susanta Adhikari, Sandip Mandal, Avijit Ghosh,
Subhajit Guria, Abhishek Pal, Arghya Adhikary, Debasis Das
PII:
S1010-6030(18)30133-3
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.04.005
JPC 11220
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
30-1-2018
31-3-2018
3-4-2018
Please cite this article as: Susanta Adhikari, Sandip Mandal, Avijit Ghosh,
Subhajit Guria, Abhishek Pal, Arghya Adhikary, Debasis Das, A FRET
based colorimetric and fluorescence probe for selective detection of Bi3+
ion and live cell imaging, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2018.04.005
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A FRET based colorimetric and fluorescence probe for selective detection of
3
+
Bi ion and live cell imaging
a*
b
a,c
a
a
Susanta Adhikari , Sandip Mandal , Avijit Ghosh , Subhajit Guria , Abhishek Pal ,
c
b*
Arghya Adhikary and Debasis Das
a
Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, West Bengal,
India., E-mail address: adhikarisusanta@yahoo.com
b
Department of Chemistry, The University of Burdwan, 713104, Burdwan, West Bengal, India. E-mail:
ddas100in@yahoo .com
c
Centre for Research in Nanoscience & Nanotechnology, (CRNN), University of Calcutta, Technology
Campus, Sector-III, Block-JD2, Salt Lake, Kolkata 700098, West Bengal,India
Graphical abstract
Highlights
1
. A Rhodamine B-Quinoline conjugate (RSQ) selectively detects Bi3+ by colorimetric and
fluorescence methods.
3
+
2
3
4
. RSQ detects Bi as low as 0.05 µM.
. The sensing mechanism is based on CHEF assisted FRET process.
3+
. RSQ efficiently images intracellular Bi in SiHa live cells.

Abstract: A Rhodamine B- quinoline conjugate (RSQ) functions as a colorimetric and fluorescence
3
+
probe for selective recognition of Bi ion based on CHEF assisted FRET process. RSQ visibly changes
3
+
its colour and fluorescence profile upon addition of Bi ion. The distinctive colour change allows facile
3
+
3+
discrimination of Bi over other common cations. Lowest concentration of Bi detected by RSQ is 0.05
3
+
µM. RSQ efficiently images intracellular Bi in live SiHa cells.
Keywords: Rhodamine-quinoline conjugate • Solvent dependent selectivity and sensitivity • DFT
calculation • Live cell imaging
1. Introduction
1
Use of bismuth in semiconductors , cosmetic products (in pigment for eye shadow, lipstick and hair
2
3
dye) , medicines (for treatment of syphilis, peptic ulcers, hypertension and dermatological disorders) ,
4
5
alloys, catalysts , metallurgical additives and preparation and recycling of uranium in nuclear fuels is
continuously increasing bismuth exposure to human beings. Traces of Bi may alter physical, mechanical
6
and magnetic properties of metallurgical materials.
Food and Agricultural Organization (FAO) and World Health Organization (WHO) do not
recommend bismuth as a trace level essential element, although several bismuth compounds have
numerous medicinal applications. Nevertheless, the mechanisms of biological activities are not well
7
established.
The toxicity of Bi found in human and other animals includes osteo-arthopathy, hepatitis and
5
neuropathology, encephalopathy and nephrotoxicity. Oral intake of Bi compounds by mice may lead its
8
accumulation in nervous system (particularly in motor neurons) . Chronic exposure to Bi causes its
aggregation at epithelial cells, leading to characteristic intra-cytoplasmic inclusions. The absorbed Bi is
transported via human serum to the cells, and finally excreted through urine.
Several methods are available for determination of traces of Bi, viz. electro thermal atomic
9
10
absorption spectrometry (ET-AAS), inductively coupled plasma mass spectrometry (ICP-MS), hydride
11
12
generation atomic absorption/fluorescence spectrometry, spectrophotometry and light scattering
13
technique. However, they involve several drawbacks like expensive instrumentation, use of toxic
organic solvents and requires pre-concentration/ separation. Stripping voltammetry also allows selective
1
4
determination of Bi .However, use of toxic mercury salts and electrodes, and presence of overlapping
3
+
2+
peaks of Bi and Cu are two serious limitations of this process.

Fluorescence based optical sensors have been emerged as valuable tools to overcome those
drawbacks. This technique involves very fast response, simple procedure and non-invasive method
allowing applications to varieties physiological systems.
3
+
Lack of Bi selective chemo-sensor inspired us to develop rhodamine B-quinoline conjugate,3',6'-
bis(diethylamino)-2-(2-((quinolin-2-ylmethyl)thio)ethyl)spiro[isoindoline-1,9'-xanthen]-3-one (RSQ).
3
+
We have not found any Bi selective fluorescence probe in literature that functions through FRET
mechanism in 1:1 DMSO: water (v/v). Rhodamine based probes are well known for red light emission,
high quantum yield, low back ground emission, low cytotoxicity, cell permeability, and good
1
5
fluorescence resonance energy transfer (FRET) acceptor. RSQ visibly changes its colour and
3
+
fluorescence profile upon addition of Bi ion that allows facile discrimination over other common
cations.
2. Experimental
2.1. Materials and equipment
1
13
Chemicals and solvents were purchased from commercial suppliers and used as received. H and C
NMR spectra were recorded on a Bruker Advance III HD (300 MHz) spectrometer. Chemical shifts were
reported in parts per million (ppm), and the residual solvent peak was used as an internal reference:
proton (chloroform δ 7.26), carbon (chloroform δ 77.16) or tetramethylsilane (TMS δ 0.00) was used as a
reference. Multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(
multiplet). Coupling constants were reported in Hertz (Hz). High resolution mass spectra were obtained
TM
on a Micro mass/Q-Toff. micro spectrometer. For thin layer chromatography (TLC), Merck precoated
TLC plates (Merck 60 F254) were used and flash chromatography separations were performed on SRL
2
30-400 mesh silica gel.
2.2. General method of UV-Vis and fluorescence titration
Path length of the cells used for absorption and emission studies was 1 cm. For UV-Vis. and
fluorescence titrations, stock solution of RSQ (10 µM) was prepared in DMSO/water (1/1, v/v) using
3
+
HEPES (0.1M, pH 7.4) buffer. Working solutions of RSQ and Bi were prepared from their respective
stock solutions. Fluorescence measurements were performed using 5 nm x 5 nm slit width. Except time
dependent spectra, all the fluorescence and absorbance spectra were taken after 5 minutes of mixing of
analyte to RSQ.
2.3. Calculation of Quantum Yield
Fluorescence quantum yields (Ф) were estimated by integrating the area under the fluorescence
curves using the equation,

Where A was the area under the fluorescence spectral curve and OD was optical density of the compound
at the excitation wavelength. The standard used for the measurement of fluorescence quantum yield was
rhodamine-B (Ф = 0.7 in ethanol). The area of the emission spectrum was integrated using the software
available in the instrument and the quantum yield was calculated according to the equation.
2.4. Job’s plot from fluorescence experiments
3
+
A series of solutions containing RSQ and Bi were prepared in such a way that the total
3
+
concentration of Bi and RSQ remain constant (10 M) in all the sets. The mole fraction (X) of RSQ
was varied from 0.1 to 0.9. The emission intensity at 597 nm was plotted against the mole fraction of the
ligand (RSQ) in solution.
2
2
.5. Synthesis
.5.1. Synthesis of 2-(bromomethyl) quinolone (1)
16, 17a-b
2
-(Bromomethyl) quinolone was prepared according to the literature procedure
. Selective
bromination of the 2-methyl quinoline (3.0 g, 32.2 mmol) with 1.5 equivalents of NBS in CCl
4
for 12
1
hours afforded 1.3 g of compound 1 in 23.2% yield as yellow solid. The solid was characterized by H
NMR (Figure S-1) (300 MHz, in ppm CDCl
3
): δ 8.187-8.159 (d, J = 8.4 Hz, 1H ), 8.104-8.076 (d, J= 8.4
Hz, 1H), 7.832-7.804 (d, J = 8.4 Hz, 1H), 7.770-7.714 (m,1H), 7.589-7.538 (t, J= 8.4 Hz, 2H), 4.730 (s,
1
3
2
1
2
H). C NMR (Figure S-2) (75 MHz, CDCl
27.31, 127.00, 121.13, 34.37.
3
): δ 156.86, 147.50, 137.25, 129.92, 129.25, 127.50, 127.43,
.5.2. Synthesis of 2-((quinolin-2-ylmethyl)thio)ethan-1-amine (2)
A mixture of 2-(bromomethyl) quinoline(1.3 g, 7.55 mmol) and 2-amino-ethanethiol (581 mg, 7.55
mmol) in dry ethanol (10 mL), was added to a in-situ solution of sodium ethanolate, prepared by mixing
sodium (166.42 mg, 7.23 mmol) to dry ethanol (15 mL) under nitrogen. The mixture was stirred for 5 h at
room temperature. After completion of the reaction as monitored by TLC, the mixture was neutralized
and extracted with ethyl acetate. The crude product was isolated by flash chromatography on silica gel
using MeOH/ DCM (1:9, v/v) to get yellow liquid, assigned as 2 with 65% yield (824 mg,). The liquid
1
was characterized by H NMR (Figure S-3) (300 MHz, CDCl
3
): δ = 1.521 (2H, s), 2.265 (2H, t), 2.505
1
3
(
2H, t), 3.665 (2H, s), 7.163 (2H, m), 7.361 (2H, m), 7.742 (2H, m). C NMR (Figure S-4) (75 MHz,
CDCl ): δ 158.49, 146.91, 136.51, 129.23, 128.89, 128.44, 127.17, 127.10, 127.03, 126.63, 126.56,
3
1
2
1
26.49, 126.43, 125.92, 121.50, 120.76, 39.86, 37.81, 30.44.
.5.3. Synthesis of 3', 6'-bis(diethylamino)-2-(2-((quinolin-2-ylmethyl)thio)ethyl)spiro[isoindoline-
,9'-xanthen]-3-one (RSQ)
1
7(c)
The acid chloride of rhodamine
in DCE (479 mg, 1 mmol) was added to the stirred solution of
amine, 2 (168 mg, 1 mmol) in DCE in presence of few drops of triethyl amine. The reaction mixture was
refluxed for 6 h. Upon completion of the reaction as monitored by TLC, the solvent was removed in

vacuum to dryness. The reaction mixture was treated with water, extracted with ethyl acetate, and dried
over anhydrous Na SO . The organic layer was removed under reduced pressure and the residue was
2
4
purified by column chromatography (EtOAc/ hexane, 3:7, v/v) to yield pink solid (290 mg, 45%), RSQ.
1
The solid was characterized by H NMR (Figure S-5) (300 MHz, CDCl
3
):δ = 1.167 (12H, t), 2.163
(2H, t), 2.310 (2H, t), 3.435 (8H, m), 4.106 (2H, s), 6.253 (2H, s), 6.350 (2H, d), 6.442 (2H, d), 7.067
1
3
(
1H, d), 7.395-7.497 (3H, m), 7.656-7.760 (3H, m), 7.933 (1H, d), 8.035 (1H, m), 8.075 (1H, m);
) δ = 12.43, 29.31, 29.83, 38.07, 39.48, 43.97, 60.00, 64.43, 97.42,
05.15, 107.78, 120.99, 122.45, 123.4, 125.73, 126.64, 127.03, 127.64, 128.51, 128.79, 129.01, 130.75,
32.03, 136.25, 147.46, 148.48, 152.91, 153.21, 158.62, 167.64; and QTOF mass (Figure S-5) m/z
C
NMR (Figure S-6) (75 MHz, CDCl
3
1
1
+
+
(
M+H) calculated for C40
H
43
N
4
O S is 643.3101, found: 643.3149.
2
2.5.4. Synthesis of N-(2-((quinolin-2-ylmethyl)thio)ethyl)acetamide (3)
To dichloromethane solution of 2 (218 mg, 1 mmol), acetic anhydride (204 mg, 2 mmol) was added
under stirring condition in presence of few drops of triethylamine. The reaction mixture was stirred for 6
h. Upon completion of the reaction as monitored by TLC, the solvent was removed in vacuum. The
residue was partitioned and extracted with ethyl acetate from water and dried over anhydrous Na
2
SO .
4
The organic layer was removed under reduced pressure and the residue was purified by column
chromatography (EtOAc/ hexane, 3:7, v/v) to yield 170 mg (65%) of 3 as a yellow liquid.
1
H NMR (300 MHz, CDCl ) (Figure S-7): δ, 8.075-8.046 (d , J = 8.7 Hz, 1H ), 7.981-7.953 (d, J = 8.4
3
Hz, 1H), 7.737-7.710 (d, J = 8.1 Hz, 1H), 7.652-7.601 (m,1H), 7.474-7.415 (m, 2H), 3.943 (s, 2H),
1
3
3
.433-3.373 (q, J = 5.7Hz, 2H), 2.588-2.546 (t, J = 6.3 Hz, 2H), 1.901 (s, 3H). C NMR (75 MHz,
CDCl ) (Figure S-8): δ, 170.29, 158.75, 147.22, 137.10, 129.71, 128.63, 127.56, 126.96, 126.41, 121.23,
9.18, 37.96, 30.87, 23.06.
3
3
3. Results and discussion
3
+
Bi has thiophilic character and thus has affinity for S-donors leading to the formation of
18
thermodynamically stable Bi-S bond. In our designed probe RSQ, we have carefully combined quinoline
and rhodamine moieties through a flexible ‘S’ donor spacer (Scheme 1). Previously, we have designed
3
+
16
Au selective probe having N, O and S donor sites based on rhodamine-quinoline conjugate. The
selectivity of rhodamine based probes can be tuned through variation of spacer and relative orientation of
3
+
2+
2+
3+
3+
19
donor sites. Generally, rhodamine based probes are selective for Al , Cu , Hg , Fe or Cr ions while
2
+
20
‘
S’ donor sites prefer Hg as predictable from SHAB principle . Literature reports suggest that nature
and length of the spacer in different rhodamine conjugates control its selectivity for different analytes, for
example, use of hydrazine and ethylenediamine as spacer have marked difference towards metal ion
21,22
selectivity.
The synthetic protocol of RSQ is shown in Scheme 1. All the intermediates have been
1
13
characterised by H NMR, C NMR and QTOF-MS spectra (Figure S-1 to S-9, ESI†).

Scheme 1Synthetic route to RSQ
3.1. Photo-physical studies
Solvent plays an important role to the selectivity of rhodamine based probes for different analyte. In
3
+
present case, DMSO: water (1:1, v/v) was used as working solvent for selective detection of Bi ion.
3
+
Initially, higher percentage of water (DMSO: water, 1: 4, v/v) was used for detection of Bi ion because
3
+
of highest emission intensity for Bi was observed in this media. Unfortunately, at higher water content
2
+
¯
2¯
of the media, Hg interfere to some extent which may be eliminated using masking agent like I /S /
1
6,23
¯
2-
3+
NCS¯.
However, both I / S masks Bi to form insoluble BiI
3
/ Bi
2
S
3
. Hence, NCS¯ is chosen which
3
+
had no adverse effect on the emission intensity of RSQ or RSQ-Bi systems (Figure 1). However, at low
2
+
water percentage (DMSO: water, 1:1, v/v), interference from Hg ion is insignificant and hence chosen
this media for entire studies (Figure S-10, ESI†).
3
+
The fluorescence response of RSQ towards Bi in presence of other cations was presented in Figure
S-11 (ESI†). Figure 2 show the selectivity of RSQ (λem = 597 nm; λex =525 nm) for common cations
while the inset presents corresponding colour changes.

Figure 1 Fluorescence spectra of RSQ (20 µM) (a) in presence of Hg2+ (1000 µM) and Bi3+ (1000 µM); (b) upon
addition of NCS¯ (2000 µM) to the system shown in (a); Medium: HEPES buffer (0.1 M, pH 7.4), DMSO: water
(1:1, v/v) (λex, 525 nm)
Figure 2 Emission intensity of RSQ (20 µM) at 597 nm (λex, 525 nm) in presence of different cations (1000 µM) in
mentioned media; Inset: colours upon irradiation with (a) visible and (b) UV light
The UV-vis. spectrum of RSQ exhibited an absorption band centered at 322 nm (Figure 3). Figure
3A show changes of absorption spectra in presence of common cations, mentioned in Figure 2.
3
+
Initially, absorbance at 322 nm increases upon gradual addition of Bi up to 1 µM. Further addition
3
+
of Bi (>1 to 1000 μM) gradually decreases this absorbance while a new band that appears at 566 nm
gradually increases, indicating spirolactam ring opening of the rhodamine unit (Figure 3B).
Figure 3 Changes in the UV-Vis. spectra of RSQ (20 μM) (A) in presence of different cations (1000 µM) and (B)
upon gradual addition of Bi3+ (0.0, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0, 50.0, 75.0, 100.0, 150.0, 200.0,
3
00.0, 500.0, 750.0 and 1000.0 μM) in same media.
Emission of free RSQ at 597 nm (λex = 525 nm) was very weak (Ф = 0.02) (Figure 4b). In presence
3
+
of Bi , ~130 fold enhancement of emission intensity along with ~30 fold increase of fluorescence

quantum yield (Ф = 0.59) were observed. As two distinct excitation wavelengths viz. 365 nm and 525 nm
were observed from two emission wavelengths (Figure S-12, ESI†), fluorescence titration was carried out
at both wavelengths (Figure 4a and 4b). RSQ show emission at 450 nm upon excitation at 365 nm,
characteristics of quinoline emission whereas the emission at 597 nm was due to open ring spirolactam
3
+
form of rhodamine unit. The emission intensity at 450 nm increases up to 1µM Bi and then decreases
3
+
gradually up to 1000 μM Bi while the emission intensity at 597 nm increases continuously upon
3
+
addition of Bi (Figure 4a). On the other hand, changing the excitation wavelength to 525 nm changes the
emission profile of the same system as presented in Figure 4b. Figure 4d shows the plot of respective
3
+
3+
emission intensity vs.Bi concentration (λem, 597 nm, λex, 525 nm). Lowest concentration of Bi detected
3
+
by RSQ is 0.05 µM. The effect of pH on RSQ in presence and absence of Bi was presented in Figure S-
3
+
1
3 (ESI†). Gradual change of the colour of RSQ with increasing Bi concentration was shown in Figure
3
+
S-14 (ESI†). Addition of Bi (up to 1µM) enhanced the emission intensity at 450 nm, ascribed as CHEF
process (λex, 365 nm) involving quinoline unit. To further strengthen the CHEF process, a model
compound, 3 devoid of rhodamine unit was synthesized (Scheme 1) that had emission maxima centered at
3
+
4
50 nm. Upon addition of Bi ion, its emission maxima red shifted to 503 nm (Figure S-18, ESI) due to
3
+
CHEF process involving quinoline unit. It is proposed that at low concentration (<1µM), Bi interact
solely with quinoline moiety whereas at higher concentration (>1µM), the spirolactam ring of rhodamine
unit opens and consequently, emission at 597 nm increases significantly along with gradual decrease of
3
+
quinoline band, indicating the commencement of FRET process. Thus the present probe recognizes Bi
1
6,23
through CHEF assisted FRET process
where quinoline unit is FRET donor and open ring rhodamine
16
unit is FRET acceptor. Figure 4c show significant overlap between donor emission (quinoline) and
acceptor absorbance (open ring rhodamine B), a basic criterion for FRET process. From Job’s plot
3
+
(
Figure S-15, ESI†), 1:1 stoichiometry between RSQ and Bi was established. The binding constant of
3
+
5
RSQ for Bi is 2.1×10 (Figure S-16, ESI†).

Figure 4 Changes in the emission spectra of RSQ (20 μM) upon gradual addition of Bi3+ (0.0, 0.05, 0.1, 0.5, 1.0,
2
λ
.0, 5.0, 10.0, 20.0, 30.0, 50.0, 75.0, 100.0, 150.0, 200.0, 300.0, 500.0, 750.0 and 1000.0 μM) (a) λex, 365 nm and (b)
ex, 525 nm; (c) spectral overlap between donor emission (quinoline; blue) and acceptor absorbance (open ring
3+
rhodamine B; pink). The dotted line (red) shows the emission spectrum of adduct [RSQ + Bi ] and (d) emission
3
+
intensity vs. Bi concentration at 597 nm (λex, 525 nm).
1
3
.2. H NMR titration
1
1
To unveil the sensing mechanism, HNMR titration was performed. Changes in the HNMR signal of
3
+
RSQ in presence of Bi were presented in Figure 5. Protons of rhodamine moiety (marked as f, g, h, i, o,
j and p) were shifted downfield significantly (marked as f´, g´, h´, i´, o´ and p´). The ‘j’ proton was shifted
up-field to j´. The protons of quinoline unit marked as k, l, n, q and r were downfield shifted to k´, l´, n´,
3
+
q´ and r´ and m was up-field shifted to m´ in presence of Bi . Mass spectrum (Figure S-17, ESI†) also
3
+
confirmed the formation of the [RSQ + Bi ] adduct having m/z at 485.6365.
.3. Theoretical studies
DFT studies theoretically support Bi assisted FRET ON process. The optimized geometries of RSQ
3
3
+
3
+
(
Table S-1, ESI†) and RSQ+Bi (Table S-2, ESI†) were generated using 6-31G/LanL2DZ and
2
4
B3LYP/LanL2DZ basis sets, respectively with Gaussian 09 software . The HOMO-LUMO energy
3
+
differences of [RSQ+Bi ] adduct was lower than that of free RSQ (Figure S6). Indicating formation of
3
+
stable adduct by RSQ with Bi ion.

1
3+
3+
Figure 5 HNMR titration: (I) free RSQ; (II) RSQ + 1.0 equiv. Bi ; (III) RSQ + 2.0 equiv. Bi in methanol-d
4
3+
Figure 6 HOMO-LUMO energy difference of free RSQ and [RSQ+Bi ] adduct
Considering all these facts, the possible sensing mechanism was portrayed in Figure 7. The increase
3
+
of absorbance and fluorescence of quinoline unit at lower Bi concentration probably indicated
3
+
3+
3+
predominant RSQ: Bi = 2:1 (mole ratio) interaction. At higher Bi concentration, RSQ: Bi mole ratio

switched to 1:1 while the spirolactam ring of rhodamine opens to initiate the FRET process, consequently
3+
quinoline emission decreases. Lack of optical probes for selective recognition of Bi ion forced us to
3+
compare the present method with other reported methods for Bi detection (Table S-3, ESI†).
Figure 7 Probable sensing mechanism of RSQ with Bi3+
3.4. Cell imaging studies
3
+
Although no cytotoxic effect of Bi is established so far, yet RSQ was explored to detect
3
+
intracellular Bi , for any possible biological applications. It is important to note that several thiol
3
+
containing biogenic moieties are present inside the cell. The thiophilic character of Bi ion may tend to
form stable complex with this bio-molecules. However, the concentration of these bio-molecules being
3
+
0
low, free intra-cellular Bi ion may be detected using the present probe. Cells were incubated at 37 C and
5
% CO in culture medium. The cells were incubated in presence and absence of 20 µM RSQ for 2h.
2
3
+
Then the cells were washed thrice with PBS buffer followed by addition of 50 μM Bi to the medium.
Fluorescence images of the cells were obtained using an EVOS® FL Cell Imaging System, Life
3
+
Technologies, USA). Figure 8 show that RSQ emits red fluorescence in presence of intracellular Bi ion.
3
+
The control experiment with cells incubated with only Bi ion shows no cytotoxic effect.
The cytotoxicity of RSQ on SiHa cells was determined by MTT assay (Figure S-19, ESI†). Upon
exposure of 20 μM RSQ for 12h, ∼90% of the cells remains viable. This nullifies the possibility of any
significant cytotoxic influence of RSQ on SiHa cells. Therefore, RSQ may be used as an effective
3
+
chemosensor for Bi in living systems.

Figure 8 Imaging of Bi3+ in SiHa cells: (a) bright field image of cells after incubating 10 μM RSQ with 50 μM
3+
Bi ; (b) fluorescence image of the cells in blue channel; (c) are merge images of a and b ; (d) fluorescence image of
the cells in red channel; (e ) merge images of a and d; (f) Bright field images of control cells after incubating only
with 10 μM RSQ; (g) fluorescence image of the cells; (h) merge images of cells f and h; (i) fluorescence images
under red channel; (j) merge images of cells f and i; RSQ was prepared in 0.3% DMSO in water. [Blue channel: 360
nm excitation, 447 nm emission; red channel: 530 nm excitation, 593 nm emission;]
3
+
Figure 8 reveals that RSQ is cell permeable to efficiently image intracellular Bi in live SiHa cells
under fluorescence microscope.
Figure 9 Imaging of Bi3+ in HeLa cells: (a) bright field image of cells after incubating with 10 μM 3; (b)
fluorescence image of the cells in green channel; (c) are merge images of a and b ; (d) Bright field images of cells
3+
after incubating with 10 μM 3 and followed by 10 μM Bi ; (e) fluorescence image of the cells; (f) merge images of
cells d and e. [green channel: 470 nm excitation, 525 nm emission]

3+
Figure 9 shows that upon addition of Bi ion, the fluorescence intensity of 3 in live cell also increase to 10
fold. Therefore, the observed CHEF process predicted for RSQ should be attributed from the initial binding
3+
of Bi ion with amine compound 2 present in RSQ.
Conclusion
In summary, we have demonstrated a FRET based rhodamine-quinoline conjugate (RSQ) as an
3+
excellent chemo-sensor for selective detection of Bi . RSQ changes its colour and fluorescence property
3
+
3+
upon addition of Bi . The LOD of RSQ for Bi is 0.05 µM, obtained by fluorescence method. RSQ can
3
+
efficiently images intra cellular Bi in SiHa cell line.
5
. References
1
. (a) J. Li, L. Jiang, B. Wang, F. Liu, J. Yang, D. Tang, Y. Lai, J. Li, Electro deposition and characterization of
copper bismuth selenide semiconductor thin films, Electrochim. Acta 87 (2013) 153–157; (b) M. A. Berding, A.
Sher, A.B. Chen, W. E. Miller, Structural properties of bismuth-bearing semiconductor alloys, J. Appl. Phys. 63
(
1988) 107-115.
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