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Article

Selective and sensitive turn-on chemosensor for arseniteion at ppb level in aqueous media applicable in cell staining

Somenath Lohar, Siddhartha Pal, Buddhadeb Sen, ManjiraMukherjee, Samya Banerjee, and Pabitra Chattopadhyay

Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503255f • Publication Date (Web): 14 Oct 2014

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Selective and sensitive turn-on chemosensor for arsenite ion at ppb

level in aqueous media applicable in cell staining

Somenath Lohar,†

Siddhartha Pal,† Buddhadeb Sen,

† Manjira Mukherjee,

† Samya Banerjee

‡ and

Pabitra Chattopadhyay*,†

† Department of Chemistry, The University of Burdwan, Burdwan-713104, West Bengal, India,

E-mail: pabitracc@yahoo.com. Fax: +91-342-2530452. ‡ Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, 560012, India

ABSTRACT. A newly designed and structurally characterized cell permeable receptor (HL) selectively senses the AsO33-

ion upto

ca. 4.1 ppb in aqueous media over the other competitive ions at biological pH through intermolecular H-bonding induced CHEF

process, established by detailed experimental and theoretical studies. This bio-friendly probe is highly competent to recognize the

existence of AsO33-

ions in living organism by developing image under fluorescence microscope and useful to estimate the amount

of arsenite ions in various water samples.

KEYWORDS. Chemosensor; AsO33-

; Cell staining

INTRODUCTION

Arsenic is a teratogenic and carcinogenic toxic element

and it being present in natural waters supports to propagate the

serious health problems allied with dermal toxicity, cardiovas-

cular disease, and neurodegenerative disorders etc.1,2

The

physical status and chemical structure of the involved com-

pound demonstrate the degree of arsenic toxicity, which is the

highest in case of the inorganic form of arsenite(III) state over

that of arsenate(V) and organic form of this element.3,4

In natural waters, especially groundwater, arsenic (As) ex-

ists as both arsenite (AsIII

) and arsenate (AsV), and the ratio of

AsV to As

III has been found to be in the range of 10-100 de-

pending on the chemical environment.5-7

Especially, high lev-

els of AsIII

(> 0.3 mg.L-1

) were found in groundwater in Bang-

ladesh, India, Laos, Cambodia and Pakistan8 and the oxidation

of AsIII

by dissolved oxygen is extremely slow. The carcino-

genic effect due to arsenite ions has already been confirmed by

the recent experimental outcome, where it has been found that

in vitro exposure of 5 μM arsenite [As3+(

OH)3] to normal stem

cells, result in cancer stem cell phenotype by 18 weeks.9

In

2001 the WHO, EPA and U.S. have already advised to lower

the maximum contamination level (MCL) of arsenic com-

pounds in drinking water to 10 ppb from 50 ppb owing to the

high toxicity of arsenicals.10

For this real urgency, the deter-

mination of ppb level arsenic species in aqueous media is to be

effectively developed in proper way.

Detection of arsenicals by colorimetric methods resulting

from the Gutzeit reaction suffers from the deadly poisonous

arsine gas (AsH3) and byproducts of mercury compoounds.11

ICP-MS being a sensitive detection strategy recommended by

the EPA is an expensive process and required an extensive

methodology for sample preparation.12

Thus, fluorescence

spectroscopy meets the requirements in developing the simple

field-effective methods to detect the arsenicals of very low

level as it has been widely utilized for numerous analytes.13-16

Till date, a few chemosensors selective for arsenate ions17

and

for AsIII

cation,18-20

including some bioluminescents for arse-

nite ion21-23

have been reported. However, AsO33–

ion selective

chemosensor is still unexplored. Herein, we report an easy to

make simple Schiff base system (HL) which senses selectively

the most hazardous AsO33-

ion as low as 54.91×10-9

M (ca.

4.12 ppb lower than MCL) in aqueous media through inter-

molecular hydrogen bonding assisted CHEF process colori-

metrically and fluorimetrically.

EXPERIMENTAL SECTION

Materials and methods

All the analytical reagent grade solvents and the other rea-

gent grade chemicals consumed in this work were procured

from commercial sources and used as received. Here, through-

out the experiments Milli-Q 18 Ω water was employed. 2,6-

diformyl-p-cresol was synthesized following the literature

procedure.24

Tetrabutylammonium salts of dihydrogen phos-

phate, acetate, halides (F-, Cl

-, Br

-, I

-); and sodium salts of

arsenate, arsenite, bicarbonate, nitrate, chlorate, monohydro-

gen phosphate, azide, cyanide, thiocyanate, sulphate, etc were

used for the selectivity study of the receptor towards different

anions.

A Perkin Elmer 2400 CHN elemental analyzer was used

for performing the elemental analyses. The electronic and

FTIR spectra were obtained on Shimadzu made UV-1800 and

Prestige-21 FTIR spectrophotometers, respectively. A Bruker

Avance DPX 500 MHz spectrometer for recording 1HNMR

spectra using DMSO-d6 solution and a Qtof Micro YA263

mass spectrometer for electrospray ionization (ESI) mass

spectra were used. To measure the pH of the solution on ad-

justing the pH of the solution using either 50 mM NaOH or

HCl solution, a Systronics pH meter (model 335) was taken. A

Hitachi F-4500 FL spectrophotometer was employed to record

the steady-state fluorescence spectra, whereas a HORIBA

JOBIN Yvon (TCSPC) spectrometer from IBH (UK) at λex=

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438 nm and MCP-PMT as a detector was used to perform the

time-resolved fluorescence lifetime measurements experi-

ments.

A HEPES buffer (1 mM, pH 7.4) (DMSO-Water 1 : 9) co-

solvent at 25 °C temperature with an HL concentration of 10

μM was used for doing all the UV−visible and Fluorescence

spectroscopic experiments (detail in the Supporting Infor-

mation). The relevant Benesi-Hildebrand equation25

was used

for calculating the apparent binding constant of the produced

HL-AsO33-

ensemble species; the estimation of the detection

limit and Job’s plot are shown in the Supporting Information.

The preparation of cells for in vitro cellular imaging and the

cell viability test with HL to determine the cytotoxicity assay

are carefully carried out and the details in the Supporting In-

formation.

A suitable single crystal was mounted on a Bruker’s Apex-

II CCD diffractometer using MoKα (λ = 0.71069) to collect the

data to solve the structure of HL, and necessary corrections

were applied using SADABS from Bruker. The direct methods

using SIR-92 duly refined by full-matrix least squares refine-

ment methods based on F2, using SHELX-97

26 was used to

solve the structure. Wingx package27,28

was taken to perform

all the calculations. Deposition of all the crystallographic data

of HL as the CCDC no. of 1008294 has been performed.

Theoretical Calculation

DFT calculations of the probe (HL), and its corresponding

HL-AsO33-

ensemble species were done with the help of

Gaussian-09 software over a Red Hat Linux IBM cluster with

the B3LYP/6-31G (d,p)29-31

functional model and basis set to

describe the understanding of the electronic configurations, the

interactions in the molecular level and the ground state charac-

ter.

RESULTS AND DISCUSSION

Synthesis of the Probe (HL)

The AsO33–

ions selective probe (HL) was synthesized by

condensation of 2,6-diformyl-p-cresol with 4-aminoantipyrine

in 1:2 mole ratio (Scheme 1) and it was characterized by spec-

troscopic and physico-chemical tools including single crystal

X-ray crystallographic analysis (Figures S-1 (a) and (b) in the

supporting information).

Scheme 1 Schematic representation of synthesis of the probe

HL

Crystals of the probe (HL) suitable for single crystal X-ray

crystallographic analyses were collected from the

DMF/acetonitrile (1:3) solution of HL on slow evaporation.

The moleculer view of HL having the monoclinic space group

'P 21/c' with sequential atom numbering is illustrated in Figure

1. Some Crystallographic parameters, bond angles and bond

distances are tabulated in Table S-1 and S-2 (Supporting in-

formation) respectively. The bond distances of two imine type

bonds C20-N4 (1.297(6) Å) and C12-N3 (1.274(6) Å) are

comparable to each other but both are shorter than that of N3-

C8 (1.391(7) Å) or N4-C21 (1.386(6) Å).

Figure 1. Solid state structure of HL with sequential atom

numbering

Synthesis of the HL-AsO33-

ensemble species

The HL-AsO33-

species (2) was isolated in the solid state

from the reaction of trisodium arsenite with methanolic solu-

tion of HL in equimolar ratio at stirring condition (Scheme S1

in the supporting information). A molecular-ion peak at m/z

701.1548 with ~20% abundance observed in the mass spec-

trum of 2 can be attributable to [M + H2O + 3H+ + Na

+]

+ (cal-

culated value at m/z, 701.1686). The 1HNMR spectrum ob-

tained in DMSO-d6 confirmed the binding mode of HL with

AsO33-

ions (Figures S-2 (a) and (b), in the supporting infor-

mation).

UV-visible Studies. UV-vis spectra of HL obtained in

HEPES buffer (1 mM, pH 7.4; water:DMSO (v/v), 9:1) at 25

°C showed the intramolecular charge transfer (CT) transition

at 356 nm. On gradual addition of AsO33-

ions (0-20 M), the

intensity of this absorption of HL at 356 nm was decreased

with simultaneous formation of a new CT absorption peak at

428 nm (Figure 2) through an isosbestic point at 380 nm and it

is reflected in the visual change from the colorless solution to

faint greenish yellow.

Figure 2. UV-vis spectral changes of HL (10 μM) on gradual

addition of AsO33-

ions (0-20 µM) in HEPES buffer.

pH studies and Selectivity. In presence and absence of

AsO33−

ions, the fluorescence intensities of HL were assessed

at various pH using HEPES buffer (Figure S-3 in the support-

ing information). This study clearly showed that the fluores-

cence of weak intensity in absence of AsO33−

ions is almost

independent over the pH range 4.0 to 10.0. But it is notewor-

thy that the fluorescence intensity of HL in presence of AsO33−

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ion is higher than that in the absence of AsO33−

ions. This ob-

servation is ascertained by the formation of HL-AsO33-

species

(2) through intermolecular H-bonding. The fluorescence fea-

tures of HL (10 μM) were also validated upon the addition of

10 equivalents in excess of different anions i.e. AsO33−

, NO3−,

Mo43-

, Br

−, F

−, HCO3

-, N3

-, ClO4

−, H2PO4

−, HPO4

2−, Cl

−, PO4

3-,

CO32-

, OAc−, SO4

2−, HSO4

- and HAsO4

2− but only AsO3

3- selec-

tively enhanced the fluorescence intensity of HL system (Fig-

ure S-4 in the supporting information).

It is remarkable to note that the presence of other competi-

tive anions does not affect the enhancement of fluorescence.

Moreover, in presence of 50 equivalents of different tested

anions in excess with HL and AsO33-

, almost no undesirable

effect on intensity was recorded (Figure S-5 (a) in the support-

ing information). It is also notable to mention that the interfer-

ence coming from metal ions is also negligible though this

Schiff base has an affinity to form complexes.32,33

Fluorescence Studies. On addition of various concentra-

tions of AsO33-

ions (0-20 µM), significant enhancement of the

fluorescence intensity at 532 nm was observed. On addition of

2.0 equivalent of AsO33-

ions, HL displayed ca. 9-fold increase

of its fluorescence intensity (Figure 3), which is also reflected

in the values of the calculated quantum yields (). The value

of increases by ca. 5.67 times due to the addition of addition

of AsO33-

ions from = 0.00228 to = 0.01294. This spectral

feature was also evidenced by the color change in fluorescence

from colorless to green in presence of light in the UV region

(Figure S-6 in the supporting information).

With the help of the fluorescence study, Job’s plot (Figure

S-7 in the supporting information) clearly demonstrates the 1:1

in stoichiometric ratio of HL and AsO33-

to form the ensemble

2. This fact of 1:1 stoichiometry is also validated by the spec-

troscopic and physico-chemical data of the isolated solid HL-

AsO33-

ensemble species. The strong binding affinity of HL

towards the AsO33-

ions has also been strengthened by the

binding constant (K) value (2.5267 x 105 M

-1) calculated from

the slope/intercept using the plot of (F∞-F0)/(Fx- F0) against 1/[

AsO33-

] (Figure S-8 in the supporting information).

Figure 3. Fluorescence spectra of HL (10 μM) as a function

of added AsO33-

ions [0-20 µM] in DMSO-water (1 : 9,

v/v) HEPES buffer (1 mM) at 25 °C [λex = 438 nm].

To validate this method the detection limit (LOD) was ob-

tained by drawing the calibration curve (Figure S-9 in the sup-

porting information) in the lower region. The detection limit

was estimated to be 54.91x10-9

M i.e. 4.12 ppb using the equa-

tion 3/S (S = slope of the calibration curve and is the zero

level standard deviation.34

This method is significantly unique

one as the LOD of HL for AsO33-

ions (4.12 ppb) is remarka-

bly lower than that of MCL.6

1H NMR Studies.

1H NMR titration was carried out in

dmso-d6 (Figure 4) in support of the formation of the HL-

AsO33−

species in solution state. This study clearly indicates

that the signal of the phenolic O-H proton shifted to upfield by

0.2856 ppm probably because of the interaction of phenolic O-

H proton with AsO33−

ions.

Figure 4. 1H NMR titration of HL upon gradual addition of

AsO33- ions in dmso-d6 (A) HL; (B) HL:AsO3

3- (1:0.5) and

(C) HL:AsO33- (1:1).

The average fluorescence life time (av) of HL was calcu-

lated to be 4.5250 ns at λem= 532 nm. The av values of 2 (at

λem= 532 nm) was enhanced from 4.7917 ns (HL : AsO3

3- = 1 :

0.5) to 4.9163 ns (HL : AsO3

3- = 1 : 1) on incremental addition

of AsO33−

ions to the HL solution (Figure 5). This observation

is in favour of the CHEF process through intermolecular hy-

drogen bonding.

Figure 5. Time resolved fluorescence decay of HL (10 μM) in the

absence and presence of added AsO33- ions (λem = 532nm).

The strong binding affinity of AsO33-

ion with HL ex-

tremely affects the PET process, which facilitates to enhance

the fluorescence intensity in favour of the selective detection

of AsO33−

ions (Scheme 2). The radiative rate constant kr and

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nonradiative rate constant knr of HL and HL-AsO33-

ensemble

(2) were calculated using the equations:16

-1

= kr + knr and kr

= f/ (viz. Table S-3 in the supporting information). This

table of the data clearly indicates that the fluorescent en-

hancement is principally attributed to the more than 5 times

increase of kr due to the binding of AsO33-

ion with HL, which

is substantiated by the large binding constant value (as men-

tioned above). Upon selective binding of AsO33-

ion, the elec-

tron lone pair on the phenolic-O atom of HL is no longer ac-

cessible for PET process, making possible to the fluorescence

enhancement.

Scheme 2 Plausible mechanism of fluorescence enhancement

of HL in presence of AsO33-

ions

Density Functional Theoretical (DFT) Studies. For under-

standing of the sort of interaction of HL with AsO33-

ion DFT

calculations were performed to attain the optimised geometry

of HL and HL-AsO33-

ensemble (2) (Figure 6). From this

study it is clear that the energy gap between HOMO-LUMO in

2 is 0.63 eV is reasonably lower than that of free HL (3.13

eV) giving rise a more stable species produced by binding the

guest (AsO33-

ion) with the host (HL) through H-bonding.

Figure 6. Geometry optimisation and theoretical calculation of

HL and 2

Cell Imaging Studies. The chemosensor (HL) was exam-

ined by applying HL to human cervical cancer HeLa cell to

explore the utility in biological system. Here, the cells of in-

terest were permitted to feed both HL and AsO33−

and the

images of the cells were acquired using the fluorescence mi-

croscopy at ex 438 nm. The distribution of the probe within

the cells with a very faint fluorescence was observed after

incubation with HL (10 μM) for 30 min. However, the cells

displayed intense fluorescence after the introduction of exoge-

nous AsO33−

into the cell via incubation with Na3AsO3 (details

in Figure 7). These cellular images obtained by adding various

concentrations of added AsO33−

ions are the interesting signa-

ture of the fluorescence response ability of the probe.

Moreover, the in vitro study demonstrated that 10 μM of

HL showed almost full cell viability in support of non-

cytotoxic effect to cell upto 6 h (Figure S-10 in the supporting

information).35

These results clearly indicate that the probe is

an excellent bio-friendly candidate for both in vivo and in vitro

purpose as AsO33−

ions sensor and imaging in both ways for

living cell staining.

Figure 7. Fluorescence images and phase contrast image of HeLa

cells after incubation for 30 min. with HL only (1, 1) and HL

plus arsenite ions (2, 2) 3 μM; (3, 3) 5 μM; (4, 4) 7 µM and

(5, 5’) 10 µM at 37 °C

In addition of the above fact, it is also noteworthy to men-

tion that the developed method was also applicable to estimate

the trace level arsenite ions in different drinking water samples

and the water of the arsenic affected zone of Purbasthali,

Burdwan, West Bengal, India36

using standard addition tech-

nique (Table S-4 in the supporting information). The result

presented in Table S4 had revealed the accuracy of the devel-

oped method in case of drinking water and the determination

of the amount of the arsenite ions in the contaminated water.

In another experiment these arsenic-contaminated water sam-

ples were treated with Fenton's reagent for 12 h maintaining

the appropriate condition37-39

to oxidise the arsenite ions to

arsenate ions and then the probe (HL) was added to these

treated water samples taking all precautions. Here, it was no-

ticed that the emission intensity was remarkably decreased

(viz. Figure S-11 in the supporting information). The results

indicated that the fluorescence due to the arsenite ions was

almost diminished because of the oxidation of arsenite ions to

the arsenate ions through Fenton's reactions. These observa-

tions clearly reveal that the developed method is highly specif-

ic for arsenite ions and useful for selectively quantitative de-

termination of arsenite ions in presence of arsenate ions in

water samples.

CONCLUSION

In conclusion, herein, a new easy to make simple Schiff

base duly structurally characterized has been successfully es-

tablished as a first report of a novel chemosensor for the most

toxic AsO33-

ions upto as low as 4.1 ppb in aqueous media at

biological pH through the intermolecular H-bonding induced

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CHEF process based on thorough experimental and theoretical

studies. This non-cytotoxic probe is a biomarker to detect the

intracellular AsO33-

ions concentrations under a fluorescence

microscope and is efficient to determine the amount of AsO33-

ions in various water samples.

AUTHOR INFORMATION

Corresponding Author

*E-mail: pabitracc@yahoo.com. Tel: +91-342-2558554 extn.

424. Fax: +91-342-2530452.

Present Addresses † Department of Chemistry, The University of Burdwan, Golap-

bag, Burdwan-713104, West Bengal, India ‡ Department of Inorganic and Physical Chemistry, Indian Insti-

tute of Science, Bangalore, 560012, India

ACKNOWLEDGMENT

The authors sincerely acknowledge Council of Scientific and

Industrial Research (CSIR), New Delhi, India for financial assis-

tance. S. Lohar wishes to thank to UGC, New Delhi, India for

offering him the fellowship. The authors are indebted to USIC,

The University of Burdwan for the single crystal X-ray diffrac-

tometer facility under PURSE program. The authors thank to Prof.

Samita Basu and Mr. Ajay Das, Chemical Science Division,

SINP, Kolkata for collecting data using TCSPC instrument.

Supporting Information. The material having experimental sec-

tion including characterization and spectral data, schemes, spec-

tra, figures, tables, crystallographic data for CCDC No.1008294,

is available free of charge via the Internet at http://pubs.acs.org.

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