Author’s Accepted Manuscript A novel FRET ‘off-on’ fluorescent probe for the selective detection of Fe3+, Al 3+ and Cr3+ ions: Its ultrafast energy transfer kinetics and application in live cell imaging Narendra Reddy Chereddy, Peethani Nagaraju, M.V. Niladri Raju, Venkat Raghavan Krishnaswamy, Purna Sai Korrapati, Prakriti Ranjan Bangal, Vaidya Jayathirtha Rao
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S0956-5663(15)00092-5 http://dx.doi.org/10.1016/j.bios.2015.01.074 BIOS7450
To appear in: Biosensors and Bioelectronic Received date: 8 November 2014 Revised date: 29 January 2015 Accepted date: 31 January 2015 Cite this article as: Narendra Reddy Chereddy, Peethani Nagaraju, M.V. Niladri Raju, Venkat Raghavan Krishnaswamy, Purna Sai Korrapati, Prakriti Ranjan Bangal and Vaidya Jayathirtha Rao, A novel FRET ‘off-on’ fluorescent probe for the selective detection of Fe3+, Al 3+ and Cr3+ ions: Its ultrafast energy transfer kinetics and application in live cell imaging, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.074 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel FRET ‘off-on’ fluorescent probe for the selective detection of Fe3+, Al3+ and Cr3+ ions: Its ultrafast energy transfer kinetics and application in live cell imaging Narendra Reddy Chereddy,*a Peethani Nagaraju,a M. V. Niladri Raju,a Venkat Raghavan Krishnaswamy,b Purna Sai Korrapati,b Prakriti Ranjan Bangal*c and Vaidya Jayathirtha Rao*a, d a
Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500
007, India b c
Biomaterials Lab, CSIR-Central Leather Research Institute, Adyar, Chennai-600 020, India
Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad-500 007, India d
AcSIR, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500 007, India
Corresponding author Tel.: +91 44 24913289; Fax: +91 44 24911589.E-Mail: [email protected]
Abstract A rhodamine-naphthalimide dyad probe, 1, that selectively responds to the addition of trivalent metal ions (Fe 3+ or Al3+ or Cr3+) via ultrafast Förster resonance energy transfer (FRET) from naphthalimide to rhodamine is designed and synthesized. 1 is highly selective to the trivalent metal ions and the presence of other monovalent or divalent metal ions do not affect its detection ability. The probe is highly sensitive and it can respond to the presence of trivalent metal ions even at sub-micromolar levels. 1 is stable over a broad range of pH, non-toxic under experimental conditions and suitable to the fluorescence bio-imaging of live cells exposed to trivalent metal ions. The trivalent metal ion induced ultrafast energy transfer kinetics of 1 is explored using time resolved fluorescence experiments.
Keywords Rhodamine-naphthalimide dyad, trivalent metal ion detection, time resolved fluorescence, ratiometric sensors, live cell imaging.
1. Introduction 1
Transition metal ions play a vital role in human life. Particularly, trivalent metal ions like Fe3+ or Al3+ or Cr3+ have their own biological significance and they directly involved in the cell function (Kawano et al., 2003). Iron (III) functions as oxygen carrier in hemoglobin, and plays vital roles in enzyme catalysis and cellular metabolism (Meneghini et al., 1997; Aisen, et al., 1999; Eisenstein et al., 2000; Rouault et al., 2006). The deficiency as well as overload of Fe3+ ion concentration may lead to several disorders (Brugnara et al., 2003; Andrews et al., 1999; Touati et al., 2000). Aluminium is the highest abundant metal in the earth crust. Extensive usage of aluminium utensils for cooking/storage and intake of aluminium based pharmaceuticals lead to increase its concentration in the human body. Excess levels of Al3+ can cause to adverse physiological effects and may result in Alzheimer’s disease, gastrointestinal problems, osteoporosis, anemia, headache, colic, rickets, memory loss and muscle ache (Nayak et al., 2002; Berthon et al., 2002). Apart from Fe3+ and Al3+, Cr3+ also has significant roles on human health. The Cr3+ deficiency may result in diabetes, cardiovascular diseases and excess levels of Cr3+ can adversely affect on cellular structures (Singh et al., 2007; Vincent et al., 2004). Development of new fluorescent probes for the detection of p-block and transition metal ions remain an important area of research owing to their fast, facile and highly sensitive detection ability over other conventional techniques. Large numbers of fluorescent probes have been reported in recent years, but majority of them are useful to detect divalent metal ions (Zheng et al., 2013; Chen et al., 2012). Recently, much effort has been devoted to develop fluorescent probes for the detection trivalent metal ions like Fe3+ or Al3+ or Cr3+ (Maity et al., 2012; Kim et al., 2010; Han et al., 2012; Yang et al., 2013; Sahoo et al., 2012; Nandre et al., 2014). Even though adequate number of probes are available for the individual detection of Fe3+ or Al3+ or Cr3+ ions, probes with the ability to detect all the three ions are few in number. In the last couple of years, Barba-Bon et al., 2012; Chen et al., 2013; 2
Goswami et al., 2013; and Venkateswarlu et al., 2014, have reported fluorescent probes for the combined detection of trivalent metal ions (Fe3+, Al3+ and Cr3+) selectively over the other divalent and monovalent metal ions. Two among them permit the ratiometric fluorescence detection of trivalent metal ions (Barba-Bon et al.,2012; Goswami et al., 2013). Ratiometric chemosensors are advantageous over the intensity based (CHEF or PET) sensors because the former allow the measurement of emission intensities at two different wavelengths and thereby eliminates the environmental effects (Lin et al., 2009). In this context, FRET based sensors are more promising over internal charge transfer (ICT) based sensors (Zhang et al., 2008). Moreover, chemosensors based on FRET mechanism have wide applications in biology owing to their superiority over turn-on, turn-off or ICT based sensors (Kurishita et al., 2010; Xia et al., 2014; Lee et al., 2015). Hence, fluorescent probes with FRET based ratiometric detection ability of trivalent metal ions (Fe3+, Al3+ and Cr3+) are commendable. However, to the best of our knowledge, no report that describes the FRET based detection of trivalent metal ions (Fe3+, Al3+ and Cr3+) is available in open literature. Keeping this in mind, in continuation to our previous efforts to develop fluorescent probes for detection of biologically relevant ions (Chereddy et al., 2015; Chereddy et al., 2013; Chereddy et al., 2012; Chereddy et al., 2012; Chereddy et al., 2011), in the present manuscript, we report a rhodamine-naphthalimide conjugate, 1, that permits ratiometric detection of Fe3+, Al3+ and Cr3+ ions present in aqueous samples and live cells utilizing FRET phenomenon. Hence, we believe this is the first ever report pertaining to the ratiometric detection of trivalent metal ions utilizing ultrafast FRET process.
2. Experimental section 2.1General
General experimental procedures are provided in the supporting information. 3
2.2 Synthesis of Rhodamine hydrazide A Rhodamine hydrazide was synthesized according to the reported procedure (Chereddy et al., 2011). 2.3 Synthesis of probe 1 Probe 1 was synthesized in a multi-step procedure. To a solution of SeO 2 (1.66 g, 15 mmol) in 1,4-dixane (20 mL), 2-methyl 8-quinolinol (1.59 g, 10 mmol) in 1,4-dixane (20 mL) was added drop wise under N2 atmosphere and the resulted mixture was stirred for 8h at 70 0C. After the complete consumption of the starting materials monitored using TLC, the reaction mixture was cooled to room temperature, filtered and subjected silica gel (100-200 mesh) column chromatography using 95:5 hexane-ethtyl acetate as eluent to get 0.87 g (50%) of 8-hydroxyquinoline 2-aldehyde (B) in pure form as yellow solid. To a solution of B (0.44 g, 2.5 mmol) in ethanol (20 mL), rhodamine hydrazide, A, (0.9 g, 2.0 mmol) was added and the mixture was refluxed for 6h. After completion of the reaction monitored using TLC, reaction mixture was cooled to room temperature, concentrated and subjected to column chromatography to obtain C (0.92 g, 75%) as white solid. To a solution of 4-bromo-1,8-naphthanoic anhydride (1.00 g, 3.6 mmol) in ethanol maintained at 80 0C, propylamine (1.0 mL) was added dropwise and the resulted mixture was stirred for 1h at 80 0C, cooled to room temperature. The precipitate formed (D) was filtered and used in the next step without further purification. To the solution of D (0.70 g, 2.20 mmol) in DMSO, piperazine (1.00 g, 11.6 mmol) and K 2CO3 (0.42 g, 3.0 mmol) were added and the resulted mixture was heated to120 0C and stirred at this temperature for 3h. After completion, the reaction mixture was extracted with DCM and subjected to column chromatography (silica gel 100-200 mesh) using ethtyl acetate as eluent to get 0.50 g (70%) of E. To 4-piperazinyl-N-propyl-1,8-naphthanamide, E, (0.48 g, 1.5 mmol) in 4
DCM (20 mL) at ice cold condition, bromoacetyl bromide (0.40 g, 2.0 mmol) was added and the mixture was stirred over night at room temperature under N 2 atmosphere. After completion of the reaction, the reaction mixture was washed with water, DCM layer was concentrated and subjected to column chromatography (silica gel 100-200 mesh, 1:9 ethtyl acetate-hexane as eluent) to afford F (0.47 g, 70%) as yellow solid. To the mixture of C (0.61 g, 1.0 mmol) and potassium carbonate (0.28 g, 2.0 mmol) in DMF, F (0.48 g, 1.1 mmol) was added and the mixture was stirred at room temperature over night. After completion, the reaction mixture was partitioned between chlroform and water, chloroform layer was collected. The aqueous layer was washed thrice with chlroform (3 x 10 mL) and the combined organic extract was washed with brine, concentrated and subjected to column chromatography to afford 1 in pure form (0.59 g, overall yield 65%). 1
H NMR (CDCl3, 500 MHz), δ (ppm): 1.00 (t, J = 7.5 Hz, 3H, NCH2CH2CH3), 1.09 (t, J
= 7.0 Hz, 12H, NCH2CH3), 1.69-1.79 (m, 2H, NCH2CH2CH3), 3.15-3.22 (m, 4H, Piperazine CH2), 3.26 (q, J = 7.0 Hz, 8H, NCH2CH3 ), 3.94 (s, 2H, Piperazine CH 2), 4.12 (t, J = 7.5 Hz, 2H, NCH2CH2CH3), 4.24 (s, 2H, Piperazine CH 2), 5.07 (s, 2H, OCH2), 6.19 (dd, J1 = 2.5 Hz, J 2 = 10 Hz, 2H, Xanthene-H), 6.45 (d, J = 2.5 Hz, 2H, XantheneH), 6.49 (d, J = 8.5 Hz, 2H, Ar-H), 7.15-7.19 (m, 2H, Ar-H), 7.24 (t, J = 4.5 Hz, 1H, ArH), 7.41 (d, J = 4.5 Hz, 2H, Ar-H), 7.49-7.57 (m, 2H, Ar-H), 7.72 (t, J =7.5 Hz, 1H, ArH), 7.94-8.04 (m, 3H, Ar-H), 8.43 (dd, J1 = 1.0 Hz, J2 = 8.5 Hz, 1H, Ar-H), 8.51 (d, J = 8.0 Hz, 1H, Ar-H), 8.57 (dd, J1 = 1.0, J2 =7.0 Hz, 1H, Ar-H), 9.38 (s, 1H, Imine-H).
13
C
NMR (CDCl3, 75 MHz), δ (ppm): 11.56, 12.57, 21.42, 41.77, 42.45, 44.31, 45.82, 52.93, 53.36, 66.63, 70.05, 97.94, 106.43, 107.92, 112.27, 115.63, 117.33, 118.78, 121.39, 123.28, 123.56, 124.15, 126.02, 126.21, 127.12, 127.96, 128.66, 1129.55, 129.74, 129.97, 131.08, 132.54, 133.70, 135.99, 139.96, 148.80, 151.00, 153.37, 153.58, 154.35, 155.17, 163.90, 164.42, 164.95, 166.89. ESI HRMS m/z: Calcd. for C59H58N8O6 974.44793; found 5
975.45633 (M+H)+. 2.4 Sample preparation for cell culture The solution of 1 for cell culture experiments was prepared in sterile DMSO with stock solution concentrations of 10 mM. Similarly, stock solutions of Fe3+, Cr3+ and Al3+ ions (10 mM) were prepared in sterile Millipore water. Freshly prepared stock solutions were used for cell culture experiment. 2.5 Cell culture experimentation W138 cells were maintained in DMEM complete media, supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37 0C humidified incubator with 5% CO2. Cells after 70% confluency were seeded into 96 well plates and 24 well plates for cyto-toxicity assays and fluorescence microscopy studies, respectively. 2.6 MTT assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay has been carried out to measure the activity of enzymes that reduce MTT to formazan dyes, giving rise to a purple colour. W138 cells (10,000) were plated in each well of a 96-well tissue culture plate with 100 μL of complete DMEM media at 37 0C humidified incubator with 5% CO2 for 24 h. Next day, the media of the each well was replaced with 100 μL fresh media and the cells were treated with different concentrations of 1 (0.1-25 µM) for another 48 h. MTT stock solution (5 mg/mL) was diluted 10 times with complete DMEM media. The DMEM media of each well was replaced with the diluted MTT solution (100 μL) and cells were allowed to incubate for 4 h. After 4 h of incubation, the media in each well was replaced with 100 μL of 1:1 DMSO-methanol mixture (v/v) to solubilize the purple formazan product. The well plate was kept on a shaker for homogeneous mixture of the solution. Finally, the 6
micro plate reader (ELx 800 MS) has been used to measure the absorbance of solution in each well of the plate at 570 nm. 2.7 Fluorescence microscopy To conduct live cell imaging experiments, W138 cells were seeded at 2x104 cells/mL/well in a 24-well tissue culture plate for 24 h at 37 0C humidified incubator with 5% CO2 in complete DMEM media. After 24 h, the cells were incubated with probe 1 (10 μM) for another 4 h. The cells were thoroughly washed with DPBS for six times to eradicate the unbound probe from the surface of cell membrane. After that, the cells treated with probe 1 were incubated with 10 μM of the respective metal ions (Fe3+ or Al3+ or Cr3+) ions for 1 h. Additionally, cells were also treated with only 1 (10 μM), and only with metal ions (10 μM) and again washed with DPBS for six times to remove the unbound materials. Finally, the fluorescence images of W138 cells treated with probe 1 and 1 and Fe3+, only probe 1 and only Fe3+ were observed under fluorescence microscope (Nikon Eclipse: TE 2000-E, Japan) through excitation of 488 nm and emission of 525 nm for green fluorescence and excitation of 518 nm and emission of 605 nm for red fluorescence. 2.8 Fluorescence Up-Conversion To perform Fluorescence Up conversion study FOG100 system (CDP System) was used. Part of the pulsed (690-1040 nm) Ti:Sapphire (Mai Tai HP, Spectra Physics) fundamental laser output (~500 mW at 800 nm) was steered into CDP2015 frequency conversion unit to have second (SH) and third harmonic (TH) as desired. A beam splitter (BS) is used to split the input beam to excitation (SH/TH pulses) and gate (fundamental residual pulses) beams. The excitation beam directed to a rotating sample cell with the help of six BSs and one mirror. A lens (f = 40 mm) was used to focus excitation beam into the sample. A ND filter was used for the excitation attenuation. The gate beam was directed by 7
two mirrors to gold-coated retro- reflector mirror connected to 8 ns optical delay line before being focused together with the fluorescence (collected by an achromatic doublet, f = 80 mm) on 0.5 mm type I BBO crystal. The angle of the crystal was adjusted to phase matching conditions at the fluorescence wavelength of interest. The up-converted signal (in the UV range) was focused with a lens (f = 60 nm) to an input slit of the monochromator (CDP2022D). The intensity of the up-converted radiation was measured with a photomultiplier tube operating in the photon counting mode. Proper filters were used before the detector to eliminate parasitic light from the up-converted signal if any. The polarization of the excitation pulses was set at magic angle relative to that of the gate pulses using Berek's variable wave plate. The sample solutions (100 µM) were placed in 1 mm rotating cell. The FWHM of the IRF in this setup was calculated about 240 fs in the 0.4 mm cell and 280 fs in the 1 mm cell. Hence, a time resolution of around 200 fs could be achieved. This time resolution is mainly limited by the optics and the duration of the laser pulses. For data analysis, the fluorescence time profile at a given emission wavelength I(λ,t) was reproduced by the convolution of a Gaussian IRF with a sum of exponential trial function representing the pure sample dynamics S(t). Gaussian term was added to account for fast non-exponential processes if any owing to vibrational or other solvent relaxation process. 2.9 TSCPC experiments The femtosecond pulses at required repetition rate were obtained from fractional part of MaiTai output passing through femtosecond Pulse Selector (3980-5S, Spectra Physics, single shot to 8 MHz). The excitation pulses at desired wavelength were generated by frequency doubling with 0.5 mm BBO crystal. This excitation pulses are focused to the sample using our Fluorescence Up-conversion set up (FOG100 system CDP System). The
8
time distribution data of fluorescence intensity were recorded on a SPC-130 TCSPC module (Becker & Hickl).
3. Results and discussion 3.1 Synthesis and spectroscopic characterization of probe 1 The synthesis of dyad 1 was achieved by coupling a FRET acceptor (8hydroxyquinilone attached rhodamine, C) to a FRET donor (piperazine attached naphthalimide, F) as shown in Scheme 1 and its formation was confirmed using NMR, ESIHRMS analytical techniques (Figs. S1-S3, Supporting Information). 3.2 Metal ion selectivity of probe 1 Metal ion sensing properties of 1 were investigated in aqueous acetonitrile (1:1 v/v 0.01 M Tris HCl-CH3CN, pH 7.4) medium using UV-Visible and fluorescence analytical techniques. As shown in Fig. 1a, dyad probe 1 (10 µM) displayed an absorption band centered at ~400 nm, in accordance with the presence of naphthalimide moiety, which is responsible to the pale yellow color of the probe’s solution. The baseline level absorbance of 1 in the rhodamine characteristic wavelength region (540-580 nm) indicated its preferential existence in spirocyclic form, under experimental conditions. Among various metal ions (10 µM) tested, the addition of Fe3+ or Al3+ or Cr3+ ions selectively enhanced the absorbance of 1 at rhodamine region, which might be resulted from the metal ion induced spirolactam ring opened form of 1 (Kim et al., 2008). The addition of Fe3+ ions enhanced the absorption intensity of 1 at 562 nm by 126- fold and changed the color of the probe’s solution from pale yellow to pink. Under similar conditions, the enhancement in the absorbance of 1 at 562 nm to the addition of Al3+ and Cr3+ ions was 18 and 20-fold, respectively (Fig. 1a). Similarly, 1 (10 µM) alone exhibited a single emission band at 532 nm corresponding to the naphthalimide moiety. Among various metal ions (10 µM) added, the addition of Fe3+ or Al3+ 9
or Cr3+ ions to 1 (10 µM) engendered a new emission band centered at 583 nm with a concomitant reduction in the naphthalimide emission at 532 nm (Fig. 1b). These observed changes are ascribed due to the energy transfer (ET) from naphthalimide donor to the pirolactam ring opened rhodamine moiety of 1 that is resulted from the metal ion chelation as shown in Fig. S4, Supporting Information (Chereddy et al., 2013). These observed variations in the absorbance and fluorescence characteristics of 1 indicated its trivalent metal ion selective detection ability. Further, metal ion competition experiments were conducted to assess the effect of the presence of other metal ions on the trivalent metal ion selective detection ability of 1. These experiments were performed by adding Fe3+ ions (10 µM) to 1 in presence of other monovalent and divalent ions (20 µM) and monitoring the variations in the probes emission profiles. The results revealed that 1 could be used to detect Fe3+ even in the presence of other metal ions. Metal ion competition experiments were also performed using Al3+ or Cr3+ ions and the results portrayed the probes selectivity towards trivalent metal (Fe3+ or Al3+ or Cr3+) ions (Fig. 2). 3.3 Sensitivity of 1 towards the trivalent metal ions Since the addition of Fe3+ or Al3+ or Cr3+ ions has significant effect on the absorption and emission characteristics of 1 (Fig. 1), the affect of the addition of incremental concentrations of these metal ions on the probe’s absorption and fluorescence characteristics was investigated. The addition of Fe3+ ions (0-30 µM) to 1 developed a new absorption band centered at 562 nm, its intensity was increased gradually with the amount of Fe3+ added and reached maximum at 20 µM of Fe3+ (Fig. 3a). At this concentration, 1 displayed 224-fold enhancement in its absorbance at 562 nm. Similar affect was observed even with Al3+ or Cr3+ ions (Figs. S5a and S6a, Supporting Information). But, the extent of increment in the absorption intensity at 562 nm was higher for Fe3+ ions compared to Al3+ or Cr3+ ions, 10
indicated the strong binding affinity of 1 with Fe3+ over Al3+ or Cr3+ ions (Fig. S7a, Supporting Information). Binding constants of the respective 1-metal ion complexes were calculated from the absorption data (Fig. S8, Supporting Information) and the values are provided in Table S1, Supporting Information. Under similar conditions, the addition of Fe3+ ions opened the spirolactam ring of the rhodamine moiety of 1 and allowed the Förster resonance energy transfer from naphthalimide to rhodamine (Fig. 3b). The ratio of the emission intensities of 1 at 583 and 532 nm (I583/I532) was linearly increased with the amount of Fe3+ added in the concentration range of 0-8 µM (R2 = 0.996) and reached maximum at 12 µM of Fe3+. Similar kind of results was obtained even with the Al3+ or Cr3+ions (Figs. S5b and S6b, Supporting Information). The detection limit of 1 for Fe3+ or Al3+ or Cr3+ ions were calculated separately from the UV-Visible and fluorescence data (Figs. S9 and S10, Supporting Information) and the values are presented in Table S1, Supporting Information. These values indicated that 1 can detect the trivalent metal ions (Fe3+ or Al3+ or Cr3+) even at sub-micromolar levels. 3.4 Job plot and ESI-MS analysis of 1-trivalent metal ion complexes The stoichiometry of 1-trivalent metal ion complexes was estimated using Job plot analysis and it was found to be 1:1 in nature (Fig. S11, Supporting Information). The 1:1 stoichiometry of these complexes was further confirmed using ESI MS analysis (Fig. S12, Supporting Information). 3.5 Effect of pH and MTT assay Stability over a broad range of pH and non-toxicity are the key criteria to a chemosensor for biological applications. The acid-base titration experiments were performed to understand the effect of pH on the absorption and emission characteristics of 1. The results revealed that either the absorption or the emission pattern of 1 were unaltered in the range of 11
pH 5-10 (Fig. S13, Supporting Information) and suggested its suitability for the detection of Fe3+ or Al3+ or Cr3+ ions over a broad range of pH. Cell viability experiment (MTT assay) was performed to assess the non-cytotoxic nature of 1 (Fig. S14, Supporting Information). The W138 cells were viable (more than 90%) even after 48 hours of their incubation with 1 at 25 µM concentrations. The observed high selectivity and sensitivity for trivalent metal ions and non-interference from other competitive metal ions, stability at physiological pH and non-toxicity under experimental conditions designated the eligibility of 1 for live cell imaging application. 3.6 Detection and imaging of metal ion contaminated live cells We further explored the possibility for the imaging of trivalent metal ion contaminated W138 (normal lung fibroblast) cells by staining with probe 1 (Fig. 4). Two sets of fluorescence images were obtained from green channel and red channel, respectively. The cells alone (a1-c1) and cells treated with Fe3+ (10 µM) alone (a2-c2) did not show significant fluorescence in both green and red channels. Whereas the cells incubated with 1 (10 µM) clearly displayed bright intracellular green fluorescence (a3-c3) indicated that probe 1 is cell membrane permeable. When cells loaded with probe 1 were exposed to exogenous Fe3+ ions (10 µM) in DMEM culture medium for 30 min at 37 0C, an intense red fluorescence was observed with a concomitant decrease in green fluorescence (a4-c4). Similar results were obtained for Al3+ and Cr3+ ions also (Figs. S15-S16, Supporting Information). Thus, the data showed in Fig. 4 and Figs. S15-S16, Supporting Information clearly demonstrated the ability of probe 1 in monitoring trivalent metal ions present in live cells. 3.7 TCSPC and femtosecond fluorescence up-conversion analysis Fluorescent probes with target induced variations in their fluorescence lifetimes are useful to monitor the intracellular metal ion concentrations using Fluorescence Lifetime 12
Imaging Microscopy (FLIM) technique (Daen et al., 2012). Since probe 1 is suitable for the detection of intracellular trivalent metal ions using fluorescence microscopy, the effect of the trivalent metal ions on its fluorescence lifetime characteristics were studied to assess applicability of 1 in fluorescence lifetime imaging microscopic analysis. Time resolved fluorescence studies employing time correlated single photon counting (TCSPC) technique is used to understand the energy transfer dynamics in 1. As shown in Figs. S17-S18, Supporting Information, the time profile of the fluorescence signals at 520 nm, remained unaffected (Fig. S18, Supporting Information) while the fluorescence counts get reduced in commensurate with the addition of trivalent metal ions in a given collection time. Interestingly, change of fluorescence counts as a function of added metal ions at a given time of the decay profile follows the same pattern as observed in steady state fluorescence quenching of naphthalimide emission (Fig. 1b). This result apparently indicates two facts: Firstly, TCSPC measurement is only recording the decay profile of effective residual emission of uncomplexed 1 and rate of reduction of fluorescence upon addition of trivalent metal ions is happening within the IRF time and it is 200 ps for our system, secondly, rhodamine is not a FRET acceptor, fluorescence quenching is resulted from the re-absorption of naphthalimide emission by rhodamine. To apprehend the real fact, we employed femtosecond fluorescence up-conversion technique monitoring the early time dynamics of 1 in absence and in presence of trivalent metal ions. As shown in Fig. 5, the time profile of fluorescence up-conversion signal of the dyad probe 1 alone at 520 nm follows bi-exponential fit with 1 ps (
PII: DOI: Reference:
www.elsevier.com/locate/bios
S0956-5663(15)00092-5 http://dx.doi.org/10.1016/j.bios.2015.01.074 BIOS7450
To appear in: Biosensors and Bioelectronic Received date: 8 November 2014 Revised date: 29 January 2015 Accepted date: 31 January 2015 Cite this article as: Narendra Reddy Chereddy, Peethani Nagaraju, M.V. Niladri Raju, Venkat Raghavan Krishnaswamy, Purna Sai Korrapati, Prakriti Ranjan Bangal and Vaidya Jayathirtha Rao, A novel FRET ‘off-on’ fluorescent probe for the selective detection of Fe3+, Al 3+ and Cr3+ ions: Its ultrafast energy transfer kinetics and application in live cell imaging, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.074 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel FRET ‘off-on’ fluorescent probe for the selective detection of Fe3+, Al3+ and Cr3+ ions: Its ultrafast energy transfer kinetics and application in live cell imaging Narendra Reddy Chereddy,*a Peethani Nagaraju,a M. V. Niladri Raju,a Venkat Raghavan Krishnaswamy,b Purna Sai Korrapati,b Prakriti Ranjan Bangal*c and Vaidya Jayathirtha Rao*a, d a
Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500
007, India b c
Biomaterials Lab, CSIR-Central Leather Research Institute, Adyar, Chennai-600 020, India
Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad-500 007, India d
AcSIR, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500 007, India
Corresponding author Tel.: +91 44 24913289; Fax: +91 44 24911589.E-Mail: [email protected]
Abstract A rhodamine-naphthalimide dyad probe, 1, that selectively responds to the addition of trivalent metal ions (Fe 3+ or Al3+ or Cr3+) via ultrafast Förster resonance energy transfer (FRET) from naphthalimide to rhodamine is designed and synthesized. 1 is highly selective to the trivalent metal ions and the presence of other monovalent or divalent metal ions do not affect its detection ability. The probe is highly sensitive and it can respond to the presence of trivalent metal ions even at sub-micromolar levels. 1 is stable over a broad range of pH, non-toxic under experimental conditions and suitable to the fluorescence bio-imaging of live cells exposed to trivalent metal ions. The trivalent metal ion induced ultrafast energy transfer kinetics of 1 is explored using time resolved fluorescence experiments.
Keywords Rhodamine-naphthalimide dyad, trivalent metal ion detection, time resolved fluorescence, ratiometric sensors, live cell imaging.
1. Introduction 1
Transition metal ions play a vital role in human life. Particularly, trivalent metal ions like Fe3+ or Al3+ or Cr3+ have their own biological significance and they directly involved in the cell function (Kawano et al., 2003). Iron (III) functions as oxygen carrier in hemoglobin, and plays vital roles in enzyme catalysis and cellular metabolism (Meneghini et al., 1997; Aisen, et al., 1999; Eisenstein et al., 2000; Rouault et al., 2006). The deficiency as well as overload of Fe3+ ion concentration may lead to several disorders (Brugnara et al., 2003; Andrews et al., 1999; Touati et al., 2000). Aluminium is the highest abundant metal in the earth crust. Extensive usage of aluminium utensils for cooking/storage and intake of aluminium based pharmaceuticals lead to increase its concentration in the human body. Excess levels of Al3+ can cause to adverse physiological effects and may result in Alzheimer’s disease, gastrointestinal problems, osteoporosis, anemia, headache, colic, rickets, memory loss and muscle ache (Nayak et al., 2002; Berthon et al., 2002). Apart from Fe3+ and Al3+, Cr3+ also has significant roles on human health. The Cr3+ deficiency may result in diabetes, cardiovascular diseases and excess levels of Cr3+ can adversely affect on cellular structures (Singh et al., 2007; Vincent et al., 2004). Development of new fluorescent probes for the detection of p-block and transition metal ions remain an important area of research owing to their fast, facile and highly sensitive detection ability over other conventional techniques. Large numbers of fluorescent probes have been reported in recent years, but majority of them are useful to detect divalent metal ions (Zheng et al., 2013; Chen et al., 2012). Recently, much effort has been devoted to develop fluorescent probes for the detection trivalent metal ions like Fe3+ or Al3+ or Cr3+ (Maity et al., 2012; Kim et al., 2010; Han et al., 2012; Yang et al., 2013; Sahoo et al., 2012; Nandre et al., 2014). Even though adequate number of probes are available for the individual detection of Fe3+ or Al3+ or Cr3+ ions, probes with the ability to detect all the three ions are few in number. In the last couple of years, Barba-Bon et al., 2012; Chen et al., 2013; 2
Goswami et al., 2013; and Venkateswarlu et al., 2014, have reported fluorescent probes for the combined detection of trivalent metal ions (Fe3+, Al3+ and Cr3+) selectively over the other divalent and monovalent metal ions. Two among them permit the ratiometric fluorescence detection of trivalent metal ions (Barba-Bon et al.,2012; Goswami et al., 2013). Ratiometric chemosensors are advantageous over the intensity based (CHEF or PET) sensors because the former allow the measurement of emission intensities at two different wavelengths and thereby eliminates the environmental effects (Lin et al., 2009). In this context, FRET based sensors are more promising over internal charge transfer (ICT) based sensors (Zhang et al., 2008). Moreover, chemosensors based on FRET mechanism have wide applications in biology owing to their superiority over turn-on, turn-off or ICT based sensors (Kurishita et al., 2010; Xia et al., 2014; Lee et al., 2015). Hence, fluorescent probes with FRET based ratiometric detection ability of trivalent metal ions (Fe3+, Al3+ and Cr3+) are commendable. However, to the best of our knowledge, no report that describes the FRET based detection of trivalent metal ions (Fe3+, Al3+ and Cr3+) is available in open literature. Keeping this in mind, in continuation to our previous efforts to develop fluorescent probes for detection of biologically relevant ions (Chereddy et al., 2015; Chereddy et al., 2013; Chereddy et al., 2012; Chereddy et al., 2012; Chereddy et al., 2011), in the present manuscript, we report a rhodamine-naphthalimide conjugate, 1, that permits ratiometric detection of Fe3+, Al3+ and Cr3+ ions present in aqueous samples and live cells utilizing FRET phenomenon. Hence, we believe this is the first ever report pertaining to the ratiometric detection of trivalent metal ions utilizing ultrafast FRET process.
2. Experimental section 2.1General
General experimental procedures are provided in the supporting information. 3
2.2 Synthesis of Rhodamine hydrazide A Rhodamine hydrazide was synthesized according to the reported procedure (Chereddy et al., 2011). 2.3 Synthesis of probe 1 Probe 1 was synthesized in a multi-step procedure. To a solution of SeO 2 (1.66 g, 15 mmol) in 1,4-dixane (20 mL), 2-methyl 8-quinolinol (1.59 g, 10 mmol) in 1,4-dixane (20 mL) was added drop wise under N2 atmosphere and the resulted mixture was stirred for 8h at 70 0C. After the complete consumption of the starting materials monitored using TLC, the reaction mixture was cooled to room temperature, filtered and subjected silica gel (100-200 mesh) column chromatography using 95:5 hexane-ethtyl acetate as eluent to get 0.87 g (50%) of 8-hydroxyquinoline 2-aldehyde (B) in pure form as yellow solid. To a solution of B (0.44 g, 2.5 mmol) in ethanol (20 mL), rhodamine hydrazide, A, (0.9 g, 2.0 mmol) was added and the mixture was refluxed for 6h. After completion of the reaction monitored using TLC, reaction mixture was cooled to room temperature, concentrated and subjected to column chromatography to obtain C (0.92 g, 75%) as white solid. To a solution of 4-bromo-1,8-naphthanoic anhydride (1.00 g, 3.6 mmol) in ethanol maintained at 80 0C, propylamine (1.0 mL) was added dropwise and the resulted mixture was stirred for 1h at 80 0C, cooled to room temperature. The precipitate formed (D) was filtered and used in the next step without further purification. To the solution of D (0.70 g, 2.20 mmol) in DMSO, piperazine (1.00 g, 11.6 mmol) and K 2CO3 (0.42 g, 3.0 mmol) were added and the resulted mixture was heated to120 0C and stirred at this temperature for 3h. After completion, the reaction mixture was extracted with DCM and subjected to column chromatography (silica gel 100-200 mesh) using ethtyl acetate as eluent to get 0.50 g (70%) of E. To 4-piperazinyl-N-propyl-1,8-naphthanamide, E, (0.48 g, 1.5 mmol) in 4
DCM (20 mL) at ice cold condition, bromoacetyl bromide (0.40 g, 2.0 mmol) was added and the mixture was stirred over night at room temperature under N 2 atmosphere. After completion of the reaction, the reaction mixture was washed with water, DCM layer was concentrated and subjected to column chromatography (silica gel 100-200 mesh, 1:9 ethtyl acetate-hexane as eluent) to afford F (0.47 g, 70%) as yellow solid. To the mixture of C (0.61 g, 1.0 mmol) and potassium carbonate (0.28 g, 2.0 mmol) in DMF, F (0.48 g, 1.1 mmol) was added and the mixture was stirred at room temperature over night. After completion, the reaction mixture was partitioned between chlroform and water, chloroform layer was collected. The aqueous layer was washed thrice with chlroform (3 x 10 mL) and the combined organic extract was washed with brine, concentrated and subjected to column chromatography to afford 1 in pure form (0.59 g, overall yield 65%). 1
H NMR (CDCl3, 500 MHz), δ (ppm): 1.00 (t, J = 7.5 Hz, 3H, NCH2CH2CH3), 1.09 (t, J
= 7.0 Hz, 12H, NCH2CH3), 1.69-1.79 (m, 2H, NCH2CH2CH3), 3.15-3.22 (m, 4H, Piperazine CH2), 3.26 (q, J = 7.0 Hz, 8H, NCH2CH3 ), 3.94 (s, 2H, Piperazine CH 2), 4.12 (t, J = 7.5 Hz, 2H, NCH2CH2CH3), 4.24 (s, 2H, Piperazine CH 2), 5.07 (s, 2H, OCH2), 6.19 (dd, J1 = 2.5 Hz, J 2 = 10 Hz, 2H, Xanthene-H), 6.45 (d, J = 2.5 Hz, 2H, XantheneH), 6.49 (d, J = 8.5 Hz, 2H, Ar-H), 7.15-7.19 (m, 2H, Ar-H), 7.24 (t, J = 4.5 Hz, 1H, ArH), 7.41 (d, J = 4.5 Hz, 2H, Ar-H), 7.49-7.57 (m, 2H, Ar-H), 7.72 (t, J =7.5 Hz, 1H, ArH), 7.94-8.04 (m, 3H, Ar-H), 8.43 (dd, J1 = 1.0 Hz, J2 = 8.5 Hz, 1H, Ar-H), 8.51 (d, J = 8.0 Hz, 1H, Ar-H), 8.57 (dd, J1 = 1.0, J2 =7.0 Hz, 1H, Ar-H), 9.38 (s, 1H, Imine-H).
13
C
NMR (CDCl3, 75 MHz), δ (ppm): 11.56, 12.57, 21.42, 41.77, 42.45, 44.31, 45.82, 52.93, 53.36, 66.63, 70.05, 97.94, 106.43, 107.92, 112.27, 115.63, 117.33, 118.78, 121.39, 123.28, 123.56, 124.15, 126.02, 126.21, 127.12, 127.96, 128.66, 1129.55, 129.74, 129.97, 131.08, 132.54, 133.70, 135.99, 139.96, 148.80, 151.00, 153.37, 153.58, 154.35, 155.17, 163.90, 164.42, 164.95, 166.89. ESI HRMS m/z: Calcd. for C59H58N8O6 974.44793; found 5
975.45633 (M+H)+. 2.4 Sample preparation for cell culture The solution of 1 for cell culture experiments was prepared in sterile DMSO with stock solution concentrations of 10 mM. Similarly, stock solutions of Fe3+, Cr3+ and Al3+ ions (10 mM) were prepared in sterile Millipore water. Freshly prepared stock solutions were used for cell culture experiment. 2.5 Cell culture experimentation W138 cells were maintained in DMEM complete media, supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37 0C humidified incubator with 5% CO2. Cells after 70% confluency were seeded into 96 well plates and 24 well plates for cyto-toxicity assays and fluorescence microscopy studies, respectively. 2.6 MTT assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay has been carried out to measure the activity of enzymes that reduce MTT to formazan dyes, giving rise to a purple colour. W138 cells (10,000) were plated in each well of a 96-well tissue culture plate with 100 μL of complete DMEM media at 37 0C humidified incubator with 5% CO2 for 24 h. Next day, the media of the each well was replaced with 100 μL fresh media and the cells were treated with different concentrations of 1 (0.1-25 µM) for another 48 h. MTT stock solution (5 mg/mL) was diluted 10 times with complete DMEM media. The DMEM media of each well was replaced with the diluted MTT solution (100 μL) and cells were allowed to incubate for 4 h. After 4 h of incubation, the media in each well was replaced with 100 μL of 1:1 DMSO-methanol mixture (v/v) to solubilize the purple formazan product. The well plate was kept on a shaker for homogeneous mixture of the solution. Finally, the 6
micro plate reader (ELx 800 MS) has been used to measure the absorbance of solution in each well of the plate at 570 nm. 2.7 Fluorescence microscopy To conduct live cell imaging experiments, W138 cells were seeded at 2x104 cells/mL/well in a 24-well tissue culture plate for 24 h at 37 0C humidified incubator with 5% CO2 in complete DMEM media. After 24 h, the cells were incubated with probe 1 (10 μM) for another 4 h. The cells were thoroughly washed with DPBS for six times to eradicate the unbound probe from the surface of cell membrane. After that, the cells treated with probe 1 were incubated with 10 μM of the respective metal ions (Fe3+ or Al3+ or Cr3+) ions for 1 h. Additionally, cells were also treated with only 1 (10 μM), and only with metal ions (10 μM) and again washed with DPBS for six times to remove the unbound materials. Finally, the fluorescence images of W138 cells treated with probe 1 and 1 and Fe3+, only probe 1 and only Fe3+ were observed under fluorescence microscope (Nikon Eclipse: TE 2000-E, Japan) through excitation of 488 nm and emission of 525 nm for green fluorescence and excitation of 518 nm and emission of 605 nm for red fluorescence. 2.8 Fluorescence Up-Conversion To perform Fluorescence Up conversion study FOG100 system (CDP System) was used. Part of the pulsed (690-1040 nm) Ti:Sapphire (Mai Tai HP, Spectra Physics) fundamental laser output (~500 mW at 800 nm) was steered into CDP2015 frequency conversion unit to have second (SH) and third harmonic (TH) as desired. A beam splitter (BS) is used to split the input beam to excitation (SH/TH pulses) and gate (fundamental residual pulses) beams. The excitation beam directed to a rotating sample cell with the help of six BSs and one mirror. A lens (f = 40 mm) was used to focus excitation beam into the sample. A ND filter was used for the excitation attenuation. The gate beam was directed by 7
two mirrors to gold-coated retro- reflector mirror connected to 8 ns optical delay line before being focused together with the fluorescence (collected by an achromatic doublet, f = 80 mm) on 0.5 mm type I BBO crystal. The angle of the crystal was adjusted to phase matching conditions at the fluorescence wavelength of interest. The up-converted signal (in the UV range) was focused with a lens (f = 60 nm) to an input slit of the monochromator (CDP2022D). The intensity of the up-converted radiation was measured with a photomultiplier tube operating in the photon counting mode. Proper filters were used before the detector to eliminate parasitic light from the up-converted signal if any. The polarization of the excitation pulses was set at magic angle relative to that of the gate pulses using Berek's variable wave plate. The sample solutions (100 µM) were placed in 1 mm rotating cell. The FWHM of the IRF in this setup was calculated about 240 fs in the 0.4 mm cell and 280 fs in the 1 mm cell. Hence, a time resolution of around 200 fs could be achieved. This time resolution is mainly limited by the optics and the duration of the laser pulses. For data analysis, the fluorescence time profile at a given emission wavelength I(λ,t) was reproduced by the convolution of a Gaussian IRF with a sum of exponential trial function representing the pure sample dynamics S(t). Gaussian term was added to account for fast non-exponential processes if any owing to vibrational or other solvent relaxation process. 2.9 TSCPC experiments The femtosecond pulses at required repetition rate were obtained from fractional part of MaiTai output passing through femtosecond Pulse Selector (3980-5S, Spectra Physics, single shot to 8 MHz). The excitation pulses at desired wavelength were generated by frequency doubling with 0.5 mm BBO crystal. This excitation pulses are focused to the sample using our Fluorescence Up-conversion set up (FOG100 system CDP System). The
8
time distribution data of fluorescence intensity were recorded on a SPC-130 TCSPC module (Becker & Hickl).
3. Results and discussion 3.1 Synthesis and spectroscopic characterization of probe 1 The synthesis of dyad 1 was achieved by coupling a FRET acceptor (8hydroxyquinilone attached rhodamine, C) to a FRET donor (piperazine attached naphthalimide, F) as shown in Scheme 1 and its formation was confirmed using NMR, ESIHRMS analytical techniques (Figs. S1-S3, Supporting Information). 3.2 Metal ion selectivity of probe 1 Metal ion sensing properties of 1 were investigated in aqueous acetonitrile (1:1 v/v 0.01 M Tris HCl-CH3CN, pH 7.4) medium using UV-Visible and fluorescence analytical techniques. As shown in Fig. 1a, dyad probe 1 (10 µM) displayed an absorption band centered at ~400 nm, in accordance with the presence of naphthalimide moiety, which is responsible to the pale yellow color of the probe’s solution. The baseline level absorbance of 1 in the rhodamine characteristic wavelength region (540-580 nm) indicated its preferential existence in spirocyclic form, under experimental conditions. Among various metal ions (10 µM) tested, the addition of Fe3+ or Al3+ or Cr3+ ions selectively enhanced the absorbance of 1 at rhodamine region, which might be resulted from the metal ion induced spirolactam ring opened form of 1 (Kim et al., 2008). The addition of Fe3+ ions enhanced the absorption intensity of 1 at 562 nm by 126- fold and changed the color of the probe’s solution from pale yellow to pink. Under similar conditions, the enhancement in the absorbance of 1 at 562 nm to the addition of Al3+ and Cr3+ ions was 18 and 20-fold, respectively (Fig. 1a). Similarly, 1 (10 µM) alone exhibited a single emission band at 532 nm corresponding to the naphthalimide moiety. Among various metal ions (10 µM) added, the addition of Fe3+ or Al3+ 9
or Cr3+ ions to 1 (10 µM) engendered a new emission band centered at 583 nm with a concomitant reduction in the naphthalimide emission at 532 nm (Fig. 1b). These observed changes are ascribed due to the energy transfer (ET) from naphthalimide donor to the pirolactam ring opened rhodamine moiety of 1 that is resulted from the metal ion chelation as shown in Fig. S4, Supporting Information (Chereddy et al., 2013). These observed variations in the absorbance and fluorescence characteristics of 1 indicated its trivalent metal ion selective detection ability. Further, metal ion competition experiments were conducted to assess the effect of the presence of other metal ions on the trivalent metal ion selective detection ability of 1. These experiments were performed by adding Fe3+ ions (10 µM) to 1 in presence of other monovalent and divalent ions (20 µM) and monitoring the variations in the probes emission profiles. The results revealed that 1 could be used to detect Fe3+ even in the presence of other metal ions. Metal ion competition experiments were also performed using Al3+ or Cr3+ ions and the results portrayed the probes selectivity towards trivalent metal (Fe3+ or Al3+ or Cr3+) ions (Fig. 2). 3.3 Sensitivity of 1 towards the trivalent metal ions Since the addition of Fe3+ or Al3+ or Cr3+ ions has significant effect on the absorption and emission characteristics of 1 (Fig. 1), the affect of the addition of incremental concentrations of these metal ions on the probe’s absorption and fluorescence characteristics was investigated. The addition of Fe3+ ions (0-30 µM) to 1 developed a new absorption band centered at 562 nm, its intensity was increased gradually with the amount of Fe3+ added and reached maximum at 20 µM of Fe3+ (Fig. 3a). At this concentration, 1 displayed 224-fold enhancement in its absorbance at 562 nm. Similar affect was observed even with Al3+ or Cr3+ ions (Figs. S5a and S6a, Supporting Information). But, the extent of increment in the absorption intensity at 562 nm was higher for Fe3+ ions compared to Al3+ or Cr3+ ions, 10
indicated the strong binding affinity of 1 with Fe3+ over Al3+ or Cr3+ ions (Fig. S7a, Supporting Information). Binding constants of the respective 1-metal ion complexes were calculated from the absorption data (Fig. S8, Supporting Information) and the values are provided in Table S1, Supporting Information. Under similar conditions, the addition of Fe3+ ions opened the spirolactam ring of the rhodamine moiety of 1 and allowed the Förster resonance energy transfer from naphthalimide to rhodamine (Fig. 3b). The ratio of the emission intensities of 1 at 583 and 532 nm (I583/I532) was linearly increased with the amount of Fe3+ added in the concentration range of 0-8 µM (R2 = 0.996) and reached maximum at 12 µM of Fe3+. Similar kind of results was obtained even with the Al3+ or Cr3+ions (Figs. S5b and S6b, Supporting Information). The detection limit of 1 for Fe3+ or Al3+ or Cr3+ ions were calculated separately from the UV-Visible and fluorescence data (Figs. S9 and S10, Supporting Information) and the values are presented in Table S1, Supporting Information. These values indicated that 1 can detect the trivalent metal ions (Fe3+ or Al3+ or Cr3+) even at sub-micromolar levels. 3.4 Job plot and ESI-MS analysis of 1-trivalent metal ion complexes The stoichiometry of 1-trivalent metal ion complexes was estimated using Job plot analysis and it was found to be 1:1 in nature (Fig. S11, Supporting Information). The 1:1 stoichiometry of these complexes was further confirmed using ESI MS analysis (Fig. S12, Supporting Information). 3.5 Effect of pH and MTT assay Stability over a broad range of pH and non-toxicity are the key criteria to a chemosensor for biological applications. The acid-base titration experiments were performed to understand the effect of pH on the absorption and emission characteristics of 1. The results revealed that either the absorption or the emission pattern of 1 were unaltered in the range of 11
pH 5-10 (Fig. S13, Supporting Information) and suggested its suitability for the detection of Fe3+ or Al3+ or Cr3+ ions over a broad range of pH. Cell viability experiment (MTT assay) was performed to assess the non-cytotoxic nature of 1 (Fig. S14, Supporting Information). The W138 cells were viable (more than 90%) even after 48 hours of their incubation with 1 at 25 µM concentrations. The observed high selectivity and sensitivity for trivalent metal ions and non-interference from other competitive metal ions, stability at physiological pH and non-toxicity under experimental conditions designated the eligibility of 1 for live cell imaging application. 3.6 Detection and imaging of metal ion contaminated live cells We further explored the possibility for the imaging of trivalent metal ion contaminated W138 (normal lung fibroblast) cells by staining with probe 1 (Fig. 4). Two sets of fluorescence images were obtained from green channel and red channel, respectively. The cells alone (a1-c1) and cells treated with Fe3+ (10 µM) alone (a2-c2) did not show significant fluorescence in both green and red channels. Whereas the cells incubated with 1 (10 µM) clearly displayed bright intracellular green fluorescence (a3-c3) indicated that probe 1 is cell membrane permeable. When cells loaded with probe 1 were exposed to exogenous Fe3+ ions (10 µM) in DMEM culture medium for 30 min at 37 0C, an intense red fluorescence was observed with a concomitant decrease in green fluorescence (a4-c4). Similar results were obtained for Al3+ and Cr3+ ions also (Figs. S15-S16, Supporting Information). Thus, the data showed in Fig. 4 and Figs. S15-S16, Supporting Information clearly demonstrated the ability of probe 1 in monitoring trivalent metal ions present in live cells. 3.7 TCSPC and femtosecond fluorescence up-conversion analysis Fluorescent probes with target induced variations in their fluorescence lifetimes are useful to monitor the intracellular metal ion concentrations using Fluorescence Lifetime 12
Imaging Microscopy (FLIM) technique (Daen et al., 2012). Since probe 1 is suitable for the detection of intracellular trivalent metal ions using fluorescence microscopy, the effect of the trivalent metal ions on its fluorescence lifetime characteristics were studied to assess applicability of 1 in fluorescence lifetime imaging microscopic analysis. Time resolved fluorescence studies employing time correlated single photon counting (TCSPC) technique is used to understand the energy transfer dynamics in 1. As shown in Figs. S17-S18, Supporting Information, the time profile of the fluorescence signals at 520 nm, remained unaffected (Fig. S18, Supporting Information) while the fluorescence counts get reduced in commensurate with the addition of trivalent metal ions in a given collection time. Interestingly, change of fluorescence counts as a function of added metal ions at a given time of the decay profile follows the same pattern as observed in steady state fluorescence quenching of naphthalimide emission (Fig. 1b). This result apparently indicates two facts: Firstly, TCSPC measurement is only recording the decay profile of effective residual emission of uncomplexed 1 and rate of reduction of fluorescence upon addition of trivalent metal ions is happening within the IRF time and it is 200 ps for our system, secondly, rhodamine is not a FRET acceptor, fluorescence quenching is resulted from the re-absorption of naphthalimide emission by rhodamine. To apprehend the real fact, we employed femtosecond fluorescence up-conversion technique monitoring the early time dynamics of 1 in absence and in presence of trivalent metal ions. As shown in Fig. 5, the time profile of fluorescence up-conversion signal of the dyad probe 1 alone at 520 nm follows bi-exponential fit with 1 ps (
