A Lysosome-Targetable Fluorescence Sensor for Ultrasensitive

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A Lysosome-Targetable Fluorescence Sensor for Ultrasensitive Detection of Hg2+ in Living Cells and Real Samples Arnab Sarkar, Sujaya Chakraborty, Somenath Lohar, Ejaj Ahmmed, Nimai Chandra Saha, Sushil Kumar Mandal, Koushik Dhara, and Pabitra Chattopadhyay Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00005 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Chemical Research in Toxicology

A Lysosome-Targetable Fluorescence Sensor for Ultrasensitive Detection of Hg2+ in Living Cells and Real Samples Arnab Sarkar,a Sujaya Chakraborty,a Somenath Lohar,a Ejaj Ahmmed,a Nimai Chandra Saha,b Sushil Kumar Mandal,c Koushik Dhara*d and Pabitra Chattopadhyay*a a Department

of Chemistry, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India.

b The c

University of Burdwan, Rajbati, Burdwan 713104, West Bengal, India.

Department of Ecological Studies & International Center for Ecological Engineering (ICEE), University of Kalyani, Kalyani, Nadia, West Bengal, India.

d Department

of Chemistry, Sambhu Nath College, Labpur, Birbhum 731303, West Bengal, India.

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Graphical Abstract:


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ABSTRACT A new lysosome-targetable fluorescence sensor, Lyso-HGP, was designed and synthesised based on 4-methyl-2,6-diformylphenol as fluorophore. Lyso-HGP displays highly sensitive fluorescent detection of Hg2+ in HEPES buffer solution (10 mM, DMSO 1%) of pH 7.0 at 37 oC

due to the formation of highly fluorescent formyl-functionalized derivative, Lyso-HGP-

CHO. The sensor triggered a “turn-on” fluorescence response to Hg2+ with a simultaneous increase of fluorescence intensity by 180-fold just after 10 min. The response is very selective over a variety of biologically relevant cations, anions, molecules, and competitive toxic heavy metal cations. The limit of detection (LOD) was calculated as low as 6.82 nM. So, it can be utilized to detect this toxic heavy metal in biology and environmental samples in aqueous buffer medium. Also, the sensor is able to monitor the subcellular distribution of Hg2+ specifically localized in lysosomes compartment in MCF7 human breast cancer cell line by fluorescence microscopy.

Keywords: Fluorescence sensor, lysosome-target, Hg2+ ion, real samples.



INTRODUCTION The designing and development of chemical probe for the detection of heavy metals has paying more attention because of their bioaccumulation, biosorption, and biomagnifications in the environment.1-3 It is well known that ions play an important role in life; especially heavy metal ions possess an essential threat to human health and the environment due to their high toxicity level.4-7 Among these heavy-metal ion like mercury (Hg) is most toxic to the most living organisms.8-12 Intake of any form of mercury is highly toxic and effectively perturbs brain, kidney, immune systems as well as nervous system at very low concentration.8-12 Besides these, the major sources of mercury toxicity are contaminated through drinkable water by inorganic mercury in the form of labile Hg2+ ions because of its effectively significant solubility in water. To protect human beings from the long-term chronic exposure to mercury toxicity, WHO has set the maximum allowable levels of total mercury in drinking water at 0.001 mg/L.13 These toxic nature of Hg2+ have gathered unparalleled attention, deepening the research of mercury in biology. Various biological and chemical aspects of Hg2+ remain undisclosed owing to having a lack of selective method for direct tracking of this ion in cellular compartment. Thus the development of the selective method for direct tracking in a targetable manner of this Hg2+ in living systems is of great scientific interest. Various traditional approaches e.g. inductively coupled plasma mass spectrometry, atomic absorption-emission spectrometry, electrochemical sensors etc. are well known to determine mercury.14-16 However, these techniques are not suitable for in vitro analysis and on-site determination of Hg2+ ions because it involves highly concentrated sample size and also sophisticated costly instrumentation. In contrast, detection by fluorescence techniques have received a lot of interest due to its simplicity, convenience, high sensitivity, and real-time non-destructive detection approach.17-21 A variety of sensor systems have been developed for detection and monitoring of Hg2+ based on chromophores and fluorophores,22-32 DNAzymes,33-35 nanosystems,36-39 oligonucleotides40 etc. Also some functionalized mesoporous materials have been developed for selective detection of Hg2+ in aqueous medium.41,42 Owing to have its high surface area and pore volume, they can act as a good platform for sensing. Moreover, the most of the methods suffer from low detection limit, poor water solubility, fast response, selectivity etc. So, there is a strong need to develop simply prepared of fast responsive selective fluorescence sensors for Hg2+ that can work in an aqueous medium. To the best of our knowledge so far, to date, there has been no description of any targetable fluorescence sensor in literature that can selectively detect lysosomal Hg2+ in living cells.


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For the first time in this work, we have designed and developed a simple and effective lysosome-targetable fluorescence sensor (Lyso-HGP) derived from the 4-methyl-2,6diformylphenol fluorophore. Here, one of the formyl group has been derivatized and/or protected by 1,2-ethandithiol as a Hg2+ reactive zone whereas the other formyl group has been tagged with morpholine fragment to behave as the lysosome-targetable moiety. Lyso-HGP shows “turn-on” selective fluorescent detection of Hg2+ ions in HEPES buffer solution (10 mM, DMSO 1%) of pH 7.0 at 37 oC through the transformation of 1,3-dithiolane into the highly fluorescent formyl-functionalized derivative, Lyso-HGP-CHO. Interestingly the sensor is also capable of detecting lysosomal Hg2+ in vitro. This research work represents a unique chemical tool that offers a selective “turn-on” response to Hg2+ over a variety of biologically relevant cations, anions, and molecules. Also, the sensor is able to monitor the changes in subcellular distribution of Hg2+ by fluorescence microscopy, and the fluorescence signals specifically localize in lysosomes compartment. Moreover it can be utilized to detect this toxic heavy metal in biology in an aqueous buffer medium as well as in environmental samples. This synthesised compound is capable of detecting trace level of Hg2+ as low as 6.82 nM (1.37 µg/L), lower than 2 µg/L which is the recommended Maximum Contaminant Level (MCL) for inorganic mercury in drinking water according to the California Department of Public Health (CDPH) as well as the United States Environmental Protection Agency (US EPA). EXPERIMENTAL SECTION Materials and Methods. All chemicals and reagents and spectroscopic grade solvents were purchased from Sigma. A HITACHI F-4600 fluorescence spectrophotometer with an attachment of a temperature controlling unit was used to obtain the fluorescence spectra in all experiments. A Qtof Micro YA263 mass spectrometer and a Bruker Advance DPX 400 were taken for recording the electron spray ionization (ESI) mass spectra and the NMR spectra respectively. FTIR measurement was done using PerkinElmer L120-00A spectrometer using KBr pellet. General Method of Spectral Studies. The stock solutions for absorption and fluorometric studies, Lyso-HGP was dissolved in dimethyl sulphoxide solvent, from which the required final concentration (10 μM) was prepared in 10 mM, pH 7.0 HEPES buffer (1 % DMSO). For all spectral measurements, the temperature of the reaction medium was maintained at 37 °C. The excitation wave length (ex) was chosen at 440 nm to obtain the



fluorescence spectra in all cases. The following equation was exercised to calculate the relative fluorescence quantum yields (ΦF) by integrating the area under the fluorescence curves:43

where, the area under the fluorescence spectral curve and the refractive index of the solvents are A and η respectively, and the optical density of the compound at the excitation wavelength is OD. Here, fluorescein (Ф = 0.79 in ethanol, λex = 425 nm) was employed as the standard for the measurement of fluorescence quantum yield.44 Cell Culture. Human breast adenocarcinoma cells (MCF7) were cultured in DMEM medium supplemented with penicillin−streptomycin antibiotic solution (1%) and FBS (fetal bovine serum, 10%) in a humidified CO2 incubator (5% CO2). The trypsinethylenediaminetetraacetic acid) solution was used to detach the cells from the culture flask and then the cells were washed in the growth medium. Followed by several times of washing, the cells were seeded on 14 mm glass coverslips into a six-well microtiter plate for the purpose of cellular imaging and permitted 24 h for complete adherence. Fluorescence Imaging Study. MCF7 cells already grown on glass coverslips were incubated with various compounds in PBS (1% DMSO) of pH 7.0 at 37 °C for 30 min. After that, cells were washed several times with PBS and were again incubated with 4, 8, and 10 μM of Hg2+ and 50 nM LysoTracker Blue for 10 min at 37 °C. After every incubation, the cells were thoroughly washed three times with PBS. Bright-field and fluorescence images of MCF7 cells were recorded by a Inverted Fluorescence Microscope, DMi8 (fully motorized widefield epifluorescence) attached with DFC7000T Peltier cooled CCD camera and LAS X Expert software associated with HCX PL FLUOTAR 40X objective with 0.6NA. Leica TCS SP8, a confocal laser scanning fluorescence microscope. Synthesis of Compound 1. To a solution of 2,6-diformyl-4-methylphenol (0.820 g, 5.0 mmol) in 20 mL CHCl3, 1,2-ethanedithiol (0.188 g, 2.0 mmol) was added drop wise under vigorous stirring at 0 °C over a period of 1 h. Then catalytic amount of iodine (0.4 mmol) was added and the resulting mixture was further stirred for 1 h at room temperature. After completion of the reaction (TLC, ethyl acetate/petroleum ether, 5/95), aqueous solution of Na2S2O3 (0.1 M, 25 mL) and NaOH (10%, 25 mL) were added to the reaction mixture. The organic layer was thus obtained and washed twice with H2O. Under reduced pressure the solvent was removed. Purification of the crude solid product so obtained was done onto silica gel column chromatography using the mixed solution of ethyl acetate and petroleum ether (5/95, v/v) as eluents to afford compound 1 as an light yellow solid in a good yield (calculated 6 ACS Paragon Plus Environment

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yield 66%). 1H NMR (400 MHz, CDCl3): δ 2.35 (s, 3H), 3.38-3.45 (m, 4H), 3.60−3.62 (t, 4H), 6.06 (s, 1H), 7.16 (s, 1H), 7.80 (s, 1H), 9.84 (s, 1H), and 11.29 (s, 1H). 13C (100 MHz, CDCl3): δ 20.6, 39.8, 47.9, 120.2, 129.2, 129.3, 133.3, 136.8, 156.8, and 196.6. HRMS (m/z) found 241.0344, calculated for [M+H]+ 241.0357, and found 273.0549, calculated for [M+MeOH+H]+ 273.0619 where M was C11H12O2S2, compound 1. Synthesis of Lyso-HGP. A 30 mL methanolic solution of 4-(2-aminoethyl)morpholine (0.240 g, 2.0 mmol) was added dropwise to a 30 mL methanolic solution of compound 1 (0.481 g, 2.0 mmol) Then the solution mixture was stirred for 30 min and allowed to reflux for another 2 h. The crude product was obtained by evaporating the solvent overnight at room temperature. The crude product was then recrystallized from MeOH/hexane (90/10, v/v) medium to afford pure crystalline compound, Lyso-HGP and finally dried in air (calculated yield 81%). Melting point 80 0C. 1H NMR (400 MHz, CDCl3): δ 2.29 (s, 3H), 2.50-2.53 (t, 4H), 2.67-2.69 (t, 2H), 3.36−3.43 (m, 4H), 3.69-3.72 (m, 6H), 6.11 (s, 1H), 6.95 (s, 1H), 7.57 (s, 1H), and 8.28 (s, 1H). 13C

(100 MHz, CDCl3): δ 20.6, 39.6, 48.8, 53.9, 56.5, 59.1, 67.0, 118.1, 127.4, 128.1, 131.1,

131.8, 156.9, and 165.6. HRMS (m/z) found 353.1359, calculated for [M+H]+ 353.1357 where M was C17H24N2O2S2, Lyso-HGP. RESULTS AND DISCUSSION Sensor Design and Synthesis. Synthesis of the targeted compound was performed following two steps (viz. Scheme 1). First of all, one of the formyl group was derivatized through the protecting by 1,2-ethanedithiol in CHCl3 using catalytic amount of iodine. After completion of the reaction, the reaction was quenched by the successive addition of an aqueous solution of Na2S2O3 and NaOH, respectively. Then the separated organic layer was evaporated off to get the crude product which was then chromatographed through a silica gel column using ethyl acetate and hexane (5/ 95, v/v) to have the pure light-yellow solid compound 1. In the second step, compound 1 was tagged through imine bond formation with 4-(2aminoethyl)morpholine in MeOH medium. The solvent was evaporated to give crude product which was again recrystallized from MeOH/hexane (90/10, v/v) medium to afford pure crystalline compound, Lyso-HGP. Characterizations of all the products were carried out with the help of physicochemical and spectroscopic tools (Supporting information Figures S1-S7). FT-IR spectrum of Lyso-HGP was carried out and presented as Figure S7 in the Supporting Information. The bands observed at 3439(b), 1636(s) and 1290(s)/1259(s) could be described due to the presence of phenolic O-H, C=N and C-N stretching frequencies respectively. Ring 7 ACS Paragon Plus Environment


stretching vibration of C=C and -CH2 groups were found at 1449(s) and 2847(m) / 2805(m) cm-1. The bands found at 1118(s), 1013(s) and 761-613(w) cm-1 could be attributed to the presence of C-O stretching, O-H bending and C-S stretching respectively. 1H NMR of LysoHGP also indicates the presence of C=N-H signal at high δ value i.e. 8.28(s), which further corroborates with the high δ value at 165.6 obtained from 13C NMR study.

Scheme 1. Synthetic Route to obtain Lyso-HGP Fluorescence Detection and Selectivity Study. The fluorescence spectrum of LysoHGP (10 μM) exhibits very weak fluorescence in aqueous medium at ex of 440 nm. The nonemissive (ΦF = 1.74 x 10-2) nature of the sensor is probably due to the presence of dithiolane moiety generated by the protection of formyl group of the fluorophore. Upon the addition of 10 μM Hg2+ into the solution, the fluorescence intensity of Lyso-HGP was dramatically enhanced 180-times with a quantum yield, ΦF = 19.24 x 10-2, and reaches its maximum value just after 10 mints incubation of the mixture at 37 oC. We have studied dose dependent “turnon” responses of Hg2+ with the concomitant addition of various concentration of Hg2+-salt (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM) after the incubation for 10 mints at 37 oC in the reaction buffer (Figure 1).

Figure 1. Fluorescence spectrum of Lyso-HGP (10 μM) upon treatment with Hg2+ (1-10 μM) incubated in reaction buffer pH 7.0 at 37 °C (ex: 440 nm).


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The fluorescence data was measured every after 2 min for 10 min for each and every concentration of Hg2+ (Figure 2). Also we have calculated the quantum yields of Lyso-HGP during the addition of Hg2+-salts (1 μM – 10 μM) of various concentrations. It was reflected from the enhanced quantum yield by more than 11-times when 10 μM of Hg2+ was used in the reaction buffer at 37 oC (Supporting information Figure S8).

Figure 2. Turn-on response of Lyso-HGP (10 μM) to Hg2+ ions in reaction buffer of pH 7.0 at 37 °C (ex: 440 nm) to various levels of Hg2+ ions concentrations plotted with the progress of time (ex: 440 nm). The selectivity of Lyso-HGP (10 μM) for Hg2+ detection towards various biologically relevant and/or competitive species and other heavy metal ions that may hinder was verified by fluorescence measurement. The result showed that fluorescence intensity was enhanced only when Hg2+-salt (10 μM) is added to the reaction buffer, and interestingly no significant changes were obtained when 100 equivalent excess of other cations, anions, and molecular species were introduced (e.g. K+, Ca2+, Mg2+, Mn2+, Fe3+, Fe2+, Cu2+, Zn2+, urea, creatinine, cell lysate, Pb2+, Cd2+, AsO43-, and AsO33-) (Figure 3). Thus, the selectivity study displays that Lyso-HGP should have possible applications to detect Hg2+ ion over a variety of biologically significant and/or competitive toxic heavy metal cations. The LOD (limit of detection) was calculated to be 6.82 nM from the calibration curve (Figure 4) using the 3σ method.45,46 A variable pH dependency study of fluorescence responses in the reaction buffer incubated (10 min) at 37 oC in presence of 10 μM Hg2+ showed that the maximum intensity was observed in the pH range of 7.0-8.0 (Supporting information Figure S9).



Figure 3. Selectivity profile of Lyso-HGP (10 μM) towards different analytes in the reaction buffer incubated at 37 °C (ex: 440 nm).

Figure 4. Plot for the determination of limit of detection (LOD) for Hg2+ ions, LOD was calculated to be 6.82 nM (at ex: 440 nm). UV-Visible Study. The absorption spectrophotometric experiment in reaction buffer at 37 oC was carried out to establish the mode of interaction of Lyso-HGP towards Hg2+. The absorption spectrum exhibited maxima centred at around 240 and 350 nm (ε = 4546 M-1cm-1) with a shoulder at ~410 nm (ε = 1778 M-1cm-1) and that is attributed to the protection of aldehyde group. Upon addition of various concentration of Hg2+, the absorption band at 240 nm gradually increases with a concomitant decrease of the absorbance at ~350 nm. Figure 5 showed the spectrum of UV-vis titration of Lyso-HGP (10 μM) with the addition of 2, 4, 6, 8, and 10 μM Hg2+ ions incubated up to 10 min at 37 oC. Moreover, the shoulder at ~410 nm was red shifted to form a new absorption bands appear at ca. 435 nm corresponding to the n→π* transition of the fluorophore generated by the deprotection of the dithiolane moiety. 10 ACS Paragon Plus Environment

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Figure 5. UV-Vis absorption titration of Lyso-HGP with various concentration of Hg2+ ions in the reaction buffer of pH 7.0 incubated at 37 °C. Proposed Mechanism for Sensing. The proposed mechanistic route for Hg2+ detection is represented in Scheme 2. The enhanced fluorescence of Lyso-HGP can be achieved up on addition of Hg2+-salts through the cleavage of thioacetal linkage47,48 of 1,3-dithiolane moiety generating the fluorophore 2-formyl-4-methyl-((2-morpholinoethyl)imino)phenol (Lyso-HGPCHO). On the basis of the fluorescence and absorption spectrum, the reaction completion time is just 10 min. To identify the proposed final product after the interaction of Hg2+, we performed the HRMS experiment and we found the peak at m/z 307.1569 in ESI (-)ve mode resembles with the formation of Lyso-HGP-CHO (calcd. m/z, 307.1658 for [Lyso-HGP-CHO+MeOHH]-, Supporting information Figure S10). 1H NMR titration of the sensor in presence of 0.5 and 1.0 equivalent Hg2+ was also carried out (Supporting Information Figure S11) to establish the possible mechanistic route. It is clearly evident from the figure that the proton signal of -CHO group was found at high δ value at 9.97 with gradual addition of Hg2+ salt.

Scheme 2. Possible mechanistic route of Hg2+ detection using Lyso-HGP through the cleavage of dithiolane moiety to formyl group.



Imaging Study. We further performed the effectiveness of Lyso-HGP for Hg2+ detection in vitro with live cells sample (MCF7) and also targetable localization in the lysosome compartment. In the control experiments, cells treated with only 10 μM of this sensor (Figure 6a) in PBS (pH 7.0, DMSO 1%) at 37 °C exhibit almost no fluorescence. For the imaging experiment, cells were incubated with 10 μM Lyso-HGP for 30 min in PBS (pH 7.0, DMSO 1%) at 37 °C and then with Hg2+ (4, 8, and 10 μM) for another 10 min in PBS under same experimental condition of the control reaction. After washed several times with PBS, cells exhibited strong contrasting fluorescence images (Figsure 6b, 6c and 6d). Moreover, the subcellular distribution of Lyso-HGP in MCF7 cells was studied. The cells were co-incubated with the sensor along with the addition of 10 μM Hg2+, and LysoTracker Blue (40 nM) in PBS (pH 7.0, DMSO 1%) at 37 °C. The blue fluorescence image in the blue channel due to the staining of the lysosomes by LysoTracker Blue was obtained (Figure 7c). And also the Figure 7b showed the fluorescence image in the green channel of sensor signal manifested due to the sensing of Hg2+. At last, the merged image (Figure 7d) implied that the green fluorescence due to Hg2+ sensing overlaps very well with the blue fluorescence coming from the LysoTracker Blue. These results establish that the newly designed fluorescence sensor, Lyso-HGP could display a brilliant lysosome-targetable property and detect Hg2+ in the lysosomes subcellular compartment. In addition to that, 10 μM of Lyso-HGP did not show any significant cytotoxic effect (Supporting information Figure S12) on the MCF7 human breast cancer cell line for at least up to 4 h of its treatment. However, significant cytotoxicity was noticed for higher doses after 4 h of incubation. These results demonstrated that the fluorescence sensor, Lyso-HGP, is a suitable and efficient candidate for monitoring changes in intracellular distribution of Hg2+.


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Figure 6. Fluorescence microscopic images of MCF7 cells for Hg2+ detection using Lyso-HGP in PBS (pH 7.0, DMSO 1%) at 37 oC. Experiment conditions: (a) 10 μM Lyso-HGP, (b) 10 μM Lyso-HGP & 4 μM Hg2+, (c) 10 μM Lyso-HGP & 8 μM Hg2+, (d) 10 μM Lyso-HGP & 10 μM Hg2+, and (e) 40 nM LysoTracker Blue. The bright-field, fluorescence, and merged images was placed in the first, second and third column respectively. Scale bar: 15 μm.



Figure 7. MCF7 cells were co-incubated with 10 μM Lyso-HGP, 10 μM Hg2+, and 40 nm LysoTracker Blue in PBS (pH 7.0, DMSO 1%) at 37 oC. (a) Bright-field image, (b) fluorescence image of the green channel due to Hg2+ sensing, (c) fluorescence image of the blue channel due to the known LysoTracker Blue, and (d) the merged image of a, b, and c. Scale bar: 15 μm. Real Sample Analysis. In addition of the above fact, it is also noteworthy to mention that the developed method was also applicable to estimate the trace level Hg2+ ions in different drinking water samples, agricultural and industrial water samples using standard addition technique (Table S1 in the supporting information). As the Tamla nalah which flows through the industrial belt of Durgapur (West Bengal, India), it carries the effluents from various industries, so the waste water samples were collected from this nalah,49 and also from Dow agrosciences Pirojsha nagar, LBS, Marg, Vikhroli, Mumbai. The result presented in Table S1 had revealed the accuracy of the developed method in case of drinking water and the determination of the amount of the Hg2+ ions in the contaminated water. These observations clearly reveal that the developed method is highly specific for Hg2+ ions and useful for selectively quantitative determination of Hg2+ ions in natural water samples in presence of other ions. CONCLUSIONS In brief, we designed and synthesized a lysosome targetable fluorescence sensor, Lyso-HGP, based on 4-methyl-2,6-diformylphenol fluorophore, for the first time, for Hg2+ ion as a lysosome-targetable fluorogenic chemosensor. One of the formyl group has been derivatized by 1,2-ethandithiol as a Hg2+ reactive zone whereas the other formyl group has been tagged 14 ACS Paragon Plus Environment

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with morpholine unit, a lysosome-targetable moiety. Lyso-HGP displays “turn-on” selective fluorescent detection of Hg2+ in HEPES buffer solution (10 mM, DMSO 1%) of pH 7.0 at 37 oC

due to the formation of highly fluorescent formyl-functionalized derivative, Lyso-HGP-

CHO. The sensor triggered a fluorescence response to Hg2+ ions with a simultaneous 180-fold increase of fluorescence intensity just after 10 min. The limit of detection (LOD) was calculated as low as 6.82 nM. The response is selective over a variety of biologically relevant cations, anions, molecules, and competitive toxic heavy metal cations. So, it can be utilized to detect this toxic heavy metal ions in biological and environmental samples in an aqueous pH medium. Also, this sensor is able to monitor the changes in subcellular distribution of Hg2+ ions by fluorescence microscopy using MCF7 human breast cancer cell line. Especially and specifically the observed fluorescence signals were localized in lysosomes compartment. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxx. 1H

NMR, 13C NMR spectrum, and HRMS of compound 1 and Lyso-HGP; HRMS of Lyso-

HGP-CHO, relative quantum yield plot, pH dependency study, cell viability (Supporting information Figures S1-S12, Table S1). AUTHOR INFORMATION Corresponding Authors Email: [email protected] (P. Chattopadhyay); [email protected] (K. Dhara) Funding This work was supported by Department of Higher Education, Science & Technology and Biotechnology (Science & Technology Branch), Govt. of West Bengal (vide project no. 698 (Sanc.)/ST/P/S & T/15-G/2015), and Department of Science and Technology (DST), Fast track research grand, New Delhi (vide project no. SB/FT/CS-142/2012). Notes The authors declare no competing financial interest.



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