5
10
R. Manjunath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 509–515
changing or suppressing their function which leads to destructive
effects. Aluminum ions have been implicated as a causative factor
of Alzheimer’s disease and associated with damages to central
nervous system in humans that can cause idiopathic Parkinson’s
Job’s plot analyses
To understand the stoichiometric nature of chemosensor L, Job’s
plot method was performed using spectrophotometer for L–Al
3+
3+
3+
disease. [11–13] Therefore, detection of Al
ion is highly
complex [25]. The total concentration of L and Al ions was kept
constant (10 M) and changing their mole fraction from 0 to 1
3+
demanding because of the potential impact of Al ion to human
health as well as environment.
l
and then absorbance of the solutions of different compositions
Now a days, there are many conventional detection methods
employed for quantitative analysis of aluminum such as high per-
formance liquid chromatography, atomic absorption spectroscopy,
inductively coupled plasma mass spectrometry, electrochemical
sensing etc.[14] However the utilization of these methods are
largely limited by tedious and time consuming procedures involves
use of sophisticated instrumentation and not suitable for real-time
monitoring. Among them non-conventional method such as
fluorescence detection method has significant advantages because
of its simplicity, high sensitivity and low cost. It can be
conveniently examined by naked eye rather than aforementioned
techniques. [15] A simple dye based compounds have widely used
for developing naked-eye diagnostic tools for detection of metal
ions. [16–18] Among the dye families, rhodamine derivatives are
ideal platforms for development of colorimetric chemosensors for
selective detection of heavy and transition metal ions due to its
spectroscopic properties such as long-wavelength emission, high
fluorescence quantum yield, and large molar extinction coefficient
and make them a good candidate. [19] The rhodamine spirolactam
form is basically colorless, while metal induced ring-opened amide
form gives rise to strong absorption within a visible range [20–24]
this feature provides a vital pathway for naked-eye detection of
metal ions. There are only few publications were reported for
was recorded. A significant increase in absorption was observed
3
+
3+
as result of L binding with Al ions. The mole fraction of Al
3
+
3+
was plotted against [Al ]/{[Al ] + [L]}. In the plot, the mole frac-
3
+
tion of the Al at which the absorption intensity is at a maximum,
gives stoichiometry of the complex.
Determination of the association constant
a
The association constants (K ) can be estimated from UV–Vis
titration according to the following Benesi–Hildebrand plot [26]
equation:
1
1
1
¼
Þ½Al3þꢃ þ
ðA ꢂ A
0
Þ
ðAmax ꢂ A Þ
0
K
a
ððAmax ꢂ A
0
A and A
0
is absorbance of L in the presence and absence of Al3+
respectively; Amax is the saturated absorbance of L in the presence
3
+
of excess amount of Al , K
mined from the slope of linear plot, [Al ] is concentration of Al
ion added during titration studies. The goodness of linear fit of
a
is association constant and was deter-
3
+
3+
3
+
0
the B–H plot of 1/(A ꢂ A ) verses 1/[Al ] for 1:2 complex formation
3
+
confirms binding stoichiometry between L and Al
.
3
+
Al ion detection through rhodamine-based receptor.
Determination of quantum yield
Experimental
The fluorescence quantum yields were determined using rhoda-
mine B as a reference with a known value of 0.69 in EtOH. The
sample and reference were excited at the same wavelength
ex = 554 nm), maintaining nearly equal absorbance. The quantum
U
Materials and instruments
(k
All the chemicals for synthesis were purchased from commer-
cial suppliers and used after further purification. Perchlorate salts
yield was calculated according to the following equation:
+
+
3+
2+
2+
2+
2+
2+
2+
2+
3+
of Na , K , Fe , Pb , Cu , Mg , Mn , Zn , Cd , Hg and Al
2
Fu As ð
g
uÞ
were purchased from Alfa Aesar, India. All the reagent-grade
solvents were purified according to standard procedures and
freshly distilled prior to use. All the reactions were monitored by
thin layer chromatography with detection by UV. Thin layer chro-
matography (TLC) was performed on silica gel 60 F254 plates
Uu ¼
U
s ꢄ
:
:
2
Fs Au
ðg
sÞ
where
U
u and Us are fluorescence quantum yields of the sample
and standard, Fu and Fs are the integrated fluorescence intensities
of sample and standard, Au and As are the absorbance of sample
(
Merck KGaA). Column chromatography was executed on silica
gel (60–120 mesh). FT-IR spectra were recorded with IR spectrom-
and standard and
standard solutions, respectively.
gu and gs are refractive index of sample and
1
13
eter Perkin–Elmer FT-IR spectrometer using KBr pellets. H and
C
NMR spectra were recorded on Bruker AVIII 300 MHz spectrometer
using tetramethylsilane (TMS) as an internal reference. Electron
spray mass spectra (ESI-MS) were carried out on a Bruker MaXis
Cell culture and fluorescence imaging
1
0,138 HRMS instrument. UV–vis spectra were recorded in room
temperature using a Shimadzu UV-1650PC spectrophotometer.
Fluorescence spectra were recorded in room temperature using
Perkin Elmer LS-45 Fluorescence spectrometer.
Breast cancer (MDA-MB-231) cell line were grown in Dulbecco’s
Modified Eagle’s Medium (DMEM, Gibco), containing 10% fetal
bovine serum (Gibco) and 1% antibiotic, at 37 °C in a humidified
2
incubator with 5% CO . Cells were grown to 80–90% confluence
for the experimental study, harvested with 0.05% trypsin (Sigma)
in phosphate buffered saline (PBS) and plated at desired cell con-
centration for fluorescence imaging. The cells were seeded into
General procedures of spectral detection
3
+
All experiments were carried out in acetonitrile/water (80/20, v/
24-well plates and experiments to assay Al uptake were per-
formed in the same media supplemented with different concentra-
ꢂ5
v) solution. A stock solution (1.0 ꢁ 10 M) of receptor L was
3
+
prepared in acetonitrile: water (80:20, v/v) mixture. The cationic
tions of a lent of 1/2 added Al . The cells were rinsed twice with
PBS and treated with DMSO-containing (10 M, DMSO:
ꢂ3
solutions (1.0 ꢁ 10 M) were prepared in ethanol/water (80/20,
L
l
v/v) for spectral analysis. The absorption and fluorescence spectra
water = 1:100) for 30 min at 37 °C. After washing twice with PBS,
the cells were imaged under a fluorescence microscope (Nikon
Eclipse-80i, Japan).
were performed by using 5
lM of receptor L in aqueous solution
with varying concentration of metal ions.
Manjunath, Rangasamy
